Lionel Rougé-«

Lionel Rouge

Travis W Bainbridge

Michael Kwok

Till Maurer

James A Ernst
email: ernst.james@gene.com

Jeremy Murray
email: murray.jeremy@gene.com

Lionel Rouge ´

Raymond Tong

Paola Di Lello

Ingrid E Wertz

P.D Adams

Afonine, P.V Bunko ´czi

Cryo-Electron Microscopy DNA: DeoxyriboNucleic Acid PCR: Polymerase Chain Reaction

Keywords: Ubiquitin proteasome system, ubiquitin, deubiquitinase (DUB), USP7 (HAUSP7), MDM2, p53, protein engineering, enzymology, x-ray crystallography, electron microscopy, negative stain, TRAF domain, catalytic domain, ubiquitin like domain, unstructured peptide, allosteric activation, switching loop, structure-based drug design, allosteric inhibitors Herpesvirus-Associated Ubiquitin-Specific Protease 7 PDB: Protein Data Bank SPR: Surface Plasmon Resonance SBDD: Structure Based Drug Discovery Fab: antigen-antibody Fragment UPS: Ubiquitin-Proteasome System ALP: Autophagy-Lysosome Pathway ATP: Adenosine Tri-Phosphate Tumor necrosis factor Receptor -Associated Factor CD: Catalytic Domain UBL: Ubiquitin-Like DUB: Deubiquitinase AMC: 7-amino-4-methylcoumarin

Lionel Rougé-« Study of the intramolecular activation mechanism of the deubiquitinase USP7 » -BioSPC -2019 Lionel Rougé-« Study of the intramolecular activation mechanism of the deubiquitinase USP7 » -BioSPC -2019 2

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26S. Ce système est basé sur l'addition de façon covalente d'une protéine de 76 résidus appelée l'ubiquitine sur la protéine substrat grâce à une succession de trois réactions enzymatiques. Par la suite, de nouvelles ubiquitines peuvent être ajoutées de façon covalente à l'ubiquitine initiale formant des chaînes de poly-ubiquitine. Ce procédé d'ubiquitination est réversible et utilise des enzymes de désubiquitination dites deubiquitinases (DUBs) afin de retirer les chaînes d'ubiquitine permettant ainsi aux substrats de retrouver leur état natif.

La protéase spécifique de l'ubiquitine (USP) n° 7 est une DUB qui joue un rôle majeur dans différents procédés cellulaires et notamment dans la régulation de p53, un facteur de transcription impliqué dans le maintien de l'intégrité du génome, ainsi que de son ubiquitine ligase MDM2.

Compte tenu de son rôle clef dans de nombreux mécanismes cellulaires, la régulation de son activité est facilitée par son organisation sous forme de multiples domaines. Dans sa région Nterminale, USP7 possède un domaine TRAF impliqué dans la reconnaissance des substrats (comme p53 et MDM2) suivi d'un domaine catalytique (CD) puis de cinq domaines similaires à l'ubiquitine (ubiquitin-like domains ou UBL) egalement impliqués dans la reconnaissance de substrats (comme DNMT1 ou ICP0). Enfin, la protéine se termine par une région peu caractérisée composée d'une vingtaine d'acides aminés et décrite comme essentielle pour récapituler l'activité enzymatique.

Une série d'études enzymatiques ont démontré que le domaine catalytique d'USP7, lorsque isolé des autres domaines, possède une faible activité. En effet, en absence de substrat, la triade catalytique n'est pas correctement orientée, empêchant toute activité protéolytique. En revanche, la structure du domaine catalytique en présence d'ubiquitine révèle un changement conformationnel important réalignant sa triade catalytique mais aussi permettant d'accommoder l'ubiquitine. Un important réarrangement est observé au niveau d'une boucle (appelée "switching loop") qui passe d'une conformation inhibitrice en absence d'ubiquitine à une conformation active lorsque l'ubiquitine est présente. Au-delà du domaine catalytique, les structures des différents domaines d'USP7 ont été déterminées et ont permis de caractériser les principaux mécanismes moléculaires utilisés par USP7 pour reconnaître ses multiples substrats. En revanche, l'organisation intra-domaines dans le contexte de la protéine dans sa totalité reste assez floue.

Mots-clés :

Protéase, deubiquitinase (DUB), USP7, MDM2, p53, ubiquitine, système ubiquitine-protéasome, régulation, allostérie, activation, domaine catalytique, changement conformationnel, inhibiteurs, ingénierie des protéines, cristallographie, enzymologie, microscopie électronique.

Abstract:

Ubiquitination is a key post translational modification that regulates the fate of proteins within the cells. It is an energetically costly event consisting of three successive enzymatic reactions. The final step results in the covalent addition of ubiquitin (a 76 residues protein) onto a lysine side chain of the substrate protein, through an isopeptide bond; this final reaction is catalyzed by enzymes called ubiquitin ligases. Depending on the signaling pathway, additional ubiquitin chains may be built upon the original monomer through covalent linkage between the Cterminal carboxylic group of the incoming ubiquitin and one of the seven lysines decorating the preceding molecule. The linkage specificity of these ubiquitin chains is a key determinant that dictates the fate of the substrate. For example, Lys48-linked poly-ubiquitination targets substrates for proteasomal degradation whereas other linkages can modulate signaling pathways (i.e. NF-kB) or protein localization. Deubiquitinating enzymes (termed deubiquitinases or DUBs) are a class of proteases that can reverse ubiquitination. To date, seven different classes of DUBs with diverse cellular roles have been described, where the largest and most well-studied class are the ubiquitin specific proteases (USPs). Structurally, USPs are papain-like proteases and possess a highly conserved catalytic triad.

Among the USP family, USP7 plays a pivotal role in regulating proteins involved in fundamental cellular processes, such as stability of the tumor suppressor p53. USP7 deubiquinates both p53 and its ubiquitin ligase, MDM2, and dysregulation of the equilibrium between p53 ubiquitination and deubiquitination has been shown to lead to cell cycle alteration, abnormal cell growth and cancer. USP7 is a 1102 amino acid protein composed of a substrate-recognition TRAF domain, a catalytic domain and five ubiquitin-like protein-protein interaction domains followed by a C-terminal unstructured region. Although a combination of biochemical, structural and enzymatic studies has provided some insights into the roles of these individual domains, the mechanisms underlying substrate recognition and catalytic activation remain poorly understood.

In this study, we elucidated the molecular mechanism by which the C-terminal 19 amino acids of USP7 (residues 1084-1102) enhance the catalytic efficiency of the catalytic domain. Our studies past 12 years. They have supported my requests to gain additional training in X-ray crystallography and cryo-electromicroscopy and have allowed me to expand the scope of my responsibilities within the department by becoming lead structural biologist on several projects. This manuscript is a manifestation of the supportive culture established at Genentech which readily promotes and encourages the career development of its employees.

I would like to thank the entire USP7 team for their great work and, in particular, Travis Bainbridge, Dr. Jeremy Murray and Dr. James Ernst for their valuable feedback on this manuscript and for allowing me to include some of their data. Travis has been instrumental in generating most of the enzymatic data, Jeremy solved all structures and both Jeremy and James's leadership helped direct this project to its completion. Jeremy is an expert crystallographer and has been a great mentor throughout the years. I would also like to thank Dr. Till Maurer, Dr. Paola Di Lello, Michael Kwok, the entire Biomolecular group (responsible for the cloning of all chimeric constructs) and all the people who participated in the collection of the crystallographic data at diverse synchrotrons. I would also like to give a special thanks to Ingrid Wertz for her great leadership as the USP7 team leader. It has been an absolute honor and pleasure to work under her guidance and to learn from her example. Ingrid's feedback has been invaluable. I would also like to thank the PAK1 and anti-BACE1 team, with special recognition for Dr. Weiru Wang, lead crystallographer on both projects, with whom I have worked in close collaboration and who taught me a lot. I would like to thank Mme Oriane Royon Da Silva for her guidance during this process and for providing me with the tools and recommendations that helped me structure this manuscript.

Her advices were instrumental in the redaction of the first part, namely reflecting upon my career and describing my development as a scientist. I would also like to thank her for coordinating many other administrative aspects related to the VAE.

Lionel Rougé-« Study of the intramolecular activation mechanism of the deubiquitinase USP7 » - BioSPC -2019 Lionel Rougé-« Study of the intramolecular activation mechanism of the deubiquitinase USP7 » -BioSPC -2019 10 I would like to thank Dr. Daniel Ladant for agreeing to guide me as my scientific supervisor for this VAE and for forming my defense committee. I really appreciate the time he has committed to help me and for his continued support.

I would like to thank the Université of Paris Diderot and l'Ecole doctorale BioSPC for giving me the opportunity to participate in this unique program. I am thankful to those who reviewed my application and to the members of the jury who will assess my work.

Finally, I thank my wife Zoe for inspiring me to continue my education many years after obtaining my last degree, for encouraging me at every step of this journey but mostly for cheering me up whenever stress got the best of me.

And thanks to my mother Olga, my father Robert and my sister Nathalie for their support (and for listening to my occasional rants).

In gratitude, The Ubiquitin -Proteasome System a)

Lionel Rougé

Dedicated to Alice, Ursula, Isidore & Laurentino

Table of contents

The Proteostasis Network

Quality control of protein integrity is implemented at several stages of the protein life cycle and spans from protein synthesis, folding and post-translational processing. This tightly regulated system controlling protein homeostasis (proteostasis) is essential to ensure a healthy proteome and is regulated by various sophisticated and coordinated biological systems processes known as the proteostasis network (PN). Alterations of the activity of the PN will prevent cells from clearing unfolded, misfolded proteins or damaged organelles and will potentially lead to disturbance in cell physiology associated with aging and age-related diseases, including neurodegeneration, cancer, and immunological and metabolic diseases [START_REF] Labbadia | The biology of proteostasis in aging and disease[END_REF][START_REF] Yerbury | Walking the tightrope: proteostasis and neurodegenerative disease[END_REF].

These PN systems are tightly linked to the translational machinery, ensure proper folding through molecular chaperones and cochaperones and are involved in protein degradation (see Figure 1). For example, during protein translation, aberrant mRNA molecules are processed and regulate degradation of the nascent polypeptide chain (U. [START_REF] Schubert | Rapid degradation of a large fraction of newly synthesized proteins by proteasomes[END_REF][START_REF] Sha | The eIF3 Interactome Reveals the Translasome, a Supercomplex Linking Protein Synthesis and Degradation Machineries[END_REF].

During polypeptide chain synthesis on ribosomes, molecular chaperones are contributing not only to proper folding of these newly synthesized chains but can also facilitate protein degradation when proper folding fails [START_REF] Mcclellan | Protein quality control: chaperones culling corrupt conformations[END_REF]. Although these systems can effectively control normal degradation of defective proteins, extensive damage exceeding intracellular refolding and degradative capacity will sequester these unfolded proteins into dedicated compartments where they await clearance through different mechanisms (i.e., inclusion bodies or aggresomes, e.g., juxtanuclear quality control compartments and insoluble protein deposits) [START_REF] Specht | Hsp42 is required for sequestration of protein aggregates into deposition sites in Saccharomyces cerevisiae[END_REF].

When protein functionality cannot be restored from misfolded and aggregated states, the cell utilizes two major degradation systems: the ubiquitin-proteasome system (UPS) [START_REF] Finley | Recognition and processing of ubiquitin-protein conjugates by the proteasome[END_REF] and the autophagy-lysosome pathway (ALP) (Zhifen [START_REF] Yang | Mammalian autophagy: core molecular machinery and signaling regulation[END_REF]. Nobel prizes were awarded in 2004 to Aaron Ciechanover, Avram Hershko, and Irwin Rose for the discovery The UPS is the primary proteolytic route that relies on degradation of short-lived, misfolded, and damaged proteins through proteasomes, large multisubunit assemblies consisting of a 19S regulatory cap recognizing and unfolding ubiquitylated substrates and a 20S proteolytic core which function is to rapidly degraded these substrates into peptides [START_REF] Coux | Structure and functions of the 20S and 26S proteasomes[END_REF][START_REF] Finley | Recognition and processing of ubiquitin-protein conjugates by the proteasome[END_REF]. Because of physical limitations of the central pore of the 20S core, clearance of large protein complexes, damaged organelles or aggregates is operated through lysosomal degradation via the ALP. In this pathway, cytoplasmic substrates are sequestered into double-membrane vesicle structure known as autophagosomes which will subsequently fuse with lysosomal vesicles leading to the formation of an autolysosome and degradation of its cargo (Zhifen [START_REF] Yang | Mammalian autophagy: core molecular machinery and signaling regulation[END_REF]. Another key function of this pathway is to provide alternative source of energy when the cell is subject to various cellular stresses (nutrient deprivation, hypoxia, oxidative stress, etc.) [START_REF] Lamb | The autophagosome: origins unknown, biogenesis complex[END_REF].

Figure 1: Overview of the proteostasis network

The proteostasis network is composed of three interconnected signaling pathways: the UPS, the ALP and the chaperone pathway. The main goal of the chaperone system is to prevent protein aggregation through a constant assessment of protein's folding state; when chaperones fail to maintain conformational integrity of their substrates (i.e. irreversible misfolding), proteins are finally directed towards the UPS or autophagy pathway for proteolytic degradation. From Hartl, Bracher, & Hayer-Hartl, 2011 (with permission). b)

The Ubiquitin Code

The UPS pathway accomplishes degradation of the majority of cellular proteins and is centered around a key protein: ubiquitin. Ubiquitin is a small, highly conserved regulatory protein composed of 76 amino acids which can be coupled to the ε-amino group of a lysine side chain;

this reversible reaction is called ubiquitination which is a multi-step process, involving consecutive action of three enzymes: during the first step, the ubiquitin-activating enzyme E1 uses ATP to form a thioester bond between a cysteine residue in its active site and the C-terminal glycine of ubiquitin. Activated ubiquitin is then transferred to a ubiquitin-conjugating enzyme E2 with the formation of second thioester intermediate. Finally, ubiquitin is transferred to a protein substrate with help of E3 ligase [START_REF] Glickman | The Ubiquitin-Proteasome Proteolytic Pathway: Destruction for the Sake of Construction[END_REF]. There are three types of E3 ligases that act through different mechanisms: the enzymatic homologous to E6-AP C terminus (HECT) domain E3s, the RING E3 domains (Really Interesting New Gene) and the RING-IBR (in between RING fingers)-RING (see Figure 2 below). Ubiquitin activating enzymes (E1, 2 known in humans) activates and conjugates ubiquitin to its active site cysteine forming a thiol-ester bond trough in an ATP dependent manner. Ubiquitin is then transferred onto the active site cysteine of an E2 enzyme (conjugating enzymes, about 40 in humans). The last step involves the transfer of ubiquitin onto its final substrate through an E3 ligase (about 1000 have been discovered so far). Three types of E3s have been described so far and differ in their mechanism of action: in the case of the RING ligases (~ 600 predicted), ubiquitin will be directly conjugated onto its substrate whereas HECT ligases will form a thioester linked intermediate with ubiquitin before transferring it to its substrate. The RING-IBR-RING have a hybrid mode of action. Finally, deubiquitinases can reverse this process by removing ubiquitin chains from their substrates.

Courtesy of Dr Tong at University of Toronto, with permission.

RING E3 function as adaptors by bringing together an E2 -ubiquitin complex and a substrate promoting the attack of the substrate lysine residue on E2 -ubiquitin thioester (Deshaies & Lionel Rougé-« Study of the intramolecular activation mechanism of the deubiquitinase USP7 » -BioSPC -2019

Lionel Rougé-« Study of the intramolecular activation mechanism of the deubiquitinase USP7 » -BioSPC -2019 18 Joazeiro, 2009;[START_REF] Joazeiro | RING finger proteins: mediators of ubiquitin ligase activity[END_REF]. E3 ligases of the HECT family receive ubiquitin from E2 with formation of the third thioester intermediate and then transfer it to the substrate [START_REF] Huibregtse | A family of proteins structurally and functionally related to the E6-AP ubiquitin-protein ligase[END_REF][START_REF] Kee | Regulation of catalytic activities of HECT ubiquitin ligases[END_REF]. Recently, the discovery of RING-IBR-RING (RBR, ~ 10 in the human genome) proteins defined a third class of ubiquitin ligases; these proteins are RING-HECT hybrid E3s defined by a RING1 domain that binds E2 Ub-conjugating enzyme and a RING2 domain that contains an active site cysteine similar to HECT-type E3s [START_REF] Dove | Molecular insights into RBR E3 ligase ubiquitin transfer mechanisms[END_REF][START_REF] Wenzel | UBCH7 reactivity profile reveals parkin and HHARI to be RING/HECT hybrids[END_REF].

Proteomics studies have shown that, even though ubiquitin is pretty small, it can be heavily post-translationally modified through phosphorylation or lysine acetylation (Choudhary, Lundly) (F. [START_REF] Ohtake | Ubiquitin acetylation inhibits polyubiquitin chain elongation[END_REF]. These PTMs add another level of regulation in order to tightly control this essential cellular pathway. One of the better understood modification is phosphorylation of Ser65 and its role in activating the E3 ligase Parkin [START_REF] Gladkova | Mechanism of parkin activation by PINK1[END_REF]; A.

F. [START_REF] Schubert | Structure of PINK1 in complex with its substrate ubiquitin[END_REF] along with its implication in mitophagy. Recently, an enzyme found is

Legionella was shown to hijack the host-cell ubiquitination pathway by adding phosphoribosyl groups to Arg42 of ubiquitin [START_REF] Dong | Structural basis of ubiquitin modification by the Legionella effector SdeA[END_REF].

The degree and type of ubiquitin linkage dictates the fate and the function of the target protein.

Protein monoubiquitination often takes place in various cellular pathways such as intracellular and membrane trafficking, DNA transcription and DNA repair [START_REF] Sigismund | Signaling through monoubiquitination[END_REF].

Addition of ubiquitin monomers to multiple lysines has also been reported and can lead to endosome formation and degradation of the substrate in the lysosome [START_REF] Herrmann | Ubiquitin and Ubiquitin-Like Proteins in Protein Regulation[END_REF].

Polyubiquitination consists of the conjugation of ubiquitin monomers attached to each other through a linkage between one of seven lysine residues (Lys6, Lys11, Lys27, Lys29, Lys33, Lys48, and Lys63) of one monomer and the C-terminal glycine of another. Chains linked through N-terminal methionine can also be formed and are called "linear" chains (Komander & Rape, 2012). A number of crystal and NMR structures of these chains (typically dimers) have been solved

over the years and have shown two types of conformations: an elongated one and a more compact (see Supplemental Figure 3 for details). Different polyubiquitin chain linkage will direct the substrate to different pathway (see Figure 3). As indicated below, substrates marked with a Lys48and Lys11-linked chains are known to target proteins to proteasome for degradation (Xu et al., ) while other linkage types are involved in distinct signaling pathways. Specifically, Lys63linked chains play a role in regulating signal transduction, DNA repair and endocytosis [START_REF] Erpapazoglou | Versatile roles of k63-linked ubiquitin chains in trafficking[END_REF][START_REF] Panier | Regulatory ubiquitylation in response to DNA double-strand breaks[END_REF], and linear polyubiquitin (Met1-linked) is important in inflammatory cytokine signaling [START_REF] Iwai | Linear ubiquitin chains: NF-κB signalling, cell death and beyond[END_REF]. Lys6-linked chains have been identified to play a role in mitophagy [START_REF] Cunningham | USP30 and parkin homeostatically regulate atypical ubiquitin chains on mitochondria[END_REF] along with Lys11 chains [START_REF] Birsa | Lysine 27 ubiquitination of the mitochondrial transport protein Miro is dependent on serine 65 of the Parkin ubiquitin ligase[END_REF]. Existence of so-called "branched" chains has also been identified by mass spectrometry and some preliminary data suggests this type of linkage might be involved in NF-𝛋𝛋B pathway activation (Fumiaki Ohtake, Tsuchiya, Saeki, & Tanaka, 2018). Recently, it was shown that mixed ubiquitin chains composed of Lys11/Lys48

were involved in the clearance of aggregation prone proteins [START_REF] Yau | Assembly and Function of Heterotypic Ubiquitin Chains in Cell-Cycle and Protein Quality Control[END_REF]. Deubiquitinases role in the UPS Protein ubiquitination can be reversed by action of deubiquitinating enzymes (DUBs) that cleave ubiquitin moieties from their substrates through their isopeptidase activity; about 100 DUBs have been identified in the human genome consisting of 90 cysteines proteases and 10 metalloproteases [START_REF] Nijman | A genomic and functional inventory of deubiquitinating enzymes[END_REF]. The zinc-dependent metalloprotease structural class, JAB1/MPN/Mov34 (JAMM), consists of 12 members [START_REF] Komander | Mechanism, specificity and structure of the deubiquitinases[END_REF].

Metalloproteases coordinate a zinc ion and a water molecule in the active site; during catalysis, a hydrogen atom from the water molecule is abstracted by a neighboring glutamate residue and the resulting hydroxyl ion attacks the carbonyl carbon of the isopeptide bond.

Outside of the JAMMs, most of the DUB belong to the superfamily of 'papain-like' cysteine proteases which consists of 6 structural classes: ubiquitin specific protease (USP or UBP in yeast), ubiquitin C-terminal hydrolases (UCH), ovarian tumor (OTU), Josephin domain (MJD), motif interacting with ubiquitin-containing novel DUB family (MINDY) and zinc finger with UFM-1 specific peptidase (ZUFSP). These last two classes were recently discovered and, to date, contain only a few members: four members for the Lys48-linked polyubiquitin selective MINDYs [START_REF] Abdul Rehman | MINDY-1 Is a Member of an Evolutionarily Conserved and Structurally Distinct New Family of Deubiquitinating Enzymes[END_REF] and only one member for the Lys63-linked polyubiquitin selective ZUFSP family [START_REF] Kwasna | Discovery and Characterization of ZUFSP/ZUP1, a Distinct Deubiquitinase Class Important for Genome Stability[END_REF].

All cysteine peptidases share a catalytic triad consisting of cysteine, histidine and aspartate/asparagine residues and their catalytic mechanism is similar to a family of plant enzymes called papains [START_REF] Storer | Catalytic mechanism in papain family of cysteine peptidases[END_REF]. The role of the histidine is to lower the pKa of the catalytic cysteine and deprotonate the thiol group in order to initiate its nucleophilic attack on the isopeptide bond, while the aspartate restricts side-chain rotation of the histidine and polarizes it (Figure 4). (Hanpude, Bhattacharya, Dey, & Maiti, 2015, with permission).

Structurally, proteins of UCH family are relatively small and do not have domains other than the catalytic core while DUBs from other families often have varying numbers of additional domains with distinct structures and functions (see Figure 5)

24

In the cell, DUBs can perform multiple tasks. As we have seen earlier, DUBs can remove polyubiquitin chains from their substrates thus reversing ubiquitin signaling. But DUBs can also edit the conjugated ubiquitin chains hence redirecting the fate of the substrate. For example, in the case of A20, Lys63-linked ubiquitin chains are disassembled so Lys48-linked chains can be formed through A20's own E3 ligase activity [START_REF] Wertz | De-ubiquitination and ubiquitin ligase domains of A20 downregulate NF-κB signalling[END_REF]. Finally, DUBs are also involved in ubiquitin homeostasis through maintaining the pool of free cellular ubiquitin and processing free ubiquitin after translation; indeed, the gene encoding for ubiquitin is expressed as a linear fusion of multiple monomers and DUBs allow release of free ubiquitin into the cellular pool (Komander et al., 2009).

b) Specificity of DUBs

Over the course of evolution, DUBs have achieved catalytic specificity by successfully distinguishing between many ubiquitin-like molecules (ubiquitin vs neuronal precursor cell expressed developmentally downregulated protein 8 or NEDD8 vs small ubiquitin-like modifier or SUMO vs interferon-stimulated gene 15 or ISG15), different peptide bonds (isopeptide vs linear peptide) but also different types of linkages and chain topology. Across all DUBs, ubiquitin binding is achieved in a similar manner; the distal ubiquitin interacts with DUBs through its canonical interaction site called the Ile44 patch while its unique and conserved C terminus "tail" (Leu71-Arg72-Leu73-Arg74-Gly75-Gly76) extends into the catalytic site (X. [START_REF] Zhu | High incidence of ubiquitin-like domains in human ubiquitin-specific proteases[END_REF], conferring specificity over the different C terminal sequences found in ubiquitin like molecules (i.e. Leu-Ala-Leu-Arg-Gly-Gly in NEDD8 or Gln-Gln/Glu-Gln-Thr-Gly-Gly in SUMO).

DUBs are also amenable to distinguish and process different bonds, either typical peptide bonds (in the case of linear chains) or isopeptide bonds (all other linkages). Work from Komander et al., showed that linear chains were most likely processed by USPs (although with low efficiency) and that OTUs, JAMMs and UCHs were catalytically unable to process peptide bonds (with the exception of OTU domain-containing deubiquitinase with linear linkage specificity (OTULIN) which specifically cleaves Met1-linked polyubiquitin chains [START_REF] Keusekotten | OTULIN antagonizes LUBAC signaling by specifically hydrolyzing Met1-linked polyubiquitin[END_REF]).

Specificity for different chain linkages have been reported for MINDY-1, USP, ZUFSP and OTU-containing DUBs. For example, the 26S proteasome-associated USP14 specifically catalyzes cleavage of Lys48 linked ubiquitin chains [START_REF] Hu | Structure and mechanisms of the proteasome-associated deubiquitinating enzyme USP14[END_REF] whereas CYLD efficiently processes Lys63 linked and linear chains [START_REF] Komander | The structure of the CYLD USP domain explains its specificity for Lys63-linked polyubiquitin and reveals a B box module[END_REF]; a structure of AMSH-LP in complex with Lys63 linked di-ubiquitin reveals how the unique sequence around Lys63 is specifically recognized by AMSH-LP allowing for selectivity over other chains. More recently, USP30 has been shown to favor Lys6-linked ubiquitin chains [START_REF] Gersch | Mechanism and regulation of the Lys6-selective deubiquitinase USP30[END_REF][START_REF] Sato | Structural basis for specific cleavage of Lys6-linked polyubiquitin chains by USP30[END_REF] and the molecular basis of Lys11-linked chains specificity of the OTU Cezanne have also been reported [START_REF] Mevissen | Molecular basis of Lys11-polyubiquitin specificity in the Lionel Rougé-« Study of the intramolecular activation mechanism of the deubiquitinase USP7 » -BioSPC -2019 Lionel Rougé-« Study of the intramolecular activation mechanism of the deubiquitinase USP7 » -BioSPC -2019 116 deubiquitinase Cezanne[END_REF]. (Swatek & Komander, 2016, with permission).

Another layer of specificity comes from the ability of the DUBs to cleave ubiquitin chains from the end (exo), exclusively generating monoubiquitins such as USP14 [START_REF] Hu | Structure and mechanisms of the proteasome-associated deubiquitinating enzyme USP14[END_REF] or internally (endo) like CYLD or A20 [START_REF] Komander | The structure of the CYLD USP domain explains its specificity for Lys63-linked polyubiquitin and reveals a B box module[END_REF][START_REF] Lin | Molecular basis for the unique deubiquitinating activity of the NF-kappaB inhibitor A20[END_REF].

c) DUBs and their regulation

Given their key role in many cellular pathways, DUB activities (just like other proteases) are often low, indicating tight regulation mechanisms requiring conformational reorganization in order to prime the catalytic triad for activation. These mechanisms include post-translational modifications (PTM), cellular recruitment to substrates and/or proteasome, inner structural rearrangements upon substrate binding and, finally, allosteric interactions with other proteins that can either activate or inhibit the enzyme (Sahtoe & Sixma, 2015).

PTM of DUBs are numerous: phosphorylation is an essential pathway controlling DUB activity with many examples of events activating DUBs such as Ser177 of DUBA (see Figure 7a) (O. W. [START_REF] Huang | Phosphorylation-dependent activity of the deubiquitinase DUBA[END_REF] or inhibiting them in the case of USP8 (Mizuno, Kitamura, & Komada, ) or CYLD [START_REF] Hutti | Phosphorylation of the tumor suppressor CYLD by the breast cancer oncogene IKKepsilon promotes cell transformation[END_REF]. CYLD can also be negatively modified through sumoylation [START_REF] Kobayashi | Deubiquitinating activity of CYLD is impaired by SUMOylation in neuroblastoma cells[END_REF]. In addition to sumoylation, ubiquitylation can also regulate DUB activity either by modulating enzyme activity [START_REF] Meray | Reversible monoubiquitination regulates the Parkinson disease-associated ubiquitin hydrolase UCH-L1[END_REF][START_REF] Todi | Lionel Rougé-« Study of the intramolecular activation mechanism of the deubiquitinase USP7 » -BioSPC -2019 Lionel Rougé-« Study of the intramolecular activation mechanism of the deubiquitinase USP7 » -BioSPC -2019 121 Ubiquitination directly enhances activity of the deubiquitinating enzyme ataxin-3[END_REF] or by affecting cellular localization. For example, ubiquitylation of BAP1 in close proximity of its nuclear localization sequence prevents its translocation to the nucleus therefore separating it from most of its targets [START_REF] Mashtalir | Autodeubiquitination protects the tumor suppressor BAP1 from cytoplasmic sequestration mediated by the atypical ubiquitin ligase UBE2O[END_REF]. Finally, some DUBs can be proteolyzed such as USP1 which can be inactivated through auto-proteolysis at an intrinsic Leu-Leu-Gly-Gly motif mimicking substrate sequence (T. T. [START_REF] Huang | Regulation of monoubiquitinated PCNA by DUB autocleavage[END_REF] or A20, which can be degraded by the MALT1 protease [START_REF] Coornaert | [END_REF]. 27 DUBs can also be allosterically regulated and physically recruited to their relevant substrates through protein-protein interactions or mediated through other domains within the DUB itself. For example, many DUBs contain ubiquitin binding domains or motifs which are essential for deubiquitinase activity [START_REF] Kayagaki | DUBA: a deubiquitinase that regulates type I interferon production[END_REF][START_REF] Mao | Deubiquitinating function of ataxin-3: insights from the solution structure of the Josephin domain[END_REF][START_REF] Meulmeester | Mechanism and consequences for paralog-specific sumoylation of ubiquitin-specific protease 25[END_REF], linkage specificity [START_REF] Winborn | The deubiquitinating enzyme ataxin-3, a polyglutamine disease protein, edits Lys63 linkages in mixed linkage ubiquitin chains[END_REF] or responsible for an increase of catalytic efficiency [START_REF] Mccullough | Activation of the endosome-associated ubiquitin isopeptidase AMSH by STAM, a component of the multivesicular body-sorting machinery[END_REF][START_REF] Row | The MIT domain of UBPY constitutes a CHMP binding and endosomal localization signal required for efficient epidermal growth factor receptor degradation[END_REF]. One interesting example consists of the binding of free ubiquitin to a Zinc-finger domain of USP5 inducing a conformational change within the catalytic domain and improving its ability to process ubiquitin chain [START_REF] Reyes-Turcu | Study of the intramolecular activation mechanism of the deubiquitinase USP7 » -BioSPC -2019 Lionel Rougé-« Study of the intramolecular activation mechanism of the deubiquitinase USP7 » -BioSPC -2019 118[END_REF]. In some cases, another layer of regulation can be added in order to not only recognize ubiquitin chains, but also the substrate to deubiquitinate. This is the case for USP15 which utilizes its ubiquitin like domain to specifically recruit the E3 ligase BRCA-1associated protein (BRAP) [START_REF] Hayes | Direct and indirect control of mitogen-activated protein kinase pathway-associated components, BRAP/IMP E3 ubiquitin ligase and CRAF/RAF1 kinase, by the deubiquitylating enzyme USP15[END_REF]. As mentioned earlier, other proteins can modulate DUB function. For example, the WD40 domain containing protein USP1-associated factor 1 (UAF1 or WDR48) has been described to positively modulate at least three different USPs (USP1, USP12 and USP46) [START_REF] Cohn | UAF1 is a subunit of multiple deubiquitinating enzyme complexes[END_REF][START_REF] Cohn | A UAF1-containing multisubunit protein complex regulates the Fanconi anemia pathway[END_REF].

Structural studies of the complex between WDR48 and ubiquitin bound to USP46 showed that WDR48 plays a scaffolding role by interacting with both ubiquitin and the DUB (respectively with a SUMO like domain and ß-propeller) seemingly stabilizing the enzyme-substrate interaction (see Figure 7b) (Yin et al., 2015). Another example is the activation of BAP1 through ASXL1 to deubiquinate mono-ubiquinated Histone 2A of the Polycomb gene repression [START_REF] Sahtoe | BAP1/ASXL1 recruitment and activation for H2A deubiquitination[END_REF].

Another form of regulation comes from substrate-mediated recognition: DUBs can be activated upon ubiquitin and/or substrate binding. For example, binding of ubiquitin to UCH-L1 at an exosite can assist realignment of the catalytic site into an active conformation (see Figure 7c) [START_REF] Boudreaux | Ubiquitin vinyl methyl ester binding orients the misaligned active site of the ubiquitin hydrolase UCHL1 into productive conformation[END_REF]. In the case of OTULIN, the substrate promotes catalytic activity; indeed, this OTU specifically senses the unique linkage of linear ubiquitin chains allowing for a reorganization of its catalytic triad and explaining how OTULIN only affects pathways where linear polyubiquitin chains are used for signaling [START_REF] Keusekotten | OTULIN antagonizes LUBAC signaling by specifically hydrolyzing Met1-linked polyubiquitin[END_REF]. Finally, USP14 has been shown to reorganize its catalytic domain upon ubiquitin binding. Indeed, the crystal structure of the isolated catalytic domain shows that the catalytic cleft leading to its active site is occluded 28 by two surface loops. Binding by ubiquitin induces a significant conformational change of these loops allowing access of the ubiquitin C-terminus to the active site [START_REF] Hu | Structure and mechanisms of the proteasome-associated deubiquitinating enzyme USP14[END_REF].

d)

Involvement in diseases

Over the years, DUBs have been described to play central roles in tightly regulated pathways often linked to pathologies, especially cancers (D'Arcy, [START_REF] D'arcy | Deubiquitinase inhibition as a cancer therapeutic strategy[END_REF]. For example, multiple DUBs have been showed to play essential roles in DNA-repair (such as USP1) [START_REF] Nijman | The Deubiquitinating Enzyme USP1 Regulates the Fanconi Anemia Pathway[END_REF] and DNA-damage response (i.e. USP28) (Huang). Apoptosis has also been shown to be regulated through about 14 DUBs such as USP9x, USP28 and CYLD [START_REF] Ramakrishna | The role of deubiquitinating enzymes in apoptosis[END_REF]; direct or indirect modulation of the tumor suppressor protein p53 (key effector in apoptosis) has also been linked to USP2, USP10, USP22, USP42 and OTUD5. Another major DUB involved in p53 homeostasis is USP7 and will be discussed more in details later.

Hallmarks of neurodegenerative diseases such as Parkinson's or Alzheimer's disease consist in the accumulation of misfolded proteins leading to their aggregation and formation of plaques and fibrils toxic to neurons. Proper clearance of these structures through the UPS is essential to maintain healthy neuronal homeostasis and has been shown to be regulated by several DUBs [START_REF] Ristic | An optimal ubiquitin-proteasome pathway in the nervous system: the role of deubiquitinating enzymes[END_REF]. One key DUB studied at Genentech for its role in mitophagy and as a potential target in the treatment of Parkinson's disease is USP30. USP9X has also been shown to be linked to Parkinson's disease through its regulation of ubiquinated α-synuclein [START_REF] Rott | Synuclein fate is determined by USP9X-regulated monoubiquitination[END_REF]. Knockdown of USP9X expression induces an increase in levels of the population of monoubiquitinated α-synuclein and toxic inclusion bodies responsible for the death of dopaminergic neurons and the formation of Lewy bodies.

3.

The Ubiquitin Specific Protease 7

Human ubiquitin-specific protease 7 (USP7), alternatively known as Herpes virus associated protease (HAUSP) belongs to the USP family of DUBs and was discovered in 1997 as a protein binding to "infected cell polypeptide 0" (ICP0, also known as Vmw110) (Everett et al., 1997). Over the years, a large number of cellular and viral proteins have been identified to interact with USP7 and it gained increasing interest for its key regulatory role in many aspects of mammalian biology and disease by controlling the stability and localization of proteins involved in DNA replication, epigenetic regulation, cell cycle mitosis and cell survival. Most importantly, USP7 has been extensively studied in tumor biology due to its regulation of the tumor suppressor p53 and the p53 ubiquitin ligase, murine double minute 2 protein MDM2 (Brooks & Gu, 2006;Cummins et al., 2004;[START_REF] Li | Deubiquitination of p53 by HAUSP is an important pathway for p53 stabilization[END_REF].

a)

USP7 is a key regulator of the p53/MDM2 axis In 2002, Li et al. identified the transcription factor p53 as the first known cellular substrate known of USP7. p53, also known as the guardian of the genome, is a critical component in DNA damage response and is a key modulator of programmed cell death or apoptosis; in this study, p53

was shown to be stabilized upon overexpression of USP7 leading to apoptosis initiation and cell cycle arrest. The opposite phenotype was observed a couple of years later by the same group

showing that reduction of USP7 levels led to p53 destabilization (Li, Brooks, Kon, & Gu, 2004).

However, a surprising observation was made when USP7 was knocked down; instead of witnessing even higher levels of p53 degradation though ubiquitination, researchers described stabilization of p53 levels (Cummins et al., 2004). Additional work revealed that the p53 ligase MDM2 was also a substrate of USP7 and was actually a preferred substrate over p53 (Hu et al., 2006). Indeed, the presence of MDM2 is necessary to control cellular levels of p53; however, when USP7 activity (or protein levels) is diminished, MDM2 is cleared through the UPS and p53 halflife increases (see Figure 9). Because of its key role in this system, several levels of regulation have been described to positively or negatively modulate its processing of ubiquitin chains linked to p53 or MDM2. ATM phosphorylation of MDM2 has been shown to reduce its E3 ligase activity but also lower its affinity for USP7 [START_REF] Meulmeester | ATM-Mediated Phosphorylations Inhibit Mdmx/Mdm2 Stabilization by HAUSP in Favor of p53 Activation[END_REF]. More recently, the two proteins TSPY-like 5 (TSPYL5) [START_REF] Epping | TSPYL5 suppresses p53 levels and function by physical interaction with USP7[END_REF] and Abraxas brother 1 (ABRO1) (J. [START_REF] Zhang | ABRO1 suppresses tumourigenesis and regulates the DNA damage response by stabilizing p53[END_REF] were identified as, respectively, reducing or improving binding affinity of USP7 towards p53 hence affecting its stability. Similarly, the two proteins death domain-associated protein (DAXX) [START_REF] Tang | Death domain associated protein (Daxx), a multifunctional protein[END_REF] and the tumor suppressor Ras association domain family 1 protein (RASSF1A) [START_REF] Song | The tumour suppressor RASSF1A promotes MDM2 self-ubiquitination by disrupting the MDM2-DAXX-HAUSP complex[END_REF]) modulate USP7 binding to MDM2 [START_REF] Bhattacharya | Emerging insights into HAUSP (USP7) in physiology, cancer and other diseases[END_REF]. These examples of regulation are unique to the p53/MDM2 axis; as we will discuss later, many more intra-or inter-molecular factors modulate the activity of this essential DUB.

b) Other functions of USP7

As mentioned above, USP7 was first discovered while studying HSV-1 replication mechanism; its ability to stabilize ICP0, a viral E3 ligase essential for lytic infection [START_REF] Boutell | Regulation of alphaherpesvirus infections by the ICP0 family of proteins[END_REF] leads to an increase in virus replication. Later studies showed that two other viruses of the Herpesviridae family hijack USP7 during their replication cycle using different viral proteins: the latency-associated nuclear antigen (LANA) [START_REF] Jäger | The ubiquitin-specific protease USP7 modulates the replication of Kaposi's sarcomaassociated herpesvirus latent episomal DNA[END_REF] and viral interferon regulatory factor 1 [START_REF] Chavoshi | Identification of Kaposi Sarcoma Herpesvirus (KSHV) vIRF1 Protein as a Novel Interaction Partner of Human Deubiquitinase USP7[END_REF] and 4 (H.-R. [START_REF] Lee | Bilateral inhibition of HAUSP deubiquitinase by a viral interferon regulatory factor protein[END_REF]) from Kaposi's sarcomaassociated herpes virus (KSHV) along with its homologue EBNA-1 from Epstein-Barr virus (EBV) (Holowaty et al., 2003). Although ICP0 has been shown to be an actual USP7 substrate [START_REF] Canning | A RING finger ubiquitin ligase is protected from autocatalyzed ubiquitination and degradation by binding to ubiquitinspecific protease USP7[END_REF], these other viral proteins such as EBNA-1 use USP7 to modulate p53 levels by competing for p53 binding on USP7 (Saridakis et al., 2005).

Finally, the E1B-55K protein from adenovirus [START_REF] Ching | A ubiquitin-specific protease possesses a decisive role for adenovirus replication and oncogene-mediated transformation[END_REF] and UL35 and UL35a from human cytomegalovirus (HCMV) [START_REF] Salsman | Proteomic profiling of the human cytomegalovirus UL35 gene products reveals a role for UL35 in the DNA repair response[END_REF] have been described as USP7 binding partners.

USP7 plays an important role in many pathways linked to DNA dynamics such as DNA damage sensing and repair, chromatin remodeling or epigenetics. During replication for example, USP7 has been found to associate with the minichromosome maintenance complex (MCM), an

Lionel Rougé-« Study of the intramolecular activation mechanism of the deubiquitinase USP7 » -BioSPC -2019

Lionel Rougé-« Study of the intramolecular activation mechanism of the deubiquitinase USP7 » -BioSPC -2019 32 important complex typically loaded onto chromatin until completion of DNA synthesis. In this context, USP7 is recruited by the MCM binding protein (MCM-BP) and promotes unloading of the MCM complex at the replication fork [START_REF] Jagannathan | A Role for USP7 in DNA Replication[END_REF] but also deubiquitinating SUMO-2, creating a "SUMO-rich" environment leading to replication progression [START_REF] Lecona | USP7 is a SUMO deubiquitinase essential for DNA replication[END_REF]. Also, when genome integrity is at risk, USP7 can deubiquitinate checkpoint kinase Chk1

(Alonso- [START_REF] Vega | USP7 controls Chk1 protein stability by direct deubiquitination[END_REF], its activator clapsin [START_REF] Faustrup | USP7 counteracts Lionel Rougé-« Study of the intramolecular activation mechanism of the deubiquitinase USP7 » -BioSPC -2019 Lionel Rougé-« Study of the intramolecular activation mechanism of the deubiquitinase USP7 » -BioSPC -2019 110 SCFbetaTrCP-but not APCCdh1-mediated proteolysis of Claspin[END_REF] along with mitotic stress checkpoint E3 ligase Chfr [START_REF] Oh | Deubiquitination of Chfr, a checkpoint protein, by USP7/HAUSP regulates its stability and activity[END_REF] in order to initiate cell cycle arrest.

USP7's role in the DNA damage response pathway is multiple. During UV-irradiation stress, it stabilizes Tat interactive protein 60 (Tip60) which subsequently deacetylates DNA damage kinase ataxia telangiectasia mutated (ATM) [START_REF] Cui | The stressresponsive gene ATF3 regulates the histone acetyltransferase Tip60[END_REF][START_REF] Khoronenkova | ATM-dependent downregulation of USP7/HAUSP by PPM1G activates p53 response to DNA damage[END_REF]; in parallel, ATM-dependent protein phosphatase PPM1G can stabilize USP7 though dephosphorylation at Ser18 leading to accumulation of p53 and cell cycle arrest [START_REF] Khoronenkova | ATM-dependent downregulation of USP7/HAUSP by PPM1G activates p53 response to DNA damage[END_REF]. USP7 can also participate in the ubiquitin dependent DNA damage signaling by regulating the RING finger protein 168 (RNF168) which functions as a regulator of histone 2A (Q. [START_REF] Zhu | USP7 deubiquitinase promotes ubiquitin-dependent DNA damage signaling by stabilizing RNF168[END_REF]. Finally, USP7 regulates two members of the DNA damage tolerance pathway: DNA polymerase η [START_REF] Qian | USP7 modulates UV-induced PCNA monoubiquitination by regulating DNA polymerase eta stability[END_REF] and the E3 ligase Rad18 [START_REF] Zlatanou | Chemistry-based functional proteomics reveals novel members of the deubiquitinating enzyme family[END_REF]. Rad18 monoubiquitinates a proliferating-cell nuclear antigen (PCNA) in response to DNA lesions; this modified state of PCNA facilitates recruitment of specialized translesion synthesis (TLS) DNA polymerases at the replication fork, which can efficiently bypass DNA lesions, thereby allowing replication to proceed [START_REF] Waters | Study of the intramolecular activation mechanism of the deubiquitinase USP7 » -BioSPC -2019 Lionel Rougé-« Study of the intramolecular activation mechanism of the deubiquitinase USP7 » -BioSPC -2019 122[END_REF].

USP7 is an essential regulator of the DNA repair pathway. It prevents degradation of the pigmentosum complementation group C (XPC) protein which is a damage recognition factor binding DNA lesions also known to initiate the nucleotide excision repair machinery (NER) [START_REF] He | Ubiquitin-specific protease 7 regulates nucleotide excision repair through deubiquitinating XPC protein and preventing XPC protein from undergoing ultraviolet light-induced and VCP/p97 proteinregulated proteolysis[END_REF]. It can also deubiquitinate another component of the NER, UV-sensitive syndrome protein (UVSSA) at Lys414 [START_REF] Higa | Inhibition of UVSSA ubiquitination suppresses transcription-coupled nucleotide excision repair deficiency caused by dissociation from USP7[END_REF][START_REF] Schwertman | UV-sensitive syndrome protein UVSSA recruits USP7 to regulate transcription-coupled repair[END_REF]. From an epigenetic perspective, many key players have been shown to interact with USP7 such as BMI1 and polycomb group RING finger 2 (MEL18), components of the multiprotein polycomp repressive complex PRC1 complex responsible for regulation of the INK4a tumor suppressor (de Bie, [START_REF] De Bie | Regulation of the Polycomb protein RING1B ubiquitination by USP7[END_REF]Maertens, El Messaoudi-Aubert, Elderkin, Hiom, & Peters, 2010). Monoubiquitinated histones H2A and H2B, key regulators of chromatin remodeling, 33 are substrates of USP7 (Faesen, Dirac, et al., 2011) along with other histone associated proteins such as acetyltransferase TIP60 [START_REF] Dar | Deubiquitination of Tip60 by USP7 determines the activity of the p53-dependent apoptotic pathway[END_REF], demethylase PHF8 (Q. [START_REF] Wang | Ubiquitin-speciic protease-7 inhibition impairs Tip60dependent Foxp3 + T-regulatory cell function and promotes antitumor immunity[END_REF], methyltransferase MLL5 [START_REF] Ding | Mixed Lineage Lionel Rougé-« Study of the intramolecular activation mechanism of the deubiquitinase USP7 » -BioSPC -2019 Lionel Rougé-« Study of the intramolecular activation mechanism of the deubiquitinase USP7 » -BioSPC -2019 109 Leukemia 5 (MLL5) Protein Stability Is Cooperatively Regulated by O-GlcNac Transferase (OGT) and Ubiquitin Specific Protease 7 (USP7)[END_REF]. DNA methyl transferase 1 (DNMT1), a well characterized oncogene, forms a ternary complex with USP7 and the E3 ligase UHRF1 and seems to be activated upon USP7 binding [START_REF] Felle | The USP7/Dnmt1 complex stimulates the DNA methylation activity of Dnmt1 and regulates the stability of UHRF1[END_REF][START_REF] Qin | Usp7 and Uhrf1 control ubiquitination and stability of the maintenance DNA methyltransferase Dnmt1[END_REF].

Recently, USP7 has been linked to the hypoxia through two mechanisms; first, these conditions induce Lys63-linked ubiquitination of USP7 leading to its association with CBP/p300 and acetylation of H3K56, key modification responsible for the upregulation of genes involved in metastasis. And second, by stabilizing HIF-1a, a key component of intratumoural hypoxia and driver of tumor growth [START_REF] Wu | K63-polyubiquitinated HAUSP deubiquitinates HIF-1α and dictates H3K56 acetylation promoting hypoxia-induced tumour progression[END_REF].

Using GNE-6640, an allosteric inhibitor of USP7, Kategaya et al. identified PIM2 as a new USP7 target. PIM2 kinase shares substrates involved in the PI3K pathway and combo therapies using both USP7 and PIM2 inhibitors showed a synergistic effect responsible for cell viability reduction (Kategaya et al., 2017).

Furthermore, USP7 functions in the immune response by stabilizing the extremely important transcription factor NF-kB responsible for expression of hundreds of genes involved in inflammation [START_REF] Colleran | Deubiquitination of NF-κB by Ubiquitin-Specific Protease-7 promotes transcription[END_REF].

Finally, p53 is not the only tumor suppressor regulated by USP7. Both monoubiquitinated PTEN (van der Horst et al., 2006) and FOXO (Song, Salmena, et al., 2008) have been shown to be regulated through deubiquitination leading to changes in their cellular localization.

c) Structural features and regulation of USP7

USP7 is highly conserved across species and is composed of 1102 residues (135 kDa). It consists of seven domains (see Figure 10) including a N-terminal poly-glutamine (poly Q) stretch (involved in protein aggregation) followed by a TRAF (tumor necrosis factor receptor -associated factor)-like domain involved in protein-protein interactions, a central USP catalytic core, five Cterminal ubiquitin-like domains (UBLs) also involved in protein-protein interactions and an unstructured C terminal region composed of 23 amino residues (residues 1080-1102) (Faesen, Dirac, et al., 2011;Holowaty et al., 2003). Multiple structures of these domains in isolation were Several structures of the TRAF-like domain (residues 53-205) were solved in isolation or in presence of substrate peptides [START_REF] Jagannathan | A Role for USP7 in DNA Replication[END_REF] H.-R. [START_REF] Lee | Bilateral inhibition of HAUSP deubiquitinase by a viral interferon regulatory factor protein[END_REF]Saridakis et al., 2005;Sheng et al., 2006). This domain adopts a conformation of an eight-stranded, antiparallel βsandwich and has been crystallized apo (1YZE, 2F1W), in presence of two p53 peptides falling between residues Pro359-Ser363 (PDB code 2F1X) or Ala364 to Ser367 (PDB code 2FOJ), two MDM2 peptides (from Glu145-Ser150, PDB code 2FOP and from Leu224 to Glu230 PDB code 2F1Y), a vif1 peptide from the Kaposi's sarcoma-associated herpesvirus viral interferon (IFN) regulatory factor 4 (vIRF4) (PDB code 2XXN), a minichromosome maintenance (MCM) complex peptide (PDB code 4KG9) and a Ub2E1 peptide (PDB code 1YY6). All peptides bind in a similar manner through a Pro/Ala-X-X-Ser consensus sequence [START_REF] Sarkari | The Herpesvirus Associated Ubiquitin Lionel Rougé-« Study of the intramolecular activation mechanism of the deubiquitinase USP7 » -BioSPC -2019 Lionel Rougé-« Study of the intramolecular activation mechanism of the deubiquitinase USP7 » -BioSPC -2019 119 Specific Protease, USP7, Is a Negative Regulator of PML Proteins and PML Nuclear Bodies[END_REF]. In 2002, crystal structures of the 40kDa catalytic domain of USP7 in an apo form (PDB code 1NB8) or covalently bound to ubiquitin aldehyde (PDB code 1NBF) were both solved at 2.3Å (Hu et al., 2002). This domain displays a typical USP fold consisting of three domains called the fingers, the palm and the thumb with the catalytic triad formed of residues Cys223, His464 and Asp481 located in a cleft between the thumb and palm (see Figure 12a andb). One key observation seems to indicate that this catalytic triad exists in an inactive conformation in absence of substrate;

indeed, in this structure, catalytic His464 is located 9.7 Å away from Cys223, too far away to abstract the proton hence preventing nucleophilic attack. Similar observation was made based in a crystal structure solved in a different space group by Molland et al.,(PDB code 4M5W crystallized in C1 21 1 vs P1 21 1 for 1NB8) [START_REF] Molland | A 2.2 Å resolution structure of the USP7 catalytic domain in a new space group elaborates upon structural rearrangements resulting from ubiquitin binding[END_REF]. This constitutively inactive state explains the poor catalytic turnover observed for the isolated catalytic domain (Hu et al., 2002). Upon ubiquitin binding, substantial conformational changes are observed; ubiquitin is being coordinated by the finger motifs along with a β-hairpin loop spanning from Phe409 to Ile419

(colored in orange in Figure 12c), which, in the apo structure, seems to be disordered. In this bound state, the catalytic triad adopts a canonical cysteine protease active site geometry. The distance between Cys223 and His464 is dramatically reduced to 3.6 Å away while hydrogen bond between His464 and Asp481 is preserved. This dramatic realignment is permitted by a conformational change of the so-called "switching loop" (residues Phe283 to Val296 colored in teal in Figure 12) located in close proximity of the catalytic triad which is speculated to occur only upon ubiquitin binding.

Recent work by Özen et al., aimed to identify and mutate key molecular determinants of USP7 catalytic domain activation and test activity of the resulting mutant proteins (Ayşegül Özen et al., 2018). Our results indicated that electrostatic interactions in the distal "switching loop" region and local packing in the hydrophobic core mediate subtle but significant conformational changes that modulate catalytic domain activation. These intramolecular rearrangements represent a hypothetical mechanism of USP7 autoregulation, which ensures the inactive state of the enzyme in the absence of a substrate. 36

The structure of a construct consisting of both TRAF and catalytic domains (from Ser64 to Glu551, PDB code 3F1Z) was solved at 3.2 Å (Hu et al., 2006) and showed little interactions between the two domains suggesting that they can behave somewhat independently.

In recent years, a number of publications have described the role of the five ubiquitin-like domains (UBL12345) in substrate recognition and several X-ray structures revealed how these UBLs in an apo form (residues Gly555 to Asn1083, PDB code 2YLM); these accessory domains adopt a characteristic β-grasp ubiquitin fold consisting of β-β-α-β-β-α-β secondary structure elements and are organized in a 2-1-2 fashion (see Figure 13a); because of their poor degree of sequence homology, charge distribution varies from domain to domain and must play a significant role in substrate recognition (Faesen, Dirac, et al., 2011). Subsequently, other groups solved various structures of these UBLs in complex with substrates (see Figure 13). Cheng et al., reported that DNMT1 could, through lysine residues located within its Lys-Gly linker, bind an acidic pocket within the UBL123 domains of USP7 (PDB code 4YOC) (J. Cheng, Yang, et al., 2015).

Superimposition of these two structures shows significant change in the UBLs overall geometry; indeed, DNMT1 binding seems to alter the "extended" conformation observed in the apo structure into a more curved organization (see Figure 13a). In 2015, Pfoh et al., and Cheng et al., solved similar structures of an ICP0 peptide from HSV-1 virus bound to different constructs of USP7

UBL domains (J. Cheng, Li, et al., 2015;Pfoh et al., 2015). Both structures revealed the presence of a cleft between UBL1 and UBL2 where the ICP0 peptide binds (see Figure 13b). Interestingly, UBL1 and 2 superimpose very well but alignment diverge for UBL3 indicating the presence of a hinge region between UBL2 and UBL3 allowing for some flexibility (see Figure 13). More recently, the polybasic region (PBR) of the ubiquitin-like, containing PHD and RING finger domains 1 UHRF1 was shown to bind the first two UBL domains of USP7 (PDB code 5C6D, see Figure 13c) (Z.-M. [START_REF] Zhang | An Allosteric Interaction Links USP7 to Deubiquitination and Chromatin Targeting of UHRF1[END_REF]. FOXO4 (van der Horst et al., 2006). But most importantly, these UBLs along with the C terminal region have been linked to USP7 activation; indeed, it was shown that the presence of the C terminal region was absolutely essential for USP7 activity (Faesen, Dirac, et al., 2011).

d) USP7 cleavage specificity

Although it was previously reported that USP7 was able to cleave Lys6, Lys11, Lys33, Lys48 and Lys63-linked di-ubiquitin chains with similar kinetics [START_REF] Faesen | The differential modulation of USP activity by internal regulatory domains, interactors and eight ubiquitin chain types[END_REF], recent NMR studies using differentially labeled di-ubiquitin chains (distal vs proximal)

suggested that USP7 preferentially binds chains with free Lys48 (Kategaya et al., 2017); this finding along with other assays revealed that USP7 sequentially depolymerizes Lys48 chains from the distal ubiquitin (exo-cleavage) whereas Lys63 chains get cleaved indiscriminately.

e) Project objectives

As mentioned previously, Faesen et al., demonstrated through biochemical characterization of a full-length USP7 construct lacking the last 23 residues at its C-terminal that this region was required for full deubiquitinase activity. However, lack of structural information prevented them from rationalizing the potential mode of action of this non-structured peptide and subsequently, to build a coherent full length USP7 model.

Using protein engineering, enzymology and structural methods, our first goal was to determine the structural relationship between the catalytic domain and the C-terminal region of USP7 responsible for enzymatic activation. Additionally, we attempted to build a full-length model of USP7 in complex with ubiquitin and/or with its substrate DNMT1 by coupling X-ray structures and lowresolution reconstruction obtained by electron microscopy.

40

B. Material and Methods

1.

Preparation and characterization of crystallography grade proteins for structural studies

a) Expression and purification of USP7CD-UBL123

The gene encoding a fragment of USP7 equivalent to residues Lys208-Asn882 (USP7CD-UBL123) was cloned into a pGEX4-T1 vector (Novagen), yielding an expression construct with an N-terminal GST tag followed by a thrombin cleavage site. Plasmid was transformed into E. coli

b) Expression and purification of USP7CD-CTP and CD-UBL45-CTP

The DNA encoding for USP7CD-CTP (Lys208-Lys554-(GGS)10-Lys1084-Asn1102) and USP7CD-UBL45-CTP (Lys208-Lys554-(GGS)10-Lys882-Asn1102) constructs were cloned into a modified pET vector with a TEV cleavable 6-histidine fusion at the N-terminal. The plasmids were transformed into E. coli BL21(DE3) Gold cells (Stratagene) and protein expression was induced by the addition of 0.5 mM IPTG and growth at 16 °C for 4 hours (note that short induction was required in order to avoid proteolysis, see Supplemental Figure S2). Cells were harvested by centrifugation and resuspended in 25 mM Tris-HCl pH 8.0, 300 mM NaCl, 0.5 mM TCEP and 10 mM imidazole (Buffer A), and homogenized before being lysed with a microfluidizer; lysates were clarified by centrifugation (15,000 x g for 45 min). Supernatants were then loaded onto a Ni-NTA Superflow column (Qiagen) pre-equilibrated with buffer A and the columns washed with 50 mL buffer A containing 40 mM imidazole and eluted with 30 mL buffer A containing 300 mM imidazole. The six-histidine tags were removed by incubating the protein with a His-tag TEV protease (prepared in-house) while dialyzed overnight against buffer A. Cleaved proteins were passed through a 3 mL Ni-NTA Superflow column (Qiagen) pre-equilibrated with 20 mL of Buffer A. Flowthrough and wash fractions were pooled, concentrated down and injected onto a gel

d) Expression and purification of C-terminal reactive ubiquitin bromoethylamine

Ubiquitin bromo-ethylamine (Ub-BEA) was produced based on previously published procedures (Borodovsky et al., 2002;Wilkinson, Gan-Erdene, & Kolli, 2005). Briefly, untagged human ubiquitin lacking Gly76 was expressed as a fusion protein with an intein and chitin binding domain (CBD). Purification over chitin-beads, followed by on-column trans-thioesterification by addition of ß-mercaptoethanol sulfonic acid (MESNa) led to the isolation of the desired thioester

f) Expression and purification of DNMT1 [Arg600-Ala1600]

DNMT1 was purified as described in Cheng et al., (J. Cheng, Yang, et al., 2015). Briefly, gene encoding sequence between Arg600-Ala1600 was cloned onto a modified pET vector with a TEV cleavable 6-histidine fusion at the N-terminal. Protein was expressed and purified using the same protocol as USP7CD-CTP and USP7CD-UBL45-CTP constructs (see section above).

b) Principle of ubiquitin-AMC assay

Ubiquitin-AMC is prepared by the C-terminal derivatization of ubiquitin with 7-amino-4methylcoumarin. Release of AMC fluorescence by DUBs can be monitored using 380 nm excitation and 460 nm emission wavelengths. Kinetic measurements were carried out using 1 nM USP7 with a series of ubiquitin-AMC (Boston Biochem) substrate titrations at 30 °C in PBS with c) Michaelis-Menten curves for USP7 3.

Structural studies

For decades, structural biology has been used in order to better understand proteins function, protein-protein interactions and overall mechanistic reactions; its ability to provide details at atomic resolution has allowed the elucidation of complicated systems and is consistently evolving.

51

Three types of radiations are routinely used to provide three-dimensional structures of macromolecules: photons, electrons and neutrons. Because of their short wavelengths (range between 0.1 Å and 100 Å for X-rays), X-ray crystallography and electron microscopy are widely used to study inter-atomic dimensions (between 1 and 2 Å). Here we have used these two methods to study the USP7 system and the basic principles of these methods are introduced in the sections below.

a) X-ray crystallography of macromolecules

When encountering atoms from a sample, X-ray photons from an incident beam are either passing through the specimen or are reflected resulting in "scattered beam". These scattered photons can be collected and provide important information about the protein structure.

Unfortunately, less than 1% of X-rays are being scattered when apply to a specimen. In order to amplify scattering signals, macromolecules are arranged into a crystalline form creating a threedimensional repetition of single molecules called a crystal lattice. X-ray crystallography consists of three steps: crystal generation, collection of resulting scattering information and model building of the three-dimensional structure of the specimen.

i Protein crystallization and optimization techniques Protein crystals are microscopic objects consisting of a regular arrangement of molecules containing between 27% to 78% solvent; their growth is controlled by multiple variables including temperature, time, humidity, pH, protein concentration. A variety of so-called "precipitant" solutions (i.e. PEGs of various molecular weights, high salt concentration, MPD, alcohols…) are used in order to promote transition of the protein solution into a supersaturated state and a metastable zone, typically favorable for crystallization (see Figure 23a). The "liquid-liquid" and "vapor" diffusion method are most commonly used in order to control the thermodynamics and kinetics of supersaturation evolution. Once generated, crystal morphology and diffraction can be optimized through addition of additives but also through a method known as dehydration which goal is to remove water from the crystal and improve overall packing (see Figure 23b). b) Method 1 depicts a serial transfer of crystals into drops containing increasing amount of precipitants or cryoprotective agents [START_REF] Esnouf | Continuous and discontinuous changes in the unit cell of HIV-1 reverse transcriptase crystals on dehydration[END_REF]. In method 2, dehydrating solution is directly added to the original crystallization drop (about 8 times original volume) and air dried for an extended period of time [START_REF] Haebel | Crystallization and Initial Crystallographic Analysis of the Disulfide Bond Isomerase DsbC in Complex with the α Domain of the Electron Transporter DsbD[END_REF]. Method 3 describes the transfer of a crystal into a drop containing a dehydrating solution; equilibration is allowed against the reservoir which contains the same percentage of dehydrating solution; this process is repeated several times. A variant of this strategy is method 4; in this case the original drop is transferred to reservoirs containing incrementing amount of dehydrating solution [START_REF] Holyoak | Malonate: a versatile cryoprotectant and stabilizing solution for salt-grown macromolecular crystals[END_REF]) (from Heras & Martin, 2005, with permission). As mentioned above, atomic structure of crystallized proteins can be determined by collecting X-ray diffraction patterns from a single crystal. In the early 1900s, Bragg developed a model of diffraction that revolutionized the field [START_REF] Bragg | The Reflection of X-rays by Crystals[END_REF]: in this model, he

proposed that scattered X-rays can yield to constructive interferences (also known as reflection)

when the angle θ between the plane and the X-ray results in a path-length difference that is an integer multiple n of the X-ray wavelength λ as described in the equation:

2d sin θ = n λ
Every reflection corresponds to an evenly spaced set of sheets running through the crystal (separated through a distance d), usually passing through the centers of the atoms of the crystal lattice and can be spatially characterized by three parameters (h,k,l) also known as Miller indices.

Using Bragg's law, each reflection can be assigned (or "indexed") allowing the characterization of the lattice's geometry by calculating its unit cell parameters (defined by both lengths and angles)

and eventually determining the specimen's symmetry within the crystal lattice out of the 65 "space groups" available.

Figure 25: Visualization of Bragg's law

Two beams with similar wavelength λ approach a crystal and are scattered off two different atoms.

Constructive interference (left) occurs when the extra length traveled by the lower beam is equal

to an integrer multiple of the wavelength of the radiation. When these conditions aren't met, the phase shift results in destructive interferences [START_REF] Contributors | Bragg's law. Wikipedia, The Free Encyclopedia[END_REF] Lionel Rougé-« Study of the intramolecular activation mechanism of the deubiquitinase USP7 » -BioSPC -2019

Lionel Rougé-« Study of the intramolecular activation mechanism of the deubiquitinase USP7 » -BioSPC -2019 55 Finally, in order to interpret the relative intensities of the reflections, a Fourier transform method is applied to generate a three-dimensional model of the electron density within the unit cell. In the next two sections, I will describe how to index and process diffraction pattern and how to use this data to generate electron density maps.

iii Processing of diffraction patterns Indexing is generally accomplished using an autoindexing routine [START_REF] Powell | The Rossmann Fourier autoindexing algorithm in MOSFLM[END_REF]; numerous algorithms and programs have been developed to perform this step: HKL2000, XDS, iMosflm... Once the space group (and therefore crystal symmetry) has been assigned, the data is integrated; this step consists in integrating all the reflections from all the recorded images with their related Miller indices and intensities. The next two steps rely on "merging" these various images by identifying which peaks appear in two or more images and "scaling" these images to a consistent intensity scale. Because of the repetitive nature of the crystallographic data collection and the potential high degree of symmetry within the crystal, symmetry-equivalent reflections can be collected multiple times. Data quality can be assessed using a reliability index measuring differences in intensities between these reflections (Rmerge) [START_REF] Kleywegt | Validation of protein crystal structures[END_REF]. Other statistical values such as I/Sigma (indicator of signal to noise ratio) can help define overall quality of the data collection.

iv Structure solution and refinement

In a diffraction experiment, the intensity of each recorded diffraction 'spot' is proportional to the square of the structure factor amplitude. The structure factor is a complex number containing information relating to both the amplitude and the phase of the wave. In order to obtain an interpretable electron density map, both amplitude and phase must be known. Unfortunately, the phase information is lost during the diffraction experiment: this is known as the phase problem.

Initial phase estimates can be obtained in a variety of ways: is collected from a crystal containing an anomalous scatterer (electron dense atom) such as naturally occurring zinc (in the case of zinc-binding protein), seleno-methionine (this nonnatural amino acid is typically added to the media during protein expression while using a methionine auxotroph competent cells) or by introduction of a heavy atom derivative (i.e. gold atom will cross link with thiol group of free cysteines). Three datasets are recorded at the following wavelengths : far below, far above and in the middle of the absorption edge; changes in the scattering amplitudes can be used to place the anomalous scatterer therefore providing initial phases which will be then used to solve the structure of the molecule [START_REF] Ealick | Advances in multiple wavelength anomalous diffraction crystallography[END_REF].

• Molecular replacement: If a related structure is known, it can be used as a search model in molecular replacement to determine the orientation and position of the molecules within the unit cell. The phases obtained this way can be used to generate electron density maps [START_REF] Taylor | The phase problem[END_REF].

• Ab initio phasing or direct methods: This is a rare method exploiting known phase relationships between certain groups of reflections which can be used to solve the phase problems for small proteins or proteins diffracting at resolutions better than 1.4 Å [START_REF] Hauptman | Phasing methods for protein crystallography[END_REF].

Having obtained initial phases, an initial model can be built. This model can be used to refine the phases, leading to an improved model; iterative rounds of refinements can be performed. To measure the agreement between the experimental X-ray diffraction data and the crystallographic model, a R-factor (or Rwork) is calculated where F is the structure factor. A similar quality criterion is Rfree, which is calculated from a subset (~10%) of reflections that were not included in the structure refinement [START_REF] Brünger | Free R value: a novel statistical quantity for assessing the accuracy of crystal structures[END_REF]; large differences between Rwork and Rfree typically suggests an over-fitting of the data and potential model bias. Finally, chemico-physical features such as stereochemistry, hydrogen bonding and distribution of bond lengths and angles are complementary measures of the model quality.

57

Below is a summary of the crystallographic data for the three structures solved: Resolution range (Å) 1 46.95 -3.31 (3.42 -3.31) 74. 03 -3.32 (3.33 -3.32) 74. 25 -3.20 (3.42 -3.20)

Space group P2 1 2 1 2 1 P 6 2 22 F222 Unit cell dimensions a, b, c (Å) α, β, δ (˚)
113. 6, 115.5, 257.8 90.0, 90.0, 90.0 148.1, 148.1, 179.9 90.0, 90.0, 120.0 104.6, 196.8, 226.9 90.0, 90. 3 Rfree = R factor calculated using 7.5% of the reflection data chosen randomly and omitted from the start of refinement

b) Electron microscopy

Transmission electron microscopy (TEM) is a microscopy technique in which an image is formed from the interaction of the electrons with the sample as the beam is transmitted through 58 the specimen [START_REF] Orlova | Structural Analysis of Macromolecular Assemblies by Electron Microscopy[END_REF]. Resulting images are projections of the object showing the distribution of density through the object. The specimen can be deposited over a thin copper grid and analyzed by "negative stain" or captured within a thin layer of vitreous ice and visualized under cryogenic condition (cryo-EM). Data processing for single-particle reconstruction relies on the alignment of thousands of images and the identification of their orientation in order to accurately build a 3D reconstruction. Below, I will briefly present some of the key aspects of this method, from sample preparation to data acquisition and image processing.

i Sample handling

The most convenient method to analyze macromolecules is negative staining, in which a drop of purified protein sample is applied to a grid and then covered with a 2% uranyl acetate solution, blotted to Whatman paper and air dried [START_REF] Saibil | Macromolecular structure determination by cryo-electron microscopy[END_REF]. This method allows visualization of the envelope of the biological assembly, suggesting information about the size, shape, and symmetry of the particle, but also regarding the homogeneity of the preparation. The method is called negative staining because the macromolecular shape is seen by exclusion rather than binding of stain (see Figure 26).

ii Principles of electron microscopy

As described earlier in this section, electrons are widely used to study macromolecules structures due to their short wavelength. Electrons can be emitted from a heated tungsten filament, a LaB6 crystal, or a tungsten tip coated with zirconium oxide, and are accelerated by an electric field using voltages between 80 and 300kV. A series of electromagnetic lenses are located throughout the length of the microscope with different functions (see Figure 27a):

-condenser lenses generate the parallel illumination necessary for high-resolution coherent imaging of the specimen.

-the objective lens, which contains the specimen, forms the first image of the specimen.

-the projector lenses magnify the image before electrons can reach the detector.

An objective aperture is also present between the objective and projector lenses, at the back focal plane of the objective aperture, preventing electrons scattered at high angles to reach the 60 image plane hence improving image contrast. By tuning the lenses below the objective (putting the microscope in so-called "diffraction mode"), the electron diffraction pattern, rather than the image of the specimen, can be projected onto the detector. 3D structures of typically small crystals can then be solved by processing these diffraction patterns, a technique called micro-ED (electron diffraction).

When incident electrons traverse a sample, three different scenarios are possible. They can either: pass through the sample without any interactions, be deflected by the electrostatic field of the nucleus or interact with the specimen. Scattering interactions can be categorized as either elastic, when electrons are scattered without energy loss, or inelastic when they transfer energy to the specimen. This energy transfer is mostly responsible for radiation damage, which limits highresolution imaging of biological molecules. Inelastically scattered electrons also impedes later image processing, which assumes elastic scattering only, and treats inelastic scattering contrast as noise. Because inelastically-scattered electrons emerge with a reduced velocity, focusing onto a higher plane within the microscope, they can be removed using an energy filters so that images are only generated using unscattered and elastically scattered electrons therefore providing improved signal-to-noise ration during later processing.

There are two important concepts in image formation: amplitude contrast and phase contrast. Contrast between unscattered electrons (which provide a "bright field" background to the image) and those scattered by a point in the specimen and which do not reach the detector (e.g.

those elastically scattered into the objective aperture, or inelastically scattered into the energy filter) is called amplitude contrast: at that point of the image, it is as though the amplitude of the incident beam had been attenuated by the corresponding point in the specimen plane. However, because the scattering power of the atoms composing biological molecules is pretty low (H, C, O and N), the intensity of the incident beam is only mildly affected after interacting with the sample and the resulting amplitude contrast is small. iii From 2D projection to 3D reconstruction: single particle EM data processing workflow

Individual images collected during an EM experiment can provide some preliminary information related to the oligomeric state and overall shape of the studied specimen. In order to protect samples from radiation damage, data acquisition is performed under low electron dosage;

unfortunately, resulting images have low signal-to-noise ratio (or SNR) and preventing interpretation of high-resolution features. Advanced computerized image processing techniques have been developed to properly interpret EM images and allow building of a three-dimensional 62 reconstruction of the particle (Y. [START_REF] Cheng | A Primer to Single-Particle Cryo-Electron Microscopy[END_REF]. In this section, the various steps composing the typical workflow used in single particle analysis will be presented (summarized in Figure 28). The second step after particle picking consists of aligning and registering these particles into 2D classes. This procedure allows for the generation of images with higher SNR, improving contrast of constant features over background signal. During grid preparation, particles are captured into different orientations and possibly different conformations; in order to group particles into homogeneous subsets, several clustering algorithms have been used, the most popular being the so-called multi-reference alignment algorithm [START_REF] Van Heel | Multivariate statistical classification of noisy images (randomly oriented biological macromolecules)[END_REF]. This algorithm relies on randomized cluster centers that grow through iterative local-search-based optimization of cluster assignments and in-plane rotations [START_REF] Reboul | A Stochastic Hill Climbing Approach for Simultaneous 2D Alignment and Clustering of Cryogenic Electron Microscopy Images[END_REF]. This method has shown several limitations (i.e. low reproducibility) and other algorithms such as maximum likelihood [START_REF] Scheres | Maximum-likelihood Multi-reference Refinement for Electron Microscopy Images[END_REF], correlation based projection matching (Zhengfan [START_REF] Yang | Iterative Stable Alignment and Clustering of 2D Transmission Electron Microscope Images[END_REF] or stochastic hill climbing [START_REF] Elmlund | PRIME: Probabilistic Initial 3D Model Generation for Single-Particle Cryo-Electron Microscopy[END_REF] have been developed. iv Cryo-EM facility at Genentech

The EM facility at Genentech consists of a Talos 200c (200 kV from Thermo Fischer Scientific) with a Ceta camera (Thermo Fischer Scientific) mostly used for negative staining and cryo-EM grids screening and a Titan Krios (300 kV, Thermo Fischer Scientific) with a K2 direct detector (Gatan) dedicated to high resolution data collection. Automated data acquisition on both microscopes is performed using the SerialEM software [START_REF] Mastronarde | Automated electron microscope tomography using robust prediction of specimen movements[END_REF]. Most of the data analysis is performed using the program cisTEM (computational imaging system for transmission electron microscopy) [START_REF] Grant | cisTEM, user-friendly software for single-particle image processing[END_REF].

66 [START_REF] Grant | Measuring the optimal exposure for single particle cryo-EM using a 2.6 Å reconstruction of rotavirus VP6[END_REF], mag_distortion_correct (Grant & Grigorieff, 2015a), CTFFIND4 [START_REF] Rohou | CTFFIND4: Fast and accurate defocus estimation from electron micrographs[END_REF], and Frealign [START_REF] Lyumkis | Likelihood-based classification of cryo-EM images using FREALIGN[END_REF]. Additionally, algorithms described previously were added for particle picking [START_REF] Sigworth | Classical detection theory and the cryo-EM particle selection problem[END_REF], 2D classification [START_REF] Scheres | Maximum-likelihood Multi-reference Refinement for Electron Microscopy Images[END_REF] and ab-initio 3D reconstruction [START_REF] Grigorieff | Frealign: An Exploratory Tool for Single-Particle Cryo-EM[END_REF] sometimes with modifications to optimize their performance.

C. Results

USP7 activation by C-terminal residues

a) C-terminal residues are critical for USP7 activity

Looking at sequence diversity of the last 19 C-terminal residues of USP7 (Lys1084 to Asn1102), we noticed that these residues were displaying a high degree of conservation across species suggesting evolutionary pressure to maintain this sequence intact (see Figure 32).

Figure 32: Sequence alignment of the C-terminal 19 residues of USP7 orthologues

In order to monitor activity of our various constructs, we established a ubiquitin-AMC enzymatic assay to measure deubiquitinase activity (see Figure 22). As positive control, we reproduced data described by Faesen et al., and showed that, as expected, deletion of the last 7 residues at the C-terminus (construct labeled "Cat-UBL1-5Δ7" in Table 2) is sufficient to prevent USP7 activation (Table 2, lanes 2 vs 3). 3). Surprisingly, several of these peptide extensions increase the activity of the catalytic domain from barely detectable to 66% of full-length USP7 activity. Based on the data, it seems like the longest extension (1084-1102) and a minimum linker of at least 5 residues are sufficient and required to partially recapitulate full length activity. Indeed, the Km for the 19-residue CTP with a linker of 5, 10 or 20 residues was identical to the full length USP7 while kcat for the 10 and 20 residues linkers was within 2-fold the activity of the full-length protein (Table 3, lanes 1, 11 and 12). We chose to further characterize the 10-residue-linker construct, hereafter referred to as USP7CD-CTP.

Point mutations at each of the nine C-terminal residues in the USP7CD-CTP construct were generated in order to identify key residues critical for USP7 activation. As seen in Table 4, alanine substitutions at residues Ile1098, Lys1099, Ile1100 and the C-terminal Asn1102 are poorly tolerated and reduce activity by 90 percent or more. Isoleucines 1098 and 1100 are particularly critical for activity and alanine mutations at these sites reduce activity to the same level as catalytic domain alone, while mutation at Lys1099 and Asn1102 result in a 7 to 8-fold reduction in kcat. which was substituted with a methionine residue. ND stands for "Not Detectable".

b) Structural understanding of catalytic activation by the Cterminal peptide of USP7

Intrigued by the ability of the C-terminal peptide to activate the catalytic domain, we sought to understand the molecular mechanism of activation. We determined the crystal structures of both 71 apo and USP7CD-CTP covalently bound to ubiquitin through its catalytic cysteine Cys223 (see Figure 34). Both datasets were indexed and integrated with XDS (Kabsch, 2010), scaled with SCALA (Evans, 2006) and the structures solved by molecular replacement using PHASER (McCoy, 2007) with structures of the USP7CD (PDB 1NB8 or 1NBF) used as search models. The final models were obtained after iterative cycles of manual rebuilding in COOT (Emsley & Cowtan, 2004) and refinement in PHENIX (Adams et al., 2010).

The crystals of apo-USP7CD-CTP diffracted to 3.0 Å and the structure solved by molecular replacement using the inactive form of USP7CD as the search model. The refined structure was similar to the inactive form of the USP7CD, with no electron density for the linker or the Cterminal peptide observed (data not shown). This suggests that without substrate, the C-terminal peptide is unstructured and doesn't directly interact with the catalytic domain. This is consistent with observations made during protein expression and purification; indeed, recombinant protein purified from pellets grown with overnight IPTG induction showed heterogeneous molecular weight by liquid chromatography mass spectrometry (see supplemental Figure S2). These observed species correspond to different proteolytic digests at the CTP of the protein indicating that the peptide is prone to processing in cell; this phenomenon is typical from sequences lacking secondary structures.

The crystals of USP7CD-CTP in complex with ubiquitin diffracted to 3.32 Å resolution and the structure solved by molecular replacement using the USP7CD-ubiquitin complex (PDB 1NBF) as the search model (see Table 1). In contrast to the apo USP7CD-CTP structure, there was a large and continuous positive Fo-Fc electron density peak in the cleft created by the conformational change of the switching loop. A model for nine residues from the C-terminal peptide (1094-LEKAIKIHN-1102) was built into the positive density peak and the structure refined (see Figure 34). There was no interpretable electron density for the 10-residue linker that tethers the catalytic domain to the C-terminal peptide leading to some ambiguity over whether the peptide is bound in cis or trans. The structure of USP7CD-CTP demonstrates that the C-terminal peptide binds into a cleft that results from the conformational rearrangement of the switching loop, subsequently referred to as the activation cleft. Productive binding of the C-terminal peptide relies on both hydrogen bonding and hydrophobic interactions (Figure 34 and Figure S3). These include the C-terminal Asn1102 engaging Asn306 and Lys310 on the α5 helix, H-bond interaction between the backbone amide nitrogen of His1101 and the carbonyl oxygen of Phe283 from the 72 switching loop, the carbonyl of Lys1099 interacts with the indole nitrogen of Trp285 from the switching loop and the carbonyl of Lys1096 is engaged in H-bond interactions with the side-chain guanidino group of Arg74 from ubiquitin. The Ile1098 and Ile1100 side-chains are buried into hydrophobic cavities on USP7CD, consistent with their critical role in catalytic activation (Table 4). Mutation of residues Asn306 and Trp285 within USP7CD further confirm the critical importance of these residues in binding to the USP7 C-terminal peptide and consistent with the function of Trp285 proposed previously (Faesen et al., 2011) (see Table S1). To gain an improved understanding of the functions of the UBL domains in terms of their role in substrate recognition and their relationship to the catalytic domain of USP7, a construct including the catalytic domain and UBL123 domains (residues Lys208-Lys882) was expressed, purified and crystallized (see Figure 14). -Protein was methylated at lysine residues in order to change chemico-physical properties of the protein surface and potentially modify key packing interactions [START_REF] Walter | Lysine methylation as a routine rescue strategy for protein crystallization[END_REF].

-A second wave of constructs with slightly shorter C-terminal boundaries were generated (Lys208-Arg871 and Lys208-Phe888).

-Poorly diffracting crystals were crushed and used as seeds in order to promote crystal growth in different conditions and potentially obtain a different crystal form [START_REF] Walter | Semi-automated microseeding of nanolitre crystallization experiments[END_REF].

-A variety of cryo-protectant solutions were tested using CryoPro kit from Hampton

Research® (low molecular weight PEGs, mineral oil, alcohols, sugars, salts and typical glycerol or ethylene glycol).

-Crystals were tested at room temperature instead of being under cryogenic conditions [START_REF] Juers | Cryo-cooling in macromolecular crystallography: advantages, disadvantages and optimization[END_REF].

Unfortunately, none of these techniques yielded any substantial improvement; the method that ended up being the most effective was dehydration. Several methods have been developed over the years with different success (see Figure 23). In our case, the diffraction properties were improved by dehydrating the crystals by sequential equilibration of the drops above wells containing increasing concentrations of PEG 3350 (method 4 in Figure 23b). The final concentration of the dehydration solution was 50% PEG 3350 and this same solution served as the cryoprotectant. Diffraction images were collected at the Diamond Light Source beamline I02 and dataset was solved using 1NB8 and 2YLM as search models for molecular replacement. Solution was found and final model was refined to 3.5 Å resolution with one molecule in the asymmetric unit (see Table 1).

In this structure, the catalytic domain adopts a conformation characteristic of the inactive form of the enzyme, very similar to other apo-USP7 crystal structures that have been determined, (Cα RMSD = 0.76 Å against PDB 1NB8); indeed, the residues of the catalytic triad are in a nonproductive conformation and the switching loop (in cyan in Figure 35) is in a conformation that would sterically hinder C-terminal peptide and ubiquitin binding (not shown). A couple of loops 75 lacking electron density in the apo structure of USP7CD and close to the active site (residues Tyr411-Asp419 and residues Asp502-Arg508) are well ordered in this structure (shown in red).

The USP7CD-UBL123 structure contains an extended α10 helix spanning 24 residues (residues 538-562) that links the catalytic domain to the first UBL domain. This extended α10 helix gives the structure an overall boomerang shape that places the UBL domains distant from the catalytic domain. domain, there are no interactions between the UBL and catalytic domains. UBL1 and UBL2 form a closely packed unit burying a large surface area of ~1200 Å 2 . In contrast, the UBL3 domain is an independent unit that is largely solvent exposed and has limited contacts with the other UBL domains.

When compared to other UBL structures adopting either an extended (PDB 4WPI and 2YLM)

or closed (PDB 4WPH, see Figure 35a) conformation, superposition of equivalent residues indicates that the UBL domains from this structure adopt a closed or "compact" conformation (RMSD with 4WPH = 1.9Å over 300 residues). However, the hydrogen bond interaction characteristic of the closed conformation between side chains of Asp587 (from UBL1) and Arg871

(from UBL3) is not observed; this could be due to low resolution of this structure.

Even though there are no direct interactions between the catalytic domain and the UBL domains within the monomer observed in the asymmetric unit, visualization of the overall crystal 77 packing organization reveals a number of potential interactions between domains from symmetry mates (see Supplemental Figure S4). Interestingly, the UBL1-3 domains of one symmetry-related molecule pack against the catalytic domain in the asymmetric unit (see Figure 35b). Residues from ß13 strand and the α8 helix in the catalytic domain and residues spanning UBL2 and UBL3 of the symmetry-related molecule make up an extensive interface that buries 1750 Å 2 and contains 24 salt bridge interactions and 12 hydrogen bond interactions. Although the biological relevance of this interface has not been tested, mapping of the ICP0 binding site onto this hypothetical dimer suggests that site could potentially be occluded by the presence of the catalytic domain hence affecting substrate binding. Finally, it is important to note that no evidence was found during purification that this construct forms potential oligomers in solution.

b) Crystal structure of USP7 catalytic domain with UBL45 in complex with ubiquitin

After successfully obtaining structures of USP7CD-CTP in complex with ubiquitin and USP7CD-UBL123, we were interested in getting a structure containing the UBL45 domains in order to potentially generate a composite model of the full-length protein. A similar approach to the one adopted for the USP7CD-CTP construct was taken; crystallization trials of constructs consisting of USP7CD-UBL345-CTP and USP7CD-UBL45-CTP with different linker lengths (Faesen, Dirac, et al., 2011) apo or in a covalent complex with ubiquitin bromo-ethylamine were set-up. Apo protein failed to crystallize but a crystal structure of USP7CD-UBL45-CTP with ubiquitin covalently attached to the catalytic cysteine was solved to 3.3 Å with P212121 symmetry (see Table 1) and four molecules in the asymmetric unit, arranged as a dimer of dimers (see Figure 36). The structure was solved by molecular replacement using models of the USP7CD-ubiquitin complex (PDB 1NBF) and the UBL45 domain (PDB 2YLM).

As expected, the catalytic domain displays all the features of an active conformation: the Cterminus of ubiquitin is covalently bound to Cys223, the catalytic triad is properly aligned and, as described earlier in the USPCD-CTP structure, the C-terminal peptide binds in the "activation cleft" stabilizing the switching loop in the "out-conformation". Similarly, as the USP7CD-CTP structure, there was no interpretable electron density for the 10-residue GGS-linker that tethers the in trans in this structure (see Figure 36b).

Full length model of USP7

In order to gain insights in the overall organization of this key DUB, we tried to crystallize a longer USP7 construct covalently bound ubiquitin or in complex with biologically relevant substrates (i.e. GMPS or DNMT1). Based on activity and structural data, we decided to work with a pseudo "full-length" USP7 construct [Lys208-Asn1102] lacking the N-terminal TRAF domain;

since this domain doesn't contribute to USP7 activity and is most likely peripheral (i.e. not involved in core packing of USP7 when bound to substrates), we decided to leave it out as we worried it could introduce an additional level of heterogeneity typically detrimental for crystallogenesis. After several failed attempts at crystallizing these large complexes, we followed two distinct approaches: first, we compiled all structural information described earlier to generate a model capturing full length USP7 complexed with ubiquitin while bound to DNMT1. And second, we used electron microscopy in order to potentially validate our preliminary model. Elucidation of the USP7CD-UBL123 and USP7CD-UBL45-CTP + ubiquitin crystal structures provided key information to better understand USP7 overall domain organization. For example, the USP7CD-UBL123 structure suggests that the catalytic domain and the UBL123 domains are not directly interacting and separated by the extended α10 helix. In contrast, the USP7CD-UBL45-CTP structure in complex with ubiquitin strongly suggests that the catalytic domain gets activated in trans by the CTP from the UBL45 domains of a non-crystallographic symmetry related molecule (Figure 36b). In order to build a coherent model for full-length USP7, several structures were combined together aiming at identifying key features involved in potential conformational changes. The centerpiece for our model consists of the USP7CD-UBL123 structure to which the catalytic domain of the NTD-USP7CD crystal structure (Hu et al., 2006) (PDB 2F1Z) has been superimposed. In this model, the TRAF domain does not seem to interact with the UBL domains. Unfortunately, adding structural information from the UBL12345 structure (Faesen, Dirac, et al., 2011) (PDB 2YLM) failed to provide a coherent model for USP7 deubiquitination of its substrates; indeed, overlay of the UBL12 domains placed Asn1084 (last residue visible by electron density) about 80 Å away from the catalytic cleft (see Figure 37a); assuming a typical amino acid measures approximately 3.5 Å, the maximum distance covered by the last 18 residues from the C terminal peptide of USP7 can only be approximately 60 Å; moreover, in the structure of the USP7CD-UBL45-CTP, the distance from the Cα of Asn1083 to the Cα of Asn1102 is 45.7 Å, confirming our estimation. The model proposed here could potentially capture an inactive state of USP7, not bound to substrate and therefore catalytically incompetent.

Instead, our proposed model for full-length USP7 relies on the structure of UBL12345 bound to DNMT1 (PDB 4YOC). Here again, the starting structure for our model is the USP7CD-UBL123 structure to which the TRAF domain has been added. Superposition of the Cα atoms of residues from the UBL12345 bound to DNMT1 structure (RMSD = 2.6 Å) reveals that the UBL45 domains curve back towards the catalytic domain. In this case, the distance from the Cα of Asn1083 to Cys223 is about 50 Å (see Figure 37b). The last step in the building of a full length USP7 model was the superimposition of the non-crystallographic symmetry related USP7CD-UBL45-CTP domains from the USP7CD-UBL45-CTP bound to ubiquitin structure allowing the placement of residues [Asn1083-Lys1102] from the C terminal peptide along with ubiquitin. This suggests the CTP could bind into the activation cleft in cis, with minor conformational changes in the UBL domains, but would require larger movements of the Cterminal peptide.

Most importantly, this full-length model is also consistent with the UBL12345-DNMT1 complex structure (PDB 4YOC) as superposition of this complex onto the equivalent residues in our full-length USP7 model reveals that DNMT1 is easily accommodated into the central cavity of the boomerang shape created by the catalytic and UBL domains; it is also interesting to note that DNMT1 binding brings the NTD of USP7 in close proximity with N-terminal domain of DNMT1 (see Figure 38). Unfortunately, full length model fails to suggest any interaction between DNMT1 exposed lysines and the C-terminal of ubiquitin. In order to validate our model, additional experimental data describing the overall shape of this complex is required. We originally attempted to obtain low resolution information of apo full-length USP7 using Small-Angle X-ray Scattering (SAXS);

b) EM studies of USP7 [Lys208-Asn1102] bound to ubiquitin and in complex with DNMT1

In order to test whether the full-length model proposed in Figure 37 was reasonable, we turned to electron microscopy. We first analyzed a sample containing USP7 covalently bound to 84 ubiquitin (Figure 39a). Rapid analysis of a few micrographs suggested that this sample was heterogeneous and adopted multiple conformations; these observations validated our decision to stop crystallographic studies as this high degree of flexibility would most certainly prevent lattice formation. Although suboptimal, we collected 2052 micrographs and perform 2D classification (see Supplemental Figure S5 and Figure 39b). Zhang, Liu, et al., 2015) suggesting that recombinant protein is homogeneous and well dispersed. Based on micrographs and SEC profile, DNMT1

behaves indeed as a well-ordered and uniform protein.

b) Best 2D classes of USP7 [Lys208-Asn1102] bound to ubiquitin (see Supplemental Figure S5 for details). A "boomerang" shaped class is highlighted with a white frame (left panel). c) Best 2D class was used to overlay model of full length USP7 bound to ubiquitin described in Figure 37b). USP7 model described earlier (see Figure 39b). Overlay of this model onto one of the class average is consistent (see Figure 39c). As expected, these classes suggest USP7 adopts a conformation less compact than originally thought even in the presence of covalently bound ubiquitin confirming the heterogenous nature of the UBL domains. Based on this suboptimal result, sample was not tested for higher resolution analysis by cryo-EM.

In order to reduce conformational heterogeneity, the complex between USP7 [Lys208-Asn1102] bound to ubiquitin and DNMT1 was formed and analyzed by negative stain (see Figure 39a). Unfortunately, even though both proteins seem to co-elute on a size exclusion chromatography (see Figure 20), particles observed on the micrographs were not consistent with the dimensions of 250kDa objects. Complexes could have been dissociated during staining and it would be interesting to repeat this experiment using a cross-linking method (i.e. glutaraldehyde, [START_REF] Strauss | Structure of glutaraldehyde cross-linked ryanodine receptor[END_REF]. Efforts to generate complexes with different binding partners are currently ongoing and preliminary results confirm complex formation between USP7 and its positive modulator GMPS (see Supplementary Figure S6).

One important caveat with the current approach is that the complexes formed here are not biologically relevant; indeed, our strategy relies on the stabilization of the catalytic domain through covalent attachment of ubiquitin and, in parallel, the addition of a biological substrate complexing would be improved by forming a stable complex of the protease with a mono or di-ubiquitinated substrate (i.e. ubiquitinated DNMT1). A catalytic dead version of USP7 would be required to prevent ubiquitin removal from the substrate. Finally, instead of using a reactive ubiquitin to complex with the catalytic domain, engineered ubiquitin with improved affinity for USP7 (Y. Zhang et al., 2013) (W. [START_REF] Zhang | Generation and Validation of Intracellular Ubiquitin Variant Inhibitors for USP7 and USP10[END_REF] could be tethered to the substrate and utilized to "lock" the complex without the need of a covalent interaction.

D. Discussion

Because of its key regulatory role in p53 homeostasis and many other pathways, USP7 has been one of the most-studied deubiquitinases. Aside from its biological relevance, one of the most interesting aspects of this protease is its tight regulation; indeed, preliminary work demonstrated that the isolated USP7 protease domain was almost catalytic dead. Multiple groups suggested that ubiquitin binding could promote rearrangement of the catalytic residues into a productive conformation (Hu et al., 2002) It is not uncommon for the activity of important cellular proteins to be autoinhibited (Pufall & Graves, 2002) and DUBs get tightly regulated as seen previously. In the absence of substrate, the catalytic domain of USP7 exists in an autoinhibited state. At the time this manuscript was published, USP7 was the only known DUB to have a catalytic domain displaying a misaligned catalytic triad; recently, a crystal structure of USP15 was solved indicating a similar organization [START_REF] Ward | The structure of the deubiquitinase USP15 reveals a misaligned catalytic triad and an open ubiquitin-binding channel[END_REF] although both DUBs are differently regulated. Our work reveals the first example of a ubiquitin-specific protease being regulated by a mechanism involving an activating peptide that stabilizes an active conformation.

In an effort to identify potential candidates displaying a similar regulation mechanism, we looked at closely-related DUBs and tried to isolate similar activating sequences within their C terminal regions. Based on Ye et al., (Yu Ye, Scheel, Hofmann, & Komander, 2009), we focused our attention towards USP47 and USP40, two DUBs sharing a switching loop consensus motif similar to USP7 (see Supplemental Figure S7). Unfortunately, these two DUBs have been poorly studied and only USP47 has been recombinantly expressed and characterized [START_REF] Piao | Production, Crystallization and Structure Determination of <em>C. difficile</em> PPEP-1 via Microseeding and Zinc-SAD[END_REF].

In this study, the full length USP47 was expressed in insect cells and tested for DUB activity in a variety of assays; in a Ub-AMC assay, USP47 performed poorly compared to USP7 whereas it shows improved processing when tested in a Lys48 or Lys63 di-Ub gel-based cleavage assay. The difference in the enzymatic properties between these two DUBs suggests that USP47 may play distinct roles in Ub-mediated cellular processes. Because these kinetics studies were performed with the full-length protein, it is unknown whether the isolated USP domain of USP47 responds to the same activation mechanism as the one of USP7. Interestingly, sequence analysis of the Cterminal region of USP47 shows a consensus motif similar to the Hyd-X-Hyd motif present in USP7 (see Figure 40). Indeed, the sequence KEKALKYIH in USP47 (residues 1357 to 1363) is highly similar to the LEKAIKIY sequence (residues 1095 to 1101) from USP7; however, this sequence is located in a region that is distal from the C-terminus of USP47 suggesting a different regulation mechanism which might not rely on the C-terminal carboxylate as seen in USP7 (see

89

In subsequent studies, we further investigated key molecular determinants within USP7 catalytic domain that help mediate its activation. Using a variety of approaches (molecular dynamics simulations, directed sampling via RosettaDesign), a number of electrostatic interactions (His294Glu) and key hydrophobic residues involved in proper packing within the hydrophobic core of USP7 (Leu299Ala) were shown to mediate substantial conformational changes improving overall USP7 activation (Ayşegül Özen et al., 2018). For example, Leu299Ala had been identified

using RosettaDesign (Kuhlman & Baker, 2000) and had an interesting effect on USP7 activity.

Leu299 belongs to the hydrophobic core (or leucine cluster) which is highly conserved amongst members of the USP family; this core typically favors bulky hydrophobic residues such as Phe or Leu (see Figure 41a). Computational design suggested that mutation of this residue into Ala would stabilizes USP7 active conformation. Even though this design appeared contradictory to overall sequence consensus for this region, enzymatic characterization (see Figure 41b) and analysis of the crystal structure of the complex between this point mutant USP7CD [Leu299Ala] in complex with Ub-BEA revealed that the switching loop adopts an intermediate conformation distinct from both the active and inactive ones (PDB code 5KYF, see Figure 41c). In this structure, the absence of the isopropyl group allows Phe283 to adopt a different rotamer conformation improving overall packing of the hydrophobic core while in the active state. (residues 224, 260, 279, 288, 299, 302) with different selectivity profiles and mechanism of action (adapted from Zhou et al., 2018, with permission).

The unique "inactive" conformation of USP7 catalytic domain has led to the discovery of inhibitors stabilizing this conformation and therefore preventing its activation. One of the most studied and advanced scaffolds was based on the hydroxy-piperidine core. This series has been 92 biochemically characterized and a number of crystal structures were solved and published over the past two years [START_REF] Gavory | Discovery and characterization of highly potent and selective allosteric USP7 inhibitors[END_REF][START_REF] Lamberto | Structure-Guided Development of a Potent and Selective Non-covalent Active-Site Inhibitor of USP7[END_REF][START_REF] O'dowd | Identification and Structure-Guided Development of Pyrimidinone Based USP7 Inhibitors[END_REF][START_REF] Turnbull | Molecular basis of USP7 inhibition by selective small-molecule inhibitors[END_REF] (see 4). These compounds act by stabilizing the inactive conformation of the catalytic site without directly engaging with Cys223, His464 or Asp481 and binding within the channel typically occupied by the C terminal tail of ubiquitin (Figure 43c). A more detailed binding mode of these compounds is shown in Supplementary Figure S8.

When this project entered Genentech's Small Molecule portfolio, our goal was to identify specific inhibitors through several methods: either by Fragment based lead discovery (FBLD) using through Surface Plasmon Resonance and Nuclear Magnetic Resonance or through our High Throughput Screening platform. These approaches combined with structure based drug design led us to the discovery of multiple scaffolds (covalent and non-covalent) including unique allosteric binders that displayed specific and on-target activity [START_REF] Di Lello | Discovery of Small-Molecule Inhibitors of Ubiquitin Specific Protease 7 (USP7) Using Integrated NMR and in Silico Techniques[END_REF]Kategaya et al., 2017).

These ligands were identified to bind the "palm" region of the catalytic domain, about 12 Å away from Cys223 and yet inhibiting its DUB activity (see Figure 44). Crystal structures of USP7 catalytic domain bound to these compounds revealed that they sterically inhibit ubiquitin binding by engaging with Asp305, an acidic residue that typically coordinating Lys48 of ubiquitin in concert with Glu308 (see Figure 44b). Although the project was discontinued for multiple reasons, our work (along with others) showed that this novel class of enzymes was druggable and that selective inhibition through allosteric site was achievable. 95 contributed to the structural elucidation of the unique activation mechanism of this DUB through its C-terminal along with a better understanding of the overall organization of its accessory domains relative to the catalytic domain. Yet, many questions remain unanswered and it will take more studies to fully decipher the different determinants that regulate its activity but also dictate its selectivity. Answering these questions will be critical to rationalize and optimize inhibitors that could be, one day, used in the treatment of life-threatening diseases in humans.

INTRODUCTION

Ubiquitin-specific protease 7 (USP7) plays a key regulatory role in many aspects of mammalian biology and disease by controlling the stability and localization of proteins involved in DNA replication, epigenetic regulation, the cell cycle, mitosis, and cell survival [START_REF] Du | DNMT1 stability is regulated by proteins coordinating deubiquitination and acetylation-driven ubiquitination[END_REF]Everett et al., 1997[START_REF] Everett | The ability of herpes simplex virus type 1 immediate-early protein Vmw110 to bind to a ubiquitin-specific protease contributes to its roles in the activation of gene expression and stimulation of virus replication[END_REF][START_REF] Kessler | Selective and reversible inhibitors of ubiquitin-specific protease 7: a patent evaluation (WO2013030218)[END_REF]Maertens et al., 2010;Song et al., 2008;[START_REF] Sowa | Defining the human deubiquitinating enzyme interaction landscape[END_REF]van der Horst et al., 2006). USP7, alternatively known as herpes associated ubiquitin-specific protease (HAUSP), belongs to a class of cysteine protease deubiquitinating enzymes (DUBs) (Komander et al., 2009) and was first identified by its ability to enhance herpesvirus replication by stabilizing infected cell polypeptide 0 (ICP0) [START_REF] Everett | The ability of herpes simplex virus type 1 immediate-early protein Vmw110 to bind to a ubiquitin-specific protease contributes to its roles in the activation of gene expression and stimulation of virus replication[END_REF]. The role of USP7 has been extensively studied in tumor biology due to its regulation of the tumor suppressor p53 and the p53 ubiquitin ligase, murine double minute 2 protein (MDM2) (Brooks andGu, 2006) (Cummins et al., 2004) (Li et al., 2004).

The complex role USP7 plays in the cell is likely facilitated by its multi-domain architecture. In addition to the catalytic domain, USP7 also contains an N-terminal TRAF domain (NTD) involved in substrate peptide binding (Hu et al., 2006;Saridakis et al., 2005;[START_REF] Sarkari | USP7/HAUSP promotes the sequence-specific DNA binding activity of p53[END_REF]Sheng et al., 2006;Zapata et al., 2007) and five C-terminal ubiquitin-like (UBL) domains (Figure 1A), which share a common b-grasp fold (Komander et al., 2009;[START_REF] Zhu | High incidence of ubiquitin-like domains in human ubiquitin-specific proteases[END_REF]. The first three UBL domains (UBL123) are involved in specific binding interactions with ICP0 (Holowaty et al., 2003;Pfoh et al., 2015), DNA methyltransferase-1 (DNMT1) (Cheng et al., 2015), ubiquitin-like, containing PHD and RING finger domains (UHRF1) (Zhang et al., 2015), MDM2 (Ma et al., 2010), and guanidine monophosphate synthetase (Faesen et al., 2011). UBL domains four and five (UBL45) have been shown to be essential for full deubiquitinase activity (Faesen et al., 2011;Ma et al., 2010). The isolated USP7 catalytic domain (USP7CD) has weak catalytic activity, consistent with the apocrystal structure that demonstrated that the catalytic domain exists in an inactive conformation with residues of the catalytic triad (Cys223, His464, and Asp481) misaligned (Hu et al., 2002). In addition, the loops close to the active site are in conformations that would sterically hinder ubiquitin binding. The crystal structure of USP7CD with ubiquitin covalently bound to Cys223 revealed that parts of USP7CD undergo significant conformational rearrangements to an active state with the catalytic residues productively aligned (Hu et al., 2002). There is a significant conformational change in an active-site loop, residues 283-295, from an inhibitory ''in-conformation'' in the apo-structure of USP7CD to an active ''out-conformation'' in the USP7CD structure in complex with ubiquitin; this loop is referred to as the switching loop (Faesen et al., 2011).

Given the importance of USP7 in biology and disease, we attempted to determine the structural relationship between the catalytic domain and UBL domains resulting in enzymatic activation. We demonstrate that a minimal C-terminal region of USP7, without the UBL domains, increases the activity of USP7CD by more than 50-fold and reveal the structural basis of this activation. In addition, we dissect which domains of USP7 are important for high-affinity binding to the substrate MDM2. This work provides novel insight into the structure of full-length USP7, reveals the molecular mechanism of activation, and shows that the entire protein is required for high-affinity MDM2 binding.

RESULTS

Activation of USP7 by C-Terminal Residues

The 19 C-terminal residues of USP7 from Lys1084 to Asn1102 are highly conserved across species (Figures 1A and1B) and are unstructured in the reported crystal structures of USP7 UBL12345 domains (Cheng et al., 2015;Faesen et al., 2011). The C-terminal residues were previously proposed to enhance/activate USP7 catalytic activity (Faesen et al., 2011). As reported previously, deletion of the residues at the C terminus of USP7 is sufficient to prevent USP7 activation (Faesen et al., 2011). Interestingly, high concentrations of the free 19-residue C-terminal peptide can substantially increase the activity of the catalytic domain (Table S1 andFigure S1). To further understand the activation of the USP7 catalytic domain by the C-terminal peptide (CTP), we engineered a series of novel constructs by tethering the catalytic domain to either a 19-residue peptide (Lys1084-Asn1102), or a 7-residue peptide (Lys1096-Asn1102) using a glycine-glycine-serine linker (GGS linker) with lengths of 5, 10, 15, or 20 residues. The activity of these constructs was assessed by a cleavage assay. Surprisingly, several of these peptide extensions increases the activity of the catalytic domain from barely detectable to 66% of full-length USP7 (Figure 1C). The K m for the 19-residue CTP with a linker of 5, 10, or 15 residues was identical to the full-length USP7, while k cat for the linkers with 10 and 20 residues was within 2-fold of the activity of the full-length protein (Table S1). We chose to further characterize the 10-residue linker construct, hereafter referred to as USP7CD-CTP.

Point mutations at each of the nine C-terminal residues in USP7CD-CTP constructs indicate that alanine substitutions at residues Ile1098, Lys1099, Ile1100, and the C-terminal Asn1102 are poorly tolerated and reduce activity by 90% or more (Table 1). Isoleucines 1098 and 1100 are particularly critical for activity, and alanine mutations at these sites reduce activity to the same level as the catalytic domain alone at 30 C, while mutation at Lys1099 and Asn1102 results in a 7-to 8-fold reduction in k cat . To determine accurate enzyme kinetics for the mutations with insufficient activity at 30 C, additional experiments were conducted at 37 C (Table S2). At 37 C, both the K m and k cat of the I1100A mutant are negatively affected, and to a lesser extent in the I1098A mutant.

Structural Understanding of Catalytic Activation by the UBL45 Domain and the C-Terminal Peptide of USP7 Intrigued by the ability of the UBL45 domain and the C-terminal peptide to activate the catalytic domain, we sought to understand the molecular mechanism of activation. We determined the crystal structures of USP7CD-UBL45 and USP7CD-CTP, both with ubiquitin covalently attached to the catalytic cysteine (Cys223), and the structure of the USP7CD-CTP construct in S1 andFigure S1B. Percent activity is given as k cat /K M relative to full-length USP7. See also Figure S1 and Tables S1 andS2.

the absence of ubiquitin. The USP7CD-UBL45 ubiquitin complex crystals diffracted to 3.3 A ˚with P2 1 2 1 2 1 symmetry and four molecules in the asymmetric unit, arranged as a dimer of dimers (Table 2). The structure was solved by molecular replacement using models of the USP7CD-ubiqutin complex (PDB: 1NBF) and the UBL45 domain (PDB: 2YLM). The catalytic domain adopts the active conformation with the C terminus of ubiquitin covalently attached to Cys223 and with the switching loop, residues 283-295, in the out-conformation. There was a large F o F c positive difference density peak in the cleft created by the conformational change of the switching loop that was continuous up to the C terminus of the UBL5 model, residue N1083.

A model for the C-terminal region, residues 1084-1102, was built into the positive F o -F c difference density and the structure refined (Figures 2A-2C). There was no interpretable electron density for the 10-residue GGS linker that tethers the catalytic and UBL45 domains, and this created some ambiguity in the connectivity of the different domains. The closest distance from the C terminus of the catalytic domain to the N terminus of the UBL4 domain is 11 A ˚, and this suggests the UBL45 domain packs against the catalytic domain of a non-crystallographic symmetry (NCS)-related molecule and therefore the C-terminal region binds in trans in this structure (Figures 2A andS3C).

The crystals of apo-USP7CD-CTP diffracted to 3.0 A ˚, and the structure was solved by molecular replacement using the inactive form of USP7CD as the search model (PDB: 1NB8). The refined structure was similar to the inactive form of the USP7CD, with no electron density for the linker or the C-terminal peptide observed (data not shown). The crystals of USP7CD-CTP in complex with ubiquitin diffracted to 3.32 A ˚resolution, and the structure was solved by molecular replacement using the USP7CD-ubiquitin complex (PDB: 1NBF) as the search model (Table 2). In contrast to the apo-USP7CD-CTP structure, there was a large and continuous positive F o F c electron density peak in the cleft created by the conformational change of the switching loop. A model for nine residues from the C-terminal peptide, 1094-LEKAIKIHN-1102, was built into the positive F o F c density peak, and the structure was refined (Figure S4). The refined structure reveals that the catalytic domain adopts the active conformation very similar to that seen in the USP7CD-UBL45 structure (root-mean-square deviation [RMSD] = 0.8). There was no interpretable electron density for the 10-residue linker that tethers the catalytic domain to the C-terminal peptide leading to ambiguity over whether the peptide is bound in cis or trans.

The structures of USP7CD-UBL45 and USP7CD-CTP demonstrate that the C-terminal peptide binds into a cleft that results from the conformational rearrangement of the switching loop, subsequently referred to as the activation cleft. Productive binding of the C-terminal peptide relies on both hydrogen bonding and hydrophobic interactions (Figures 2C andS4). These include the C-terminal Asn1102 engaging Asn306 and Lys310 on the a5 helix, 3.31 (3.42-3.31) 74.03-3.32 (3.33-3.32) 74. 25-3.20 (3.42-3.20) Space group P2 1 2 1 2 1 P6 2 22 F222 Unit cell dimensions a, b, c (A ˚) 113. 6, 115.5, 257.8 148.1, 148.1, 179.9 104.6, 196.8, 226.9 a, b, g ( ) 90.0, 90. (legend continued on next page)

H-bond interacting between the backbone amide nitrogen of His1101 and the carbonyl oxygen of Phe283 from the switching loop, the carbonyl of Lys1099 interacting with the indole nitrogen of Trp285 from the switching loop, and the carbonyl of Lys1096 engaging in H-bond interactions with the side-chain guanidino group of Arg74 from ubiquitin. The Ile1098 and Ile1100 side chains are buried into hydrophobic cavities on USP7CD, consistent with their critical role in catalytic activation (Figure S1 and Table S2).

Mutation of residues Asn306 and Trp285 further confirm the critical importance of these residues in binding to the USP7CTD (Table S1) and are consistent with the function of Trp285 proposed previously (Faesen et al., 2011).

The binding of the C-terminal peptide does not significantly perturb the structure of the catalytic domain as demonstrated by the excellent agreement of equivalent residues of the USP7CD-UBL45, USP7CD-CTP, and PDB: 1NBF structures (RMSD = 0.78 and 0.57 A ˚, respectively). Instead, the observed increase in catalytic activity is likely due to the stabilization of this active conformation. Furthermore, the structure of the USP7CD-UBL45 indicates the improved activity observed in this construct compared with the USP7CD-CTP construct is likely due to the more optimal positioning of the C-terminal residues of the USP7CD-UBL45 so that the C-terminal residues of ubiquitin are locked in the substrate channel of USP7 (Figure 2A).

NMR Evaluation of C-Terminal Peptide Extension on Ubiquitin Binding

To further assess the solution dynamics of conformational changes of USP7CD, 1 H/ 15 N correlated nuclear magnetic resonance (NMR) spectra of USP7CD and USP7CD-CTP were measured and compared. These spectra show good superposition of all peaks except the C-terminal Lys554 and, to a lesser extent, the preceding three residues. Additional peaks in the C-terminally extended construct are visible and indicate a largely unstructured linker with characteristic peaks in the region of the Gly residues. This lack of differences in the backbone amide proton and nitrogen chemical shift between the two constructs indicate that the overall structures are similar and that the presence of the C-terminal extension has no effect on the overall properties of USP7CD including its solution dynamics. The overlay is shown in Figure S2 for clarity. Not all signals of USP7CD could be assigned with approximately 30% of the expected number of peaks missing and/or broadened due to intermediate exchange between substructures. This is particularly the case in the region near the active site stretching from residues Leu216 to Lys240.

In contrast to the similarities in the spectra of USP7CD and USP7CD-CTP alone, the spectra of the ubiquitin-bound state show extensive differences. We have classified two types of change upon addition of ubiquitin. Chemical shift perturbations are observed with the addition of ubiquitin to the catalytic domain, and the gradual shift changes indicate fast exchange. Based on these chemical shift changes, the affinity of ubiquitin binding to the USP7CD can to be assigned in the range of 120-140 mM (Figure S2C and Table S3). These fast exchange shifts map to the USP7CD palm-domain ubiquitin interface and align well with the bound state in the covalent complex solved by X-ray crystallography. Perturbations of the same residues are observed upon addition of ubiquitin to the USP7CD-CTP, one difference being that these are now in intermediate exchange, reflecting the increased binding affinity also observed in the biochemical assays. Shown in Figure S2E, the same residues in USP7CD and USP7CD-CTP are affected and indicate that the USP7CD-CTP-ubiquitin complex has the same interface and structure, consistent with X-ray crystallographic data.

A second set of changes, marked with asterisks in Figure 2E, are observed in the 15 N/ 1 H correlated NMR spectra of the USP7CD-CTP construct. These approximately 80 new signals, previously not observed in the spectra of USP7CD alone (Figure S2), by far outnumber the signals expected from the C-terminal extension alone, and hence most likely stem from the catalytic domain. Taken together, this suggests that the C-terminal peptide is stabilizing the ubiquitin-bound state of USP7, reducing the conformational averaging of chemical shifts in the catalytic domain and, at the same time, changing the dynamics of parts of the USP7CD.

The Crystal Structure of USP7 Catalytic Domain with UBL Domains 1-3, USP7CD-UBL123, Reveals a Modular Arrangement of the Catalytic Domain and UBL Domains

To gain an improved understanding of the functions of the UBL domains in terms of their role in substrate recognition and their relationship to the catalytic domain of USP7, we crystallized a construct that includes the catalytic domain and UBL123 domains (UDP7CD-UBL123), residues 208-882 (Figure 3). The final model was refined to 3.2 A ˚resolution and includes residues 211-881 (Table 2).

In the context of the USP7CD-UBL123 structure, the catalytic domain (residues 208-554) adopts a conformation characteristic of the inactive form of the enzyme, very similar to other apo-USP7 crystal structures that have been determined (Ca RMSDs = 0.76 A ˚against PDB: 1NB8). The residues of the catalytic triad, Cys223, His464, and Asp481, are in a non-productive conformation with the side chains of His464 and Asp481 within hydrogen bonding distance of each other, but the side chain of His464, the proposed catalytic base, is over 10 A ˚distant from the sulfhydryl group of the catalytic cysteine, Cys223. The residues of blocking loop 1 (residues 408-429), blocking loop 2 (residues 459-462), as well as the switching loop (residues 285-291), are in conformations that would sterically hinder ubiquitin binding, which is similar to the other crystal structures of apo-USP7CD. The crystal packing observed in all apo-catalytic domain structures are very similar, but the crystal packing of the current USP7CD-UBL123 structure reveals different packing interactions indicating that the active-site loop conformations are not caused by crystal packing effects and suggest that USP7CD exists in an inactive conformation.

(D) The effect of addition of unlabeled ubiquitin onto 2 H/ 13 C/ 15 N-labeled USP7CD. (E) The effect of addition of unlabeled ubiquitin onto 2 H/ 13 C/ 15 N-labeled USP7CD GGS 10 -CTP. In both (D) and (E), a zoomed region of the 15 N TROSY-HSQC spectra of USP7CD (pink on the left) and USP7CD GGS 10 -CTP (blue on the right) on their own and after addition of unlabeled native ubiquitin (from bovine erythrocytes; Sigma) in a 10-fold access (light blue and green) is shown. Arrows show the chemical shifts observed from the direct interaction. Asterisks mark the appearance of new signals in the USP7CD GGS 10 -CTP spectra. See also Figure S2.

The USP7CD-UBL123 structure contains an extended a10 helix spanning 24 residues (residues 538-562) that links the catalytic domain to the first UBL domain (Figure 3C). This extended a10 helix gives the structure an overall boomerang shape that places the UBL domains distant from the catalytic domain. Although UBL2 and UBL3 curve back toward the catalytic domain, there are no interactions between the UBL and catalytic domains (Figure 3). The extended helical conformation of the a10 helix has also been observed in the crystal structure of UBL12 domains alone (PDB: 4PYZ, residues 537-793) and UBL123 alone (PDB: 4WPH and 4WPI, residues 535-888). The superposition of equivalent residues from these structures on the USP7CD-UBL123 structure indicates they are very similar (RMSD = 0.62, 2.02, and 2.22 A respectively).

The three UBL domains, residues 563-881, each adopt a b-grasp ubiquitin-like fold and exist in an elongated conformation. UBL1 and UBL2 form a closely packed unit burying a large surface area of 1200 A ˚2. In contrast, the UBL3 domain is an independent unit that is largely solvent exposed and has limited contacts with the other UBL domains. The elongated arrangement of the UBL123 domains observed in this structure is similar to the crystal structure of the isolated UBL1-5 domains (PDB: 2YLM), (Figure 4B). The Ca atoms for equivalent residues in UBL domains 1-2 superimpose with an RMSD = 0.6 A ˚, and the Ca atoms for equivalent residues in UBL domains 1-3 superimpose with an RMSD = 2.6 A ˚. The larger RMSD when UBL3 is included in the superposition is a result of a difference in the relative orientation of UBL3 with respect to the UBL12 domain in the two structures. This larger RMSD may be due to crystal packing effects or alternatively to the dynamic nature of the UBL3 domain. Interestingly, crystal packing indicates that the UBL1-3 domains pack around the base of a symmetry-related catalytic domain. Residues from the b13 strand and the a8 helix in the catalytic domain and residues spanning UBL2 and UBL3 of the symmetry-related molecule make up an extensive interface that buries 1750 A ˚2 and contains 24 salt-bridge interactions and 12 hydrogen bond interactions. Although there is no evidence that USP7 forms oligomers in solution in the absence of protein substrate, the crystal packing interactions might influence the boomerang shape of the protein.

Molecular Model of Full-Length USP7

The crystal structure of USP7CD-UBL123 reveals the catalytic domain in the inactive conformation with the UBL123 domains prevented from interaction with the catalytic domain by an extended a10 helix. In contrast, the USP7CD-UBL45 structure in complex with ubiquitin, the catalytic domain, is in the active conformation, and this structure reveals a fully ordered C-terminal region with the UBL45 domain interacting with the catalytic domain in trans (Figure 2). In attempting to build a coherent model for full-length USP7 by incorporating structural information from USP7CD-UBL123, USP7CD-UBL45, NTD-USP7CD (Hu et al., 2006) (PDB: 2F1Z), and UBL12345 in complex with DNMT1 (Cheng et al., 2015) (PDB: 4YOC), we were unable to propose a plausible model using single-chain protomers from our individual crystal structures, in which the C-terminal activates in cis. Instead our proposed model for full-length USP7 relies on the non-crystallographic symmetry relationship observed in the USP7CD-UBL45 structure. The centerpiece for our model is the USP7CD-UBL123 structure. Superposition of the equivalent Ca atoms from the catalytic domain of the NTD-USP7CD crystal structure (PDB: 2F1Z) results in a model that suggests that the NTD does not interact with the UBL domains. Superposition of the equivalent Ca atoms from the DNMT1-UBL1-5 complex structure (PDB: 4YOC), residues 562-881, onto the USP7CD-UBL123 structure (RMSD = 2.6 A ˚) reveals that the UBL45 domains curve back toward the catalytic domain. In the UBL1235-DNMT1 complex structure, the C-terminal region, residues 1084-1102, was not observed, and the C-terminal residue of the UBL12345 domain is Asn1083. The distance from the Ca of Asn1083 in the transformed model to the switching loop region of the USP7CD-UBL123 structure is 47 A ˚(Figure 4C). Next, we superposed the UBL45 domain from the USP7CD-UBL45 structure onto equivalent residues from the transformed PDB: 4YOC structure. In the structure of the USP7CD-UBL45, the distance from the Ca of Asn1083 to the Ca of Asn1102 is 45.7 A ˚. This suggests the C terminus of USP7 could bind into the activation cleft in cis, with minor conformational changes in the UBL domains, but would require larger movements of the C-terminal peptide (Figure 4). The proposed flexibility of the C-terminal region is consistent with the two crystal structures that contain UBL12345 (PDB: 2YLM and 4YOC) since, in both these structures, the C-terminal region is disordered from residues 1084-1102. Our model of fulllength USP7 is also consistent with the UBL12345-DNMT1 complex structure (PDB: 4YOC) as superposition of this complex onto the equivalent residues in our full-length USP7 model reveals that DNMT1 is easily accommodated into the central cavity of the boomerang shape created by the catalytic and UBL domains of USP7, and DNMT1 binding brings the NTD of USP7 in close proximity with N-terminal domain of DNMT1 (Figure S3).

An alternative model for full-length USP7 in which the C-terminal peptide activates in trans is also possible. In this model of the non-crystallographic symmetry relationship of the UBL45 domain (UBL45 0 ) observed in the USP7CD-UBL45 structure (Figure 2), the catalytic domain and the symmetry-related UBL123 domain (UBL123 0 ) from the USP7CD-UBL123 structure serve as the centerpiece. This positions the C terminus of UBL3 0 in close proximity to the N terminus of the trans interacting UBL4 0 domain. Superposition of the equivalent Ca atoms from the catalytic domain of the NTD-USP7CD crystal structure (PDB: 2F1Z) results in a model that suggests that the NTD and UBL domains are in close proximity. There are a few clashes in this model that can be relieved by energy minimization (Figure S3). Although the trans activation model requires a mix and match approach from different structures and does not fit together as easily as the cis activation model, a trans activation mechanism is possible based on the ability of the isolated 19-residue peptide to activate the catalytic domain in trans (Figure S2). However, multi-angle light scattering data suggest that both USP7CD-UBL45 and USP7CD-CTP behave as monomers in solution (data not shown), but it is possible that cis and/or trans activation could occur depending on the nature of the substrate.

MDM2 Recognition by USP7

Previous work has shown that fragments of known USP7 substrates bind weakly to both the USP7-NTD domain (Hu et al., 2006;Saridakis et al., 2005;Sheng et al., 2006) and USP7-UBL123 domains (Holowaty et al., 2003;Ma et al., 2010). However, no studies have addressed the question of whether the USP7 domains could recognize substrate in an additive manner. To address this question, we evaluated the ability of the USP7 deletion constructs characterized in Table S1 to bind MDM2 by surface plasmon resonance (SPR). Consistent with the previous reports of peptide binding to the USP7-NTD domain alone, we observed a weak interaction of full-length MDM2 with a USP7 construct containing NTD-USP7CD (Figure 5B). However, we found that full-length USP7 binds MDM2 with nanomolar affinity (Figure 5A), although no direct interaction of USP7CD-UBL12345 with MDM2 was observed at concentrations of USP7 up to 500 nM (Figure 5C). Although multiple interaction sites have been proposed between the MDM2 dimer and USP7 (Faesen et al., 2011;Ma et al., 2010;[START_REF] Cheng | Regulation of MDM2 E3 ligase activity by phosphorylation after DNA damage[END_REF], the chi-squared/Rmax goodnessof-fit criteria are satisfied using a singlesite model, suggesting the multiple MDM2-USP7 interaction sites act cooperatively and cannot be experimentally deconvoluted. This model of cooperative binding is supported by the observation that the binding of USP7-NTD and USP7CD-UBL12345 when added together do not reproduce the binding of full-length USP7 (Figure S4). This experiment cannot determine if one or two protomers of USP7 bind to the MDM2 dimer.

DISCUSSION

Using mutagenesis, enzyme kinetics, biophysical binding experiments, and X-ray crystallography, we have shown how the C terminus of USP7 acts to enhance deubiquitinase activity by stabilizing the ubiquitin binding conformation of the catalytic domain. Individual point mutations at residues I1100 or I1098 in the C-terminal peptide are able to abolish this enhancement in deubiquitinase activity (Table 1 andFigure S1), supporting previous work with the I1100S mutant (Faesen et al., 2011), the specificity of the interaction and the importance of the Hyd-X-Hyd motif, represented by Ile 1098 -Lys-Ile 1100 in USP7. The binding of the C-terminal peptide into an activation cleft in the USP7 catalytic domain is made possible by the conformational transition of the switching loop as it moves from an inactive in-conformation to an active out-conformation. These data, combined with the other crystal structures of USP7, enable us to propose an activation mechanism in which the binding of ubiquitin initiates a series of conformational changes not only in the catalytic domain but also the C terminus of the UBL domains. These conformational changes include the conversion of the catalytic loop, containing the catalytic Cys223, to an a-helical conformation and the movement of the switching loop into the out-conformation thus exposing the activation cleft, which allows binding of the C-terminal peptide, stabilizing the active conformation and the catalytic triad. These new crystal structures of USP7 provide insight into the role of the UBL domains. We propose a model for full-length USP7 that suggests the UBL domains do not interact with the catalytic domain but rather the C-terminal region is in close proximity to the catalytic domain and is able to stabilize the active conformation (Figure 4D). Due to the low resolution of the crystal structures, the disorder of the engineered linker, and the conformational flexibility of the UBL domains, we are unable to determine definitively whether the C terminus of USP7 binds into the activation cleft in cis or in trans. However, since we are able to activate the catalytic domain with the addition of peptide, trans activation is certainly possible and could be employed as a regulatory mechanism of USP7 on some substrates.

Although both the individual NTD and UBL domains have previously been reported to have some interaction with MDM2, it is clear from the SPR data that both the NTD and the UBL domains are necessary for high-affinity binding of USP7 to MDM2. In addition, MDM2 binding is not affected by removal of the USP7 C-terminal peptide, suggesting that MDM2 binding and ubiquitin cleavage activity are independently regulated. The affinities we measured for the USP7-MDM2 interaction are, however, consistent with MDM2 as a specific substrate of USP7, and the affinity of USP7 for MDM2 is similar to the binding affinity of UBL12345 to DNMT1 (Cheng et al., 2015). The MDM2 binding data are in agreement with the cis activation model, and this model suggests that MDM2 may bind in a manner similar to that depicted in the full-length USP7-DNMT1 complex model (Figure S3).

In the absence of substrate, the catalytic domain of USP7 exists in an autoinhibited state. It is not uncommon for the activity of important cellular proteins to be autoinhibited (Pufall and Graves, 2002), and there are many examples of the tight control of protease activity by an activating peptide; this activation mechanism is especially common among viral proteases (Bartenschlager et al., 1994(Bartenschlager et al., , 1995)). However, to our knowledge, this is the first example of a ubiquitin-specific protease being regulated by a mechanism involving an activating peptide that stabilizes an active conformation. Multiple sequence alignment of all human USPs indicates that only USP47 and USP40 contain switching loop consensus motifs that are similar to USP7 (Ye et al., 2009). Intriguingly, USP47 has a conserved seven-residue activation sequence containing a Hyd-X-Hyd motif that is not at the very C terminus but is instead located in a region that is distal from the C terminus of USP47. No corresponding activation motif was found in USP40, suggesting that its activity is either not regulated, as is observed for other DUBs, or it is regulated by an alternate mechanism, or the conservation of an activation motif in the USP40 sequence is too low to be detected. In future work, it will be interesting to understand how USP7 interacts with other protein substrates, and how this may regulate USP7 activity and substrate stability.

EXPERIMENTAL PROCEDURES

Expression and purification of the USP7 constructs used in the biochemistry experiments and enzyme kinetics with ubiquitin-AMC are described in the Supplemental Experimental Procedures.

Expression and Purification of USP7CD-UBL123, USP7CD-UBL45, and USP7CD-CTP for Crystallography The gene encoding a fragment of USP7 equivalent to residues 208-882 (USP7CD-UBL123) was cloned into the NdeI/BamHI sites of pGEX4-T1 (Novagen), yielding an expression construct with an N-terminal GST tag followed by a thrombin cleavage site. The USP7CD-UBL45 (K208-N1102-DR555-L881) and USP7CD-CTP (K208-K554-DR555-N1083-K1084-N1102) constructs were cloned into a modified pET vector with a TEV cleavable 6-histidine fusion at the N terminus. The plasmids were transformed into Escherichia coli BL21(DE3) Gold cells (Stratagene) and expression was induced by the addition of 0.5 mM isopropyl b-D-1-thiogalactopyranoside and growth at 16 C for 4 hr. The cells were harvested by centrifugation and resuspended in 25 mM Tris-HCl (pH 8.0), 300 mM NaCl, 0.5 mM tris(2-carboxyethyl)phosphine (TCEP), 10 mM imidazole, and 5% (v/v) glycerol (buffer A) and homogenized before being lysed with a microfluidizer; the lysate was clarified by centrifugation (15,000 3 g for 45 min) and no imidazole was added to buffer A for the USP7CD-UBL123 construct purification. The supernatants from the USP7CD-UBL45 and USP7CD-CTP lysis were loaded onto an Ni-NTA Superflow column (QIAGEN) pre-equilibrated with buffer A, and the column was washed with 50 mL of buffer A containing 40 mM imidazole and eluted with 30 mL of buffer A containing 300 mM imidazole. The supernatant from the USP7CD-UBL123 was loaded onto a 5 mL GST Sepharose column (GE Healthcare) pre-equilibrated with 20 mL of buffer A. The column was washed with 50 mL of buffer A and eluted with 30 mL of buffer A containing 10 mM reduced glutathione (Sigma). The six-histidine tags were removed by TEV protease (Sigma) and the GST tag was removed by thrombin (Calbiochem).

Cleaved USP7CD-UBL45 and USP7CD-CTP were passed through a 3 mL Ni-NTA Superflow column (QIAGEN) pre-equilibrated with 20 mL of buffer A. The column was washed with 25 mL of buffer A containing 40 mM imidazole. All three proteins were subjected to gel filtration using a HiLoad 16/60 Superdex 75 prep grade column (GE Healthcare) pre-equilibrated with 25 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 0.5 mM TCEP. The peak fractions were pooled and the final protein concentration was determined by absorbance at 280 nm.

Production of Ubiquitin Bromoethylamine and Complex Formation with USP7CD-UBL45 and USP7CD-CTP Ubiquitin bromoethylamine (Ub-BEA) was produced according to published procedures (Borodovsky et al., 2002;Wilkinson et al., 2005). The ubiquitin thiolester intermediate (3 mL at 3 mg/mL) was mixed with 450 mg of bromoethylamine (MP Biomedicals) and 600 ml of 2 N NaOH. The reaction was incubated at room temperature for 30 min and quenched by loading it onto a HiLoad 16/60 Superdex 75 prep grade column (GE Healthcare) pre-equilibrated with 25 mM Tris-HCl (pH 7.5), 300 mM NaCl, and 0.5 mM TCEP. The fractions corresponding to the desired molecular weight for Ub-BEA were collected; 10 mg of purified USP7CD-UBL45 or USP7CD-CTP were immediately added and the reaction was incubated overnight at 4 C. The resultant complexes were dialyzed against 25 mM Tris-HCl (pH 8.0), 75 mM NaCl, and 0.5 mM TCEP (buffer B) before loading onto a Mono Q 10/100 GL column (GE Healthcare) pre-equilibrated with 40 mL of buffer B. The column was washed with buffer B followed by elution of the complex with a gradient of 0-0.75 M NaCl in the same buffer. Peak fractions were pooled and complex formation was confirmed by analysis on native PAGE gels. The complexes were concentrated to a final concentration of 10 mg/mL.

Crystallization and Structure Determination of USP7CD-UBL45 in

Complex with Ubiquitin, USP7CD-CTP in Complex with Ubiquitin, and USP7CD-UBL123 Crystals of the USP7CD-UBL45-ubiquitin complex were obtained at 4 C by the hanging-drop vapor-diffusion method by mixing equal volumes of protein at 10 mg/mL with well solution containing 0.2 M K chloride, 0.05 M HEPES (pH 7.5), and 35% v/v pentaerythritol propoxylate. Crystals of the USP7CD-CTPubiquitin complex were obtained at 19 C by the hanging-drop vapor-diffusion method by mixing equal volumes of protein at 10 mg/mL with well solution containing 0.05 M calcium chloride, 0.1 M 2-(N-morpholino)ethanesulfonic acid (pH 6.0), and 45% PEG 200. Diffraction images were collected at the Advanced Photon Source beamline ID22.

Crystals of USP7CD-UBL123 were obtained at 19 C by the hanging-drop vapor-diffusion method by mixing equal volumes of USP7CD-UBL123 at 15 mg/mL with a well solution containing 0.2 M tri-potassium citrate (pH 8.3) and 20% PEG 3350. Crystals typically appeared after a couple of days, and initial diffraction was poor. The diffraction properties were improved by dehydrating the crystals by sequential equilibration of the drops above wells containing increasing concentrations of PEG 3350. The final concentration of the dehydration solution was 50% PEG 3350, and this same solution served as the cryoprotectant. The crystals were harvested and flash frozen in liquid nitrogen. Diffraction images were collected at the Diamond Light Source beamline I02.

All datasets were indexed and integrated with XDS (Kabsch, 2010), scaled with SCALA (Evans, 2006), and the structures were solved by molecular replacement using PHASER (McCoy, 2007) with structures of the USP7CD (PDB: 1NB8 or 1NBF) and a truncated form of the UBL domains as the search models (PDB: 2YLM). The final models were obtained after iterative cycles of manual rebuilding in COOT (Emsley and Cowtan, 2004) and refinement in PHENIX (Adams et al., 2010).

Surface Plasmon Resonance

Binding experiments were carried out by SPR measurements on a Biacore 3000 (GE Healthcare) instrument at 25 C. Affinity purified, E. coli expressed hMDM2 was purchased from Millipore (catalog no. 23-033 M). To avoid avidity effects from the multimeric protein, hMDM2 was immobilized at the sensor at a surface density of 1,000 RU on an activated Biacore CM5 sensor chip using standard amine coupling procedures as described by the manufacturer. All experiments were conducted in HBS-P (0.01 M HEPES [pH 7.4], 0.15 M NaCl, 0.005% v/v Surfactant P20). Association rates (k a ) and dissociation rates (k d ) were calculated using a one-to-one Langmuir binding model by simultaneous fitting of the association and dissociation sensorgrams (BIAevaluations version 4.1). All fits have a chi-square/Rmax value of less than 10%, satisfying accepted goodness-of-fit criteria. The equilibrium dissociation constant (K D ) was calculated as the ratio of k d /k a .

NMR Spectroscopy

All NMR spectra were recorded on Bruker Avance 600 and 800 MHz spectrometers operating at 14.1 and 18.8 T using triple resonance cryogenic probes optimized for proton detection. All 3D spectra were acquired with a spectral width of 16 ppm and 1,024 data points in the direct proton dimension, 22 ppm (HNCA) and 59 ppm (CBCA(CO)NH), and 128 data points in the 13 C dimension using constant time evolution, States-TPPI acquisition [START_REF] Marion | Application of phase sensitive two-dimensional correlated spectroscopy (COSY) for measurements of 1H-1H spin-spin coupling constants in proteins[END_REF], and 28 ppm and 56 sample points in the 15 N dimension with echo-antiecho type selection [START_REF] Cavanagh | Sensitivity improvement in isotropic mixing (TOCSY) experiments[END_REF]. The resulting spectral resolutions were 10, 28, and 60 Hz/data points, respectively. Maximum entropy linear prediction [START_REF] Laue | Reconstruction of phase-sensitive two-dimensional NMR spectra by maximum entropy[END_REF] was used to increase the resolution in the indirect dimensions. All spectra were recorded at 298 K. In order to avoid chemical shift deviations due to differences in temperature, all spectrometers were calibrated using the methanol/glycol method [START_REF] Findeisen | A 1H-NMR thermometer suitable for cryoprobes[END_REF]. The pH was set to 7.2 without correction due to isotope shifts. The backbone resonances were assigned with 3D HNCA [START_REF] Grzesiek | An efficient experiment for sequential backbone assignment of medium-sized isotopically enriched proteins[END_REF][START_REF] Stonehouse | Minimisation of sensitivity losses due to the use of gradient pulses in tripleresonance NMR of proteins[END_REF] 3D CBCA(CO)NH [START_REF] Grzesiek | Amino acid type determination in the sequential assignment procedure of uniformly 13C/15N-enriched proteins[END_REF], and 3D HNCO [START_REF] Schleucher | Coherence selection by gradients without signal attenuation: application to the three-dimensional HNCO experiment[END_REF] as well as [ 15 N, 1 H] nuclear Overhauser effect spectroscopy (NOESY)-heteronuclear single quantum coherence (HSQC) (Bax et al., 1990) spectra. 2D data were collected in the phase-sensitive mode using the echo-antiecho method [START_REF] Findeisen | A 1H-NMR thermometer suitable for cryoprobes[END_REF] with 4,000 sampling points in the direct dimension and 512 data points in the indirect dimension.

NOESY spectra were recorded with a mixing time of 150 ms. Interaction of USP7 with ubiquitin was monitored by the chemical shifts changes in the [ 15 N, 1 H]TROSY spectra. The Bruker software package TOPSPIN 3.2 was used for data processing. All data evaluation was done in NMR view and CCPN [START_REF] Vranken | The CCPN data model for NMR spectroscopy: development of a software pipeline[END_REF]. Visualization and presentation of the 3D tertiary USP7, as well as related protein structures from the PDB, were done in PyMOL (The PyMOL Molecular Graphics System, Version 1.7.4; Schro ¨dinger, LLC). The sequential assignment of USP7 wild-type is described elsewhere. 2 H, 15 N, 13 C isotopically labeled NMR samples were prepared for the USP7CD (208-554) and the USP7CD (208-554)-CTP (1084-1102). All samples contained 137 mM NaCl, 10 mM Na 2 HPO, 27 mM KCl, and 1.8 mM KH 2 PO 4 adjusted to a pH of 7.2 and contained 7% (w/w) 2 H 2 O and 0.5 mM NaN 3 . Ubiquitin titration experiments were done by addition of purified bovine ubiquitin (from erythrocytes; Sigma) for a stock solution of 20 mM in the same buffer. All proton chemical shifts were referenced to internal DSS (50 mM) and 13 C/ 15 N referenced indirectly using the 1 H chemical shift of the methyl group in DSS and multiplying this value by 0.25144953 for 13 C and 0.101329118 for 15 N (Wishart et al., 1995).

ACCESSION NUMBERS

The peak assignments are accessible in the BMRB (http://www.bmrb.wisc. edu) using the accession code BMRB: 26766. USP7 is a genetically validated deubiquitinase for MDM2, a ubi quitin ligase that promotes degradation of the tumour suppressor p53. USP7 depletion enhances ubiquitination of USP7 substrates, including MDM2. Subsequent MDM2 proteasomal degradation stabilizes p53, thus promoting cell cycle arrest and apoptosis 3,6,7 . USP7 inhibition enhances ubiquitination of additional substrates that also regulate cellular physiology (Fig. 1a) 3,7-11 . To identify USP7 inhibitors, we esta blished an activitybased highthroughput screening cascade (Extended Data Fig. 1a). In parallel we developed a nuclear magnetic resonance (NMR) screen to identify USP7 catalytic domainbinding fragments (Extended Data Fig. 1b). A hittolead selection cascade was established to guide optimization of highthroughput screening and NMR hits into more potent USP7 inhibitors. Selectivity assays and structural biology directed medicinal chemistry design at the apex of the cascade. Assays to measure total and ubiquitinatedMDM2 levels were implemented as proximal indicators of USP7 activity, followed by viability assays using inactive compounds from matched chemical series as negative controls (Fig. 1b).

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Progression of highthroughput screening and NMR screen hits through the hittolead selection cascade identified a series of fragments having favourable properties (Extended Data Figs 1c-e, 2a-e). Optimization of the parent fragment GNE2916 yielded GNE6640 and GNE6776 (Extended Data Fig. 3a). These USP7 inhibitors enhanced endogenous MDM2 ubiquitination while achieving selectivity over USP47 (which has high USP7homology) and USP5, a highly active deubiquitinase 12 . Both compounds accelerated endogenous MDM2 turnover with cycloheximide, a necessary co treatment as MDM2 is a p53 target gene (Fig. 1a,c and Extended Data Fig. 3b). Accordingly, both GNE6640 and GNE6776, but not controls GNE2118 and GNE6641, promoted endogenous MDM2 ubiquitination with Lys48 (K48)linked polyubiquitin chains, which direct proteasomal degradation 13 (Fig. 1d and Extended Data Fig. 3c). Active USP7 inhibitors stabilized p53 and upregulated p21 in wildtype cells, but not in p53null or USP7null cells (Fig. 1e and Extended Data Fig. 3b). These data were corroborated by significantly decreased viability of wildtype, but not USP7 null cells, treated with active inhibitors (Extended Data Fig. 3d,e). Thus GNE6640 and GNE6776 target cellular USP7, MDM2, and p53 signalling pathways.

Previously reported deubiquitinase inhibitors are neither potent nor selective when evaluated in a massspectrometrybased activity assay 4 . In this assay, GNE6640 and GNE6776 selectively inhibited recombi nant USP7 relative to 36 other deubiquitinases (Fig. 2a and Extended Data Fig. 4a). GNE6776 remained selective even at 100 μ M, a more than sixfold higher concentration than used in cellular assays (Fig. 2a and Extended Data Fig. 4b). Because deubiquitinases are regulated by numerous cellular mechanisms 14 , we developed an activitybased probe assay to evaluate effects on endogenous deubiquitinases (J.H. et al., manuscript in preparation). GNE6640 or GNE6776 signifi cantly inhibited USP7 while remaining selective against 44-47 other detected deubiquitinases (Fig. 2b and Extended Data Fig. 4c,d). Given the favourable features of these inhibitors, we investigated their efficacy in animal models. Pharmacodynamic and pharmacokinetic studies indicated that GNE6776 is orally bioavailable and promotes ontarget pathway modulation in human xenografts (Extended Data Fig. 4e-i). Although efficacious exposure was only transiently achieved, GNE6776 caused modest, although significant, EOL1 xenograft growth delay (Extended Data Fig. 4j). Developing USP7 inhibitors that have improved druglike properties will be necessary to comprehen sively evaluate USP7 inhibition in vivo.

USP7 and MDM2 regulate proteins other than p53 (refs 3, 7-11, 15) (Fig. 1a). Having confirmed the selectivity and cellular activity of GNE 6640 and GNE6776, we prescreened 441 cell lines in 3day viability assays to enable an unbiased analysis of cellular factors that regulate USP7 inhibitor sensitivity (Extended Data Fig. 5a). GNE6641 was inactive in all cell lines whereas GNE6640 decreased viability of 108 cell lines with halfmaximal inhibitory concentration (IC 50 ) ≤ 10 μ M (data not shown). Next, we selected a subset of 181 cell lines displaying a range of sensitivities for additional analysis. GNE6641 remained inactive in 5day viability assays, whereas GNE6640 and GNE6776 decreased viability of 54 and 6 cell lines, respectively, with IC 50 ≤ 10 μ M (Extended Data Fig. 5b and Supplementary Table 1). Viability data and exome sequencing analysis were integrated to identify features associated with compound sensitivity. Acute myeloid leukaemia (AML) cell lines had increased sensitivity to GNE6640 (Extended Data Fig. 5c,e). Loss offunctionmutant TP53 lines were, in general, less responsive than wildtype TP53 lines, but the trend was not significant (Extended Data Fig. 5d). Indeed, the TP53 R175 hotspot was associated with increased sensitivity, whereas the Y220 hotspot was associated with decreased sen sitivity (Extended Data Fig. 5c, e and Supplementary Table 1). Although both compounds decreased proliferation and activated caspases in TP53 wildtype cell lines, these effects were reduced, but not abolished, in TP53null cell lines (Extended Data Fig. 6a-c). Combining GNE6640 with doxorubicin or cisplatin, DNAdamaging agents that activate the p53 response 16 , enhanced USP7 inhibitor efficacy (Extended Data Fig. 6d,e). Thus the MDM2 or p53 pathway is a mediator of GNE6640 and GNE6776 cellular activity but is not the only determinant. These findings corroborate genetic studies that have crossed TP53 -/-and USP7 -/-mice, which minimally rescues USP7 -/-embryonic lethality 7 .

The enhanced efficacy of GNE6640 in combination with DNA damaging agents suggested a strategy to identify signalling pathways that intersect with USP7 inhibition. We therefore screened EOL1 AML cells with GNE6640 or GNE6776 in combination with 589 chemo therapeutic or targeted agents. Top hits included PI3K and PIM kinase inhibitors (Extended Data Fig. 6f, g and Supplementary Table 2). PIM kinases share substrates with PI3K pathway kinases including TSC1/2 and Bad (Extended Data Fig. 7a) 17 . Given the role of PIM2 in hae matological malignancies 17 , we confirmed combination efficacy with Bliss analysis using PIM inhibitors GDC0339 (ref. 18) or GDC0570 (Fig. 2c and Extended Data Fig. 7b-d). Both inhibitors share struc tural similarities and are selectively potent against PIM kinases (X.W. et al., manuscripts in preparation). In mechanistic studies, GNE6776 promoted a modest elevation of p21 levels in AML cell lines whereas PIM2 levels were reduced. The GNE6776induced decrease in PIM2 levels was rescued by inhibition of the ubiquitinactivating enzyme UAE1 or the proteasome inhibitor bortezomib 13 . PIM2 levels were lower in USP7null cells, which was rescued by proteasome inhibition, collectively indicating that USP7 stabilizes PIM2 levels via a ubiquitin and proteasomedependent mechanism (Extended Data Fig. 7e,f). A treatment time course with GNE6776, GDC0570 or both compounds revealed that, as previously reported 17 , GDC0570 reduced phospho rylation of Bad and S6 and decreased expression of shortlived proteins p21 and Mcl1. GNE6776 decreased PIM2 levels and, remarkably, also reduced S6 and Bad phosphorylation and total Mcl1 levels, similar to PIM inhibition. In combination, GNE6776 and GDC0570 treat ments enhanced cleavage of poly(ADPribose) polymerase (PARP) and caspase3, mechanistically confirming the cell viability studies (Fig. 2d). We therefore hypothesized that PIM2 is a USP7 substrate. In support of this idea, endogenous USP7 and PIM2 coimmunoprecipitated and GNE6776 treatment enhanced PIM2 ubiquitination (Fig. 2e and Extended Data Fig. 7g). Recombinant USP7, but not the C223S 13 17 18 67 92 97 99 99 99 99 4 5 7 15 61 91 99 99 99 99 2 2 5 9 35 78 97 99 100100 2 2 5 8 25 69 95 99 100100 -4 -4 -1 2 18 66 94 99 100100 0 0 3 6 22 65 94 99 100100 -4 -4 -1 3 21 67 94 99 100100 -2 2 5 8 22 66 94 99 100100 [GDC-0570] (μM) 3 5 6 6 5 2 0 0 0 0 -2 16 34 38 31 14 3 0 0 0 2 2 2 49 60 27 5 0 -1 -1 4 -1 1 7 37 24 5 0 -1 -1 2 -3 0 2 12 12 3 0 0 0 3 -3 0 1 2 3 1 0 0 0 -3 -8 -6 -5 -5 0 1 0 0 0 1 -4 -2 -1 -1 -1 0 0 0 0 -3 -8 -5 -4 -2 1 1 0 0 0 0 -3 0 0 -1 -1 0 0 0 tool compounds inhibit USP7 was of interest. Enzymatic studies verified inhibition of intrinsic USP7 activity (turnover rate, k cat ) achieved by attenuating substrate binding (Michaelis constant, K m ) (Extended Data Fig. 8a). NMR analysis confirmed that USP7 inhibitors weaken USP7 catalytic domain interactions with native ubiquitin (Fig. 3a and Extended Data Fig. 8b,c). Analysis of cocrystal structures revealed that both compounds bind into a pocket approximately 12 Å from the catalytic triad that is located at the interface of the USP7 catalytic domain palm, fingers, and thumb subdomains (Fig. 3b and Extended Data Fig. 8d,e). The phenol rings of the phenolaminopyridine moieties are buried in the pocket and the hydroxyl groups are engaged in hydrogenbond interactions with USP7H403. The 2aminopyridine is directed out of the pocket towards solvent with the 2amino group engaging USP7D349 (Extended Data Fig. 8d). The substituents at the 5position of the aminopyridine-the pyridinecarboxamide (GNE6776) and indazole (GNE6640)-are largely solventexposed and lie between the α 5 and α 6 helices. The NH atoms of the GNE6776 carboxamide moiety engage USP7D305 on the α 5 helix, whereas the indazole of GNE6640 makes extensive van der Waals contacts in the crevice between α 5 and α 6 (Fig. 3c and Extended Data Fig. 8d). Comparison of the cocrystal structures of GNE6776 and GNE6640 with the USP7-ubiquitin complex (Protein Data Bank (PDB) accession number 1NBF) reveals that the compounds sterically inhibit ubiquitin binding and prevent transition of the USP7 catalytic domain α 5 helix to the active conformation 5 (Fig. 3c and Extended Data Fig. 8d). However, given the extensive interactions between USP7 catalytic domain and ubiquitin 5 , we investigated the importance of the USP7 residues that interact with the ubiquitinK48 side chain. The ubiquitin K48A mutant has reduced affinity for USP7 catalytic domain (Extended Data Fig. 9a); similarly, recombinant D305A/E308A USP7 catalytic domain failed to cleave tetraK48 or tetraK63linked polyubiquitin conju gated to a TAMRAlabelled peptide 19 . This apparent inactivity was due to a significant increase in K m (ref. 20) (Extended Data Fig. 9b,c). Expression of a D305A/E308A USP7fulllength mutant increased K48linked polyubiquitination on endogenous MDM2, comparable to expression of catalytically inactive USP7 (Extended Data Fig. 9d). Thus ubiquitinK48 and USP7D305/E308 residues are critical for USP7-ubiquitin binding and substrate deubiquitination.

The GNE6640 and GNE6776 binding modes suggested that USP7 may preferentially associate with ubiquitin moieties that have free K48 side chains. Mechanistically, this could direct USP7 to dis tal subunits of K48linked polyubiquitin, resulting in sequential dis taltoproximal chain depolymerization (exocleavage). However, in nonK48linked chains, USP7 may bind and cleave proximally to any ubiquitin moieties having accessible K48 side chains (exo, endo, or ) 15 NThr or proximal (Prox.) 15 NLeulabelled diubiquitins (see also Extended Data Fig. 10b,c). Middle/bottom rows: two dimensional bandselective optimized flipangle short transient (SOFAST) spectra overlays and onedimensional traces of labelled diubiquitin. Left/middle: 15 NThr or 15 NLeu peaks from K48linked diubiquitin without (green) and with (orange) USP7. Right/middle: 15 NThr or 15 NLeu peaks from K63linked diubiquitin without (purple) and with (orange) USP7. Bottom row: 15 NThr or 15 basecleavage) (Extended Data Fig. 10a). To evaluate this idea, we engi neered K48 and K63linked diubiquitin with differentially labelled distal and proximalubiquitin subunits using spectroscopically dis tinct amino acids. To select ubiquitin residues for isotopic labelling, we evaluated chemical shift perturbations induced by USP7 binding (Extended Data Fig. 10b). The cluster of residues L8, T12, T14, and L15 broaden upon USP7 catalytic domain binding and are well resolved in the spectra; thus Leu residues were 15 Nlabelled in the proximal diubiquitin and Thr residues were 15 Nlabelled in the distal subunit (Fig. 4a and Extended Data Fig. 10b,c). NMR analysis showed preferen tial USP7 catalytic domain binding to the distal subunit of K48linked diubiquitin, whereas USP7 catalytic domain binding was similar to both proximal and distal K63linked di ubiquitin subunits (Fig. 4a and Extended Data Fig. 10c). Importantly, both D305A USP7 and D305A/ E308A USP7 catalytic domain mutants demonstrated similar binding to both proximal and distal K48linked diubiquitin subunits (Fig. 4a).

On the basis of these data, we evaluated whether differential di ubiquitin binding affects the dynamics of ubiquitin chain depolyme rization by USP7. A previous study reported that USP7 cleaves K6, K11, K33, K48, and K63linked diubiquitins with similar k cat and K m values, indicating that different isopeptide bond conformations do not substantially impact cleavage efficiency 20 . Remarkably, our studies reveal that USP7 sequentially depolymerizes tetraK48linked ubiqui tin from the distal residue (exocleavage) whereas tetraK63ubiquitin chains are cleaved via a combination of exo, endo, or basecleavage, as nearly complete depolymerization occurs within 4 min (Fig. 4b and Extended Data Fig. 10d,e). Thus USP7 preferentially binds ubiquitin moieties that have free K48 side chains, resulting in protracted and sequential depolymerization of K48linked polyubiquitin.

Herein we describe GNE6640 and GNE6776, selective USP7 inhibitors that possess a structurally defined mechanism of inhibi tion. Establishing stringent screening cascades was critical for selecting and optimizing ontarget inhibitors. Combination studies revealed a previously undescribed intersection between USP7 deubiquitinase activity and PIM kinases in regulating cell viability. Cocrystal struc tures of GNE6640 or GNE6776 pointed to the importance of the complementary charged interactions between USP7D305/E308 and ubiquitinK48 side chains, which we confirmed using mutational analysis. Notably, D305G has been identified as a somatic lossof function mutant in patients with acute lymphoblastic leukaemia 21 . NMR analysis of USP7 binding to native monoubiquitin and differ entially labelled diubiquitins revealed that USP7 preferentially inter acts with ubiquitin moieties having free K48 side chains. It has been proposed that the inefficiency of some deubiquitinases to depolymerize longer substrateconjugated K48linked chains enables a threshold for proteasometargeting polyubiquitination 22 ; our studies substantiate this idea and provide a biophysical mechanism. Numerous proteins, includ ing other deubiquitinases, ubiquitin ligases, DNA repair and endocyto sis machinery, and epigenetic regulators are functionally dependent on ubiquitin binding 23 . Developing selective inhibitors that attenuate ubiq uitin binding is an effective strategy for USP7 inhibition. Our studies demonstrate the feasibility of this approach, which may have broader applications for inhibiting other classes of ubiquitinbinding proteins.

Online Content Methods, along with any additional Extended Data display items and Source Data, are available in the online version of the paper; references unique to these sections appear only in the online paper.

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MethOdS

High-throughput screen and counterscreen assays. USP7 UbA10 time-resolved fluorescence energy transfer (TR-FRET) activity assay. Potential inhibitors of USP7 were identified in a TRFRETbased enzyme activity assay with UbA10 as substrate. UbA10 is a fragment of the naturally occurring ubiquitin precursor Ub52; it retains the tenaminoacid segment of Ub52 that extends beyond the ubiquitin C terminus. The primary screening assay is a novel TRFRETbased activity assay that measures cleavage by fulllength USP7 of a doubly tagged peptide substrate. The peptide is tagged with glutathione Stransferase (GST) on the N terminus and with eight histidine residues on the C terminus (GST-UbA10His). The tags are detected by antiGSHd2 (TRFRET acceptor) and antiHis europium (TRFRET donor), respectively. Cleavage of this substrate by USP7 at the ubiquitin C terminus results in separation of these two tags and loss of TRFRET signal. Compounds were dispensed into 1,536well black plates (MaKO, Aurora Microplates) followed by 2 μ l fulllength recombinant USP7 in assay buffer (50 mM HEPES pH 7.5, 0.1% Prionex (Pentapharm), 0.01% Triton X100, and 10 mM dithiothreitol (DTT)). After a 10min incubation, the reaction was started by the addition of 2 μ l GST-UbA10His substrate in assay buffer. After 75 min of reaction, 2 μ l of a detection antibody reagent, containing antiHiseuropium (Life Technologies) and antiGSTd2 (CISbio) in assay buffer were added. After 60 min, the fluorescence at 618 nm and 671 nm with excitation at 340 nm was read on a ViewLux reader (PerkinElmer). Cleavage of the doubly tagged substrate resulted in loss of TRFRET signal, whereas inhibition of USP7 by compound restored the signal. The TRFRET ratio was calculated as fluorescence at 671 nm divided by fluorescence at 618 nm. TRFRET ratios were normalized to controls to determine the percentage inhibition for the single concentration screen. For confirmation in concentration-response mode (ten points with n = 2), percentage inhibition was plotted against compound concentration, and the data were fitted to a four parameter curve with Screener Assay Analyzer (Genedata) to determine IC 50 values. The assay buffer was optimized to maintain enzyme stability and to maximize assay signal to background: Triton X100 was included to prevent nonspecific adsorption of the enzyme and/or substrate to the assay plate and/or to compound aggregates, Prionex carrier protein was included to help stabilize the enzyme and to prevent nonspecific adsorption to container and tubing surfaces as well as to minimize nonspecific inhibition by library compounds, and DTT served to maintain good USP7 activity and minimize the impact of inhibitors that act through redox cycling. Reagent concentrations were optimized for good assay performance at approximately 50% substrate conversion in the signal decrease assay with a key aim of minimizing the required concentration of USP7. The GST-UbA10His concentration was adjusted to maximize assay signal, antiGSTd2 concentration was adjusted approximately in parallel with that of the substrate, and antiHiseuropium was used at a concentration that represented a minimum that was compatible with robust detection on the ViewLux plate reader. Timecourse evaluations were conducted to confirm that the enzyme concentration (10 nM) and reaction time (75 min) were in the linear range of enzyme activity. Additionally, extended time courses for the detection reaction were used to demonstrate that the 60min incubation was sufficient to reach equilibrium. Under the final assay conditions, reagent stability studies indicated greater than 20 h of acceptable performance. Over the course of the screen, Z′ values averaged 0.76. USP7 di-ubiquitin FRET activity assay. Potential USP7 inhibitors were confirmed in an orthogonal activity assay with an internally quenched K63linked diubiquitin substrate (U310, Boston Biochem). Conditions were similar to those used for the UbA10 TRFRET activity assay. Compounds dispensed into 1,536well plates were preincubated with fulllength recombinant USP7 in assay buffer for 10 min, and the reaction was started by the addition of 2 μ l of the diubiquitin substrate. During the 60min incubation, cleavage of the substrate by USP7 resulted in the release of quenching of the TAMRA tag by the QXl tag and thus an increase in fluorescence. The fluorescence intensity was read with excitation at 540 nm and emission at 585 nm. Concentration-response assay methods and data analyses were conducted as for the UbA10 TRFRET assay. USP7 Ubiquitin/Rho110 activity assay. Potential USP7 inhibitors were also confirmed in an orthogonal activity assay with ubiquitin/rhodamine110 as substrate. Compounds were preincubated for 10 min with 2 μ l fulllength recom binant USP7 in assay buffer, and the reaction was started by the addition of 2 μ l of ubiquitin/rhodamine110 (U555, Boston Biochem). After a 60min reaction in which USP7 cleaved the rhodamine110 from the ubiquitin and thus increases fluorescence, the fluorescence intensity was read with excitation at 485 nm and emission at 535 nm. General assay conditions and data analyses were as described above. USP7 di-ubiquitin cleavage assay with mass spectrometric detection. To further validate compounds that met the initial highthroughput screen confirma tion criteria, the ability of compounds to inhibit diubiquitin cleavage was assessed by monitoring the conversion to ubiquitin by mass spectrometry. Compounds were dispensed into 384well polypropylene plates (Greiner BioOne), and 10 μ l USP7 were added and allowed to incubate for 10 min. The reaction was started by the addition of 10 μ l of K48linked diubiquitin substrate (UC200, Boston Biochem) and allowed to progress for 70 min; then it was stopped by the addition of 20 μ l 2% formic acid. The assay plates were stored frozen at -80 °C until analysis by mass spectrometry at Agilent Technologies. Before quantification, the enzyme reaction was passed over a RapidFire cartridge to remove buffer components. Both di ubiquitin substrate consumption and ubiquitin product formation were monitored by mass spectrometry using multiple reaction monitoring (MRM) in positive ion mode with parent ion/daughter ion transitions of 1142.1/260.1 and 770.8/817.7, respectively. The percentage conversion of substrate was plotted as a function of compound concentration to generate the IC 50 values as indicated above. NMR screen and binding studies. All NMR spectra were recorded on Bruker Avance 600 MHz and 800 MHz spectrometers operating at 14.1 and 18.8 T using tripleresonance cryogenic probes optimized for proton detection. All two dimensional spectra were acquired with a spectral width of 16 p.p.m. and 2,048 (TROSY) or 954 (SOFAST) data points in the direct proton dimension and 28 p.p.m. and 192 sample points in the 15 N dimension with echo/antiecho (TROSY) or StatesTPPI (SOFAST) type selection. The resulting free induction decay resolution was 12.52 and 19.05 Hz per data point for the TROSY and 20.08 and 17.10 Hz per data point for the SOFAST spectra, respectively. All spectra were recorded at 300 K. The pH was adjusted to 7.2 without correction owing to isotope shifts. For data processing, NMRPipe/NMRDraw and the BRUKER software package TOPSPIN 3.2 were used. All data evaluation was done in NMR view and CCPN. Visualization and presentation of the threedimensional tertiary USP7 as well as related protein structures from the RSCB Protein Database were done in PyMOL (PyMOL Molecular Graphics System,version 1.7.4,Schrödinger). All samples contained 137 mM NaCl, 10 mM Na 2 HPO 4 , 27 mM KCl, and 1.8 mM KH 2 PO 4 adjusted to a pH of 7.2 and contained 7% (w/w) 2 H 2 O and 0.5 μ M NaN 3 . Ubiquitin titration experiments were done by addition of purified bovine ubiquitin (from erythro cytes, Sigma) from a stock solution of 20 mM in the same buffer. All proton chemical shifts were referenced to internal DSS (50 μ M) and 15 N referenced indirectly using the 1 H chemical shift of the methyl group in DSS by multiplication with a factor of 0.101329118. NMR screening conditions. The primary fragment screen was performed on 4,871 fragments in mixtures of 5. The individual compound concentration was 500 μ M and the concentration of unlabelled USP7 was 7 μ M. Binders were identified by the presence of signals stemming from the individual ligand in the proton satura tion transfer difference spectra recorded at 284 K. The criterion used to identify a binder was a signaltonoise ratio of above 5. All primary binders were re measured as a single compound to confirm binding. Confirmed binders were defined as compounds with a saturation transfer difference signaltonoise ratio of above 10. All confirmed binders were tested again at 2-2.5 mM, by proteinobserved 1 H/ 15 N TROSY experiments in the presence of 15 Nlabelled USP7 at 220-340 μ M. NMR binding studies. The concentrationdependent NMR shift perturbations caused by the interaction of unlabelled ubiquitin with 15 Nlabelled USP7 in the absence and presence of USP7 inhibitors were fitted to the function for a twostate fast exchanging equilibrium based on concentrationdependent chemical shift perturbations:

δ δ Δ = Δ + + - + + - K K [P] [L] ( [P] [L]) 4[P][L] 2[P] max d d 2
where Δ δ is the chemical shift change at various protein/ligand ratios, Δ δ max is the chemical shift change at saturation, K d is the dissociation constant, and [L] and [P] are the ligand and protein concentrations, respectively. The chemical shift change is the root mean square of the

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Compound synthesis. Synthesis of GNE-6640 is representative of the syntheses for GNE-2118, GNE-6776, GNE-6641, GNE-2931, GNE-2917, and GNE-2916 and GNE-9603.

3,5Dibromo4ethylpyridin2amine: into a 500ml threenecked round bottom flask purged and maintained with an inert atmosphere of nitrogen were placed 4ethylpyridin2amine (10 g, 81.85 mM), tetrahydrofuran (200 ml), and NBS (29 g, 162.94 mmol) at 0 °C. The resulting solution was stirred at room temperature for 15 min and then concentrated under vacuum. The residue was purified on a silica gel column eluting with DCM/MeOH (100:1 to 20:1), to afford 18 g (79%) of the title compound. 1 H NMR (400 MHz, CDCl 3 ) δ 8.04 (s, 1H), 7.33-7.23 (m, 0H), 4.93 (s, 2H), 2.93 (q, J = 7.5 Hz, 2H), 1.60 (s, 0H), 1.17 (t, J = 7.5 Hz, 3H). Liquid chromatography-mass spectrometry (LC-MS) (electro spray ionization (ESI) m/z): 264.1 (M + H + ).

3Bromo4ethylpyridin2amine: into a 500ml threenecked roundbottom flask purged and maintained with an inert atmosphere of nitrogen was placed 3,5dibromo4ethylpyridin2amine (18 g, 64.29 mmol) in tetrahydrofuran (300 ml). To this was added a solution of nbutyllithium (in hexane) (58 ml, 2.2 mol l -1 ) at -78 °C. The resulting solution was stirred at -78 °C for 1 h, quenched by the addition of 450 ml NH 4 Cl and then extracted with ethyl acetate (2 × 500 ml). The combined organic layers were washed with brine (2× 500 ml), dried over anhydrous sodium sulfate, and concentrated under vacuum. The crude product was purified by flash and preparative HPLC to afford 12 g (93%) of the title compound as a white solid. 1 H NMR (400 MHz, CDCl 3 ) δ 7.89 (d, J = 5.1 Hz, 1H), 6.60-6.48 (m, 1H), 4.93 (s, 2H), 2.69 (q, J = 7.6 Hz, 2H), 1.33-1.14 (m, 5H). LC-MS (ESI m/z): 264.1 (M + H + ). 4Ethyl3(4methoxyphenyl)pyridn2amine: into a 500ml threenecked roundbottom flask purged and maintained with an inert atmosphere of nitrogen was placed a solution of 3bromo4ethylpyridin2amine (12 g, 59.68 mmol) in CH 3 CN (100 ml), (4methoxyphenyl)boronic acid (11 g, 72.39 mmol), Na 2 CO 3 (120 ml, sat.), and Pd(dppf)Cl 2 (1.2 g, 1.64 mmol). The resulting solution was stirred at 110 °C for 1 h, diluted with of 500 ml of ethyl acetate, and then extracted with ethyl acetate (2 × 500 ml). The combined organic layers were washed with brine (3 × 200 ml), dried over anhydrous sodium sulfate, and concentrated under vacuum. The residue was purified on a silica gel column eluting with ethyl acetate/petroleum ether (1:100 to 1:10) to afford 10 g (73%) of the title compound. 5Bromo4ethyl3(4methoxyphenyl)pyridin2amine: into a 250ml three necked roundbottom flask purged and maintained with an inert atmosphere of nitrogen were placed 4ethyl3(4methoxyphenyl)pyridn2amine (10 g, 43.80 mmol), tetrahydrofuran (THF) (100 ml), followed by NBS (7.8 g, 43.83 mmol) at 0 °C. The resulting solution was stirred at room temperature for 15 min, diluted with 500 ml EtOAc and 500 ml H 2 O. The resulting solution was extracted with ethyl acetate (2 × 500 ml). The organic layers were combined, washed with brine (2 × 500 ml), and concentrated under vacuum. The residue was purified on a silica gel column eluting with ethyl acetate/petroleum ether (1:20 to 1:10) to afford 8 g (59%) of the title compound. 1 H NMR (400 MHz, CDCl 3 ) δ 8.09 (s, 1H), 7.22-7.09 (m, 2H), 7.09-6.98 (m, 2H), 4.55 (s, 2H), 2.47 (q, J = 7.5 Hz, 2H), 1.10-0.84 (m, 3H). LC-MS (ESI m/z): 264.1 (M + H + ).

4(2Amino5bromo4ethylpyridin3yl)phenol: into a 250ml threenecked roundbottom flask purged and maintained with an inert atmosphere of nitrogen was placed 5bromo4ethyl3(4methoxyphenyl)pyridin2amine as a white solid (8 g, 26.04 mmol), dichloromethane (100 ml), followed by tribromoborane (19.6 g, 78.24 mmol) at 0 °C. The resulting solution was stirred at room temperature for 1 h and then quenched by the addition of 100 ml of NaHCO 3 (1 M) at 0 °C. The solids were collected by filtration and then washed with 100 ml H 2 O and 300 ml of ethyl acetate/petroleum ether (1:1) to afford 6.3 g (83%) of the title compound as a white solid. 1 H NMR (400 MHz, CDCl 3 ) δ 8.09 (s, 1H), 7.22-7.09 (m, 2H), 7.09-6.98 (m, 2H), 4.55 (s, 2H), 2.47 (q, J = 7.5 Hz, 2H), 1.10-0.84 (m, 3H). LC-MS (ESI m/z): 264.1 (M + H + ). 4[2Amino4ethyl5(1Hindazol5yl)3pyridyl]phenol (GNE6640): into a 250ml threenecked roundbottom flask purged and maintained with an inert atmosphere of nitrogen were placed 4(2amino5bromo4ethylpyridin3yl) phenol (1.0 g, 3.41 mmol,), 6(tetramethyl1,3,2dioxaborolan2yl)2Hindazole (880 mg, 3.41 mmol,), potassium carbonate (3.3 g, 23.88 mmol), water (30 ml), 1,4dioxane (25 ml), and Pd(dppf)Cl 2 (200 mg, 0.3 mmol). The resulting solu tion was stirred at 80 °C for 16 h, and diluted with 500 ml H 2 O and 500 ml ethyl acetate. The organic layer was washed twice with brine (2 × 250 ml) and concen trated under vacuum. The residue was purified on a silica gel column eluting with DCM/CH 3 OH (20:1-10:1) to afford the title compound. 1 H NMR (400 MHz, DMSOd 6 ) δ 13.07 (s, 1H), 9.52 (s, 1H), 8.07 (d, J = 1.0 Hz, 1H), 7.74 (s, 1H), 7.65 (dd, J = 1.6, 0.8 Hz, 1H), 7.56 (dt, J = 8.6, 0.9 Hz, 1H), 7.28 (dd, J = 8.5, 1.6 Hz, 1H), 7.10-7.04 (m, 2H), 6.92-6.86 (m, 2H), 4.94 (s, 2H), 2.26 (q, J = 7.4 Hz, 2H), 0.60 (t, J = 7.4 Hz, 3H). 13 C NMR (150 MHz, DMSOd 6 ) δ 15. 0, 24.0, 111.3, 117.4, 122.2, 122.3, 124.0, 127.8, 129.3, 130.2, 132.2, 135.3, 140.2, 147.2, 151.8, 157.2, 157.4 5[6Amino4ethyl5(4hydroxyphenyl)3pyridyl]Nmethylpyridine2 carboxamide (GNE6776): 1 H NMR (400 MHz, DMSOd 6 ) δ 9.56 (s, 1H), 8.80 (q, J = 4.7 Hz, 1H), 8.59 (dd, J = 2.3, 0.9 Hz, 1H), 8.07 (dd, J = 8.0, 0.9 Hz, 1H), 7.96 (dd, J = 8.0, 2.2 Hz, 1H), 7.79 (s, 1H), 7.11-7.02 (m, 2H), 6.94-6.86 (m, 2H), 5.19 (s, 2H), 2.84 (d, J = 4.8 Hz, 3H), 2.26 (q, J = 7.4 Hz, 2H), 0.62 (t, J = 7.4 Hz, 3H).

13

C NMR (150 MHz, DMSOd 6 ) δ 29. 1, 38.8, 41.4, 131.6, 136.6, 137.1, 138.8, 141.5, 146.4, 152.5, 154.1, 161.1, 163.1, 164.2, 171.3 [START_REF] Nicholson | The multifaceted roles of USP7: new therapeutic opportunities[END_REF].40 (d, J = 27.5 Hz, 4H), 2.29 (q, J = 7.4 Hz, 2H), 0.68 (t, J = 7.5 Hz, 3H). 13 C NMR (150 MHz, DMSOd 6 ) δ 14. 1, 26.6, 57.7, 73.4, 115.0, 119.5, 127.0, 128.3, 129.4, 129.7, 130.5, 135.7, 137.5, 147.0, 148.4, 4[2Amino4ethyl5(2methylindazol6yl)3pyridyl]phenol (GNE6641): 1 HNMR (400 MHz, DMSOd 6 ) δ 9.54 (s, 1H), 8.34 (s, 1H), 7.76 (s, 1H), 7.71 (d, J = 9 Hz, 1H), 7.45 (d, J = 0.9 Hz, 1H), 7.09 (d, J = 9 Hz, 2H), 6.99 (d, J = 1.2 Hz, 1H), 6.96 (d, J = 1.2 Hz, 2H) 4.99 (s, 2H), 4.17 (s, 3H), 2.51 (q, J = 1.8 Hz, 2H), 0.60 (t, J = 7.5 Hz, 3H). 13 C NMR (150 MHz, DMSOd 6 ) δ 14.3, 22.8, 116.2, 116.7, 120.0, 120.2, 120.6, 124.0, 124.7, 126.7, 127.8, 131.0, 136.3, 146.7, 148.9, 156.8, 157 4[2Amino4ethyl5(2methoxyphenyl)3pyridyl]phenol (GNE2931): 1 H NMR (400 MHz, DMSOd 6 ) δ 9.54 (s, 1H), 7.58 (s, 1H), 7.34 (ddd, J = 8.2, 7.4, and 1.8 Hz, 1H), 7.17-6.94 (m, 5H), 6.88 (d, J = 7.7 Hz, 2H), 4.89 (s, 2H), 3.71 (s, 3H), 2.09 (q, J = 7.2 Hz, 2H), 0.55 (t, J = 7.5 Hz, 3H). 13 C NMR (150 MHz, DMSOd 6 ) δ 13.8, 23.1, 55.2, 111.0, 116.0, 119.6, 120.2, 123.6, 126.7, 127.6, 128.8, 130.8, 131.6, 146.7, 149.5. 156.7, 156.8, 156.9. HRMS m/z 321.1598 (M + H + , C 20 H 21 O 2 N 2 requires 321.1650).

3[6Amino4ethyl5(4hydroxyphenyl)3pyridyl]benzamide (GNE2917): 1 H NMR (400 MHz, DMSOd 6 ) δ 9.52 (s, 1H), 7.99 (s, 1H), 7.84 (dtd, J = 7.1, 3.6, 1.8 Hz, 2H), 7.73 (s, 1H), 7.54-7.43 (m, 2H), 7.35 (s, 1H), 7.10-6.94 (m, 2H), 6.93-6.74 (m, 2H), 5.02 (s, 2H), 2.23 (q, J = 4.4 Hz, 2H), 0.61 (t, J = 4.4 Hz, 3H). 13 C NMR (150 MHz, DMSOd 6 ) δ 14. 1, 22.6, 115.9, 116.0, 119.8, 125.9, 126.4, 126.5, 128.2, 128.5, 130.7, 130.8, 132.3, 133.2, 134.2, 139.1, 146.7, 148.4, 156.7, 157.2 ), 2.33 (q, J = 7.4 Hz, 2H), 0.61 (t, J = 7.5 Hz, 3H). 13 C NMR (150 MHz, DMSOd 6 ) proton correlated carbon chemical shifts δ 14.9, 42.6, 113.9, 116.7, 116.7, 129.0, 131.3, 131.4, (15.0, 25.9, 29.0. impurities) 7.43-7.31 (m, 3H), 7.14-6.95 (m, 2H), 6.93-6.74 (m, 2H), 5.04 (s, 2H), 2.33 (q, J = 7.4 Hz, 2H), 2.06 (s, 2H), 0.61 (t, J = 7.5 Hz, 3H). 13 C NMR (150 MHz, DMSOd 6 ) δ 13.7, 23.9, 24.0, 120.0, 121.2, 125.6, 127.4, 129.2, 129.7, 130.2, 134.7, 136.9, 140.1, 140.3, 157.5, 168.8 Synthesis of 9chloroN(5chloro2,4dimethoxyphenyl)N(cyanomethyl) 5,6,7,8tetrahydroacridine3carboxamide (GNE6831): 9Chloro5,6,7,8 tetrahydroacridine3carboxylic acid (100 mg, 0.38 mmol) was dissolved in 2 ml of N,Ndimethylformamide (DMF) and charged with 1[bis(dimethylamino)methy lene]1H1,2,3triazolo [4,5b]pyridinium 3oxide hexafluorophosphate (HATU) (144 mg, 0.38 mmol). After stirring at room temperature for 30 min, the mixture was then charged with 3((5chloro2,4dimethoxyphenyl)amino)propanenitrile (91 mg, 0.38 mmol). The resulting solution was stirred at 110 °C for 1 h, diluted with of 50 ml of ethyl acetate, and then extracted with ethyl acetate (2 × 100 ml). The combined organic layers were washed with brine (3 × 200 ml), dried over anhydrous sodium sulfate, and concentrated under vacuum. The residue was puri fied on a silica gel column eluting with ethyl acetate/petroleum ether (1:100 to 1:10) to afford the title compound (110 mg, 0.30 mmol, 78% yield) of the title compound. Letter reSeArCH 27.0, 33.5, 37.4, 56.4, 56.4, 98.1, 111.7, 116.3, 122.7, 123.0, 125.1, 125.8, 127.8, 129.7, 130.3, 135.6, 139.8, 144.8, 154.5, 155.3, 160.6, 170.0 13 C NMR (150 MHz, DMSOd 6 ) δ 21.8, 21.8, 22.2, 27.0, 33.5, 40.2, 54.6, 56.1, 56.3, 80.0, 80.2, 97.8, 111.4, 122.7, 124.3, 124.4, 125.8, 126.7, 129.6, 129.8, 137.7, 139.8, 145.0, 154.2, 154.7, 160.3, 170.2 13 C NMR (150 MHz, DMSOd 6 ) proton correlated carbon chemical shifts δ: 22.4, 22.4, 27.5, 34.1, 56.6, 56.7, 56.8, 64.7, 98.1, 98.3, 123.2, 126.4, 127.4, 129.6, 129.8 13 C NMR (150 MHz, DMSOd 6 ) δ 22.0, 27.0, 33.5, 49.7, 52.6, 57.9, 58.4, 123.5, 125.5, 125.9, 126.5, 137.9, 139.9, 145.5, 160.4, 170.0 13 C NMR (150 MHz, DMSOd 6 ) δ: 21.9, 27.0, 33.6, 34.5, 44.5, 48.5, 52.3, 66.0, 123.9, 125.4, 126.0, 126.5, 129.8, 137.0, 140.0, 145.4, 160.6, 166,0 169.7. HRMS m/z 402.1579 (M + H + , C 21 H 25 O 3 N 3 Cl requires 402.1725). Synthesis of GNE-0300 is representative of the syntheses for GNE-8735.

Synthesis of (1S)1(5bromo1Hindol2yl)N(2phenylethyl)ethanamine (GNE0300): 1(5bromo1Hindol2yl)ethan1one (90 mg, 0.38 mmol) was dis solved in 2 ml of DMF and charged with (S)phenethylamine (46 mg, 0.38 mmol). After stirring at room temperature for 30 min, the mixture was diluted with 50 ml ethyl acetate and 50 ml water. The mixture was partitioned and the organic was collected. The aqueous layer was then extracted with ethyl acetate (2 × 100 ml). The combined organic layers were washed with brine (3 × 200 ml), dried over anhydrous sodium sulfate and concentrated under vacuum. The residue was puri fied on a silica gel column eluting with ethyl acetate/petroleum ether (1:100 to 1:10) to afford the title compound (120 mg, 0.35 mmol, 92% yield). 1 H NMR (400 MHz, DMSOd 6 ) δ 11.05 (s, 1H), 7.59 (d, J = 1.9 Hz, 1H), 7. 30-7.19 (m, 3H), 7.20-7.06 (m, 6H), 6.21 (d, J = 1.8 Hz, 1H), 3.93 (q, J = 6.6 Hz, 1H), 2.77-2.56 (m, 4H), 1.36 (d, J = 6.6 Hz, 3H). 13 C NMR (150 MHz, DMSOd 6 ) δ 22.5, 36.5, 49.3, 51.6, 98.0, 111.5, 113.4, 122.0, 123.1, 126.3, 128.7, 129.0, 130.3, 135.1, 140.8, 146 25.7, 28.3, 32.6, 36.0, 49.3, 55.6, 110.7, 112.8, 119.7, 122.1, 125.8, 128.2, 128.6, 130.3, 132.5, 140.5 Synthesis of GDC-0570 followed similar procedures as described for GDC-0339 as below.

(Z)2,3,6,7tetrahydro1Hazepine hydrochloride: 4 N hydrogen chloride in 1,4dioxane (250 ml; 1 mol) was added over 5 min to a stirred, icecooled solution of (Z)tertbutyl 2,3,6,7tetrahydro1Hazepine1carboxylate (50 g; 0.254 mol) in methanol (250 ml). On complete addition, the ice bath was removed and stirring continued at room temperature for 3.75 h. Volatiles were removed under reduced pressure and the residue triturated twice with diethyl ether (300 ml) to afford (Z)2,3,6,7tetrahydro1Hazepine hydrochloride as a pale pink solid (32.3 g; 95%). 1 HNMR (DMSOd 6 , 400 MHz) δ 9.54 (br s, 2H), 6.40-6.25 (m, 2H), 3.1-3.05 (m, 4H), 2.55-2.40 (m, 4H).

(Z)1(1methyl4nitro1Hpyrazol5yl)2,3,6,7tetrahydro1Hazepine: a mixture of (Z)2,3,6,7tetrahydro1Hazepine hydrochloride (32.3 g; 0.24 mol), 5chloro1methyl4nitro1Hpyrazole (37.2 g; 0.23 mol), potassium fluoride (56.24 g; 0.96 mol), and diisopropylethylamine (64 ml; 0.362 mol) in anhydrous DMSO (650 ml) was heated at 75 °C for 21 h. On cooling, the mixture was poured into water (1,500 ml), extracted with ethyl acetate (4 × 500 ml), and the combined organics washed with water (2 × 400 ml) and brine (300 ml) then dried (MgSO 4 ). The solvent was removed under reduced pressure to afford (Z)1(1methyl4 nitro1Hpyrazol5yl)2,3,6,7tetrahydro1Hazepine as a light brown solid (50.74 g; 99%). 1 HNMR (400 MHz, CDCl 3 ) δ 8.00 (s, 1H), 5.95-5.85 (m, 2H), 3.80 (s, 3H), 3.30-3.20 (m, 4H), 2.45-2.35 (m, 4H)

. LC-MS (ESI m/z): 223.1 [M + H + ].
4(1Methyl4nitro1Hpyrazol5yl)8oxa4azabicyclo [5.1.0]octane: seventy seven per cent metachloroperbenzoic acid (77 g; 0.343 mol) was added portionwise over 10 min to a stirred, icecooled solution of (Z)1(1methyl 4nitro1Hpyrazol5yl)2,3,6,7tetrahydro1Hazepine (50.74 g; 0.23 mol) in dichloromethane (1,000 ml). An ice bath was used to control the minor exotherm observed during a smallerscale reaction. On complete addition, the ice bath was removed and stirring continued at room temperature for 18 h. The reaction mixture was washed with saturated sodium hydrogen carbonate (750 ml), 1 N sodium hydroxide (2 × 500 ml), and brine (350 ml). The organics were dried (MgSO 4 ) and the solvent removed under reduced pressure to afford 4(1methyl 4nitro1Hpyrazol5yl)8oxa4azabicyclo [5.1.0] Rel(4R,5R)5azido1(1methyl4nitro1Hpyrazol5yl)azepan4ol: to a stirred solution of 4(1methyl4nitro1Hpyrazol5yl)8oxa4azabicyclo [5.1.0] octane (29.28 g) in methanol (350 ml) and water (90 ml) was added ammonium chloride (16.5 g; 0.308 mol) followed by sodium azide (20 g; 0.307 mol). The mixture was heated behind a blast screen at 70 °C for 22 h, cooled, then concen trated to 100 ml under reduced pressure at 40 °C. The concentrated solution was poured into water (1,300 ml), extracted with dichloromethane (4 × 400 ml), and the combined organics dried (MgSO 4 ). Evaporation under reduced pressure at 35 °C gave rel(4R,5R)5azido1(1methyl4nitro1Hpyrazol5yl)azepan4ol (antiisomer and a racemic mixture) as a pale yellow oil (34.5 g; 96% over two steps). Rel(4R,5R)4azido5fluoro1(1methyl4nitro1Hpyrazol5yl)azepane: fifty per cent deoxofluor in THF (111 ml; 0.307 mol) was added slowly over 20 min to a stirred, icecooled solution of rel(4R,5R)5azido1(1methyl4 nitro1H pyrazol5yl)azepan4ol (32.5 g; 0.115 mol) in dichloromethane (500 ml). On complete addition, the ice bath was removed and stirring continued at room temperature for 20 h. The reaction mixture was recooled in an ice bath and saturated sodium hydrogen carbonate (400 ml) added dropwise (note this reac tion is effervescent). After stirring for 30 min, the layers were separated and the aqueous layer extracted with dichloromethane (2 × 500 ml). Pooled organics were dried (MgSO 4 ) and the solvent removed under reduced pressure. Flash column chromatography on silica eluting with 0-100% ethyl acetate in isohexane gradient afforded rel(4R,5R)4azido5fluoro1(1methyl4nitro1Hpyrazol5yl)aze pane as a pale orange oil (26 g; 80%). 1 HNMR (400 MHz, CDCl 3 ) δ 8.03 (s, 1H), 4.90-4.65 (m, 1H), 4.00-3.85 (m, 1H), 3.77 (s, 3H), 3.40-3.10 (m, 4H), 2.35-2.05 (m, 3H), 1.95-1.75 (m, 1H). LC-MS (ESI m/z):

284.3 [M + H + ].
Rel(4R,5R)5fluoro1(1methyl4nitro1Hpyrazol5yl)azepan4amine: a mixture of rel(4R,5R)4azido5fluoro1(1methyl4nitro1Hpyrazol5yl) azepane (26 g; 91.8 mmol) and triphenylphosphine (24.1 g; 92 mmol) in tetrahy drofuran (400 ml) and water (80 ml) was heated at 60 °C for 20 h, cooled and con centrated to approximately 80 ml under reduced pressure. Ethyl acetate (500 ml) was added and the mixture extracted with 1 N HCl (4 × 125 ml). Pooled acidic extracts were washed with ethyl acetate (500 ml), basified to pH 14 with 6 N NaOH, and extracted with dichloromethane (3 × 400 ml). Combined extracts were dried (MgSO 4 ) and the solvent removed under reduced pressure to give rel(4R,5R)5 fluoro1(1methyl4nitro1Hpyrazol5yl)azepan4amine as a pale yellow oil (22 g; 93%). tertButyl (4R,5R)5fluoro1(1methyl4nitro1Hpyrazol5yl)azepan4 ylcarbamate: ditertbutyl dicarbonate (28 g; 128.3 mmol) was added to a stirred, icecooled solution of rel(4R,5R)5fluoro1(1methyl4nitro1Hpyrazol 5yl)azepan4amine (22 g; 85.6 mmol) and diisopropylethylamine (22.4 ml; 128.6 mmol) in dichloromethane (600 ml). On complete addition, the ice bath was removed and stirring continued at room temperature for 20 h. The reaction mixture was washed with saturated sodium hydrogen carbonate (500 ml) and the aqueous layer reextracted with dichloromethane (2 × 300 ml). Pooled organics were dried (MgSO 4 ) and the solvent removed under reduced pressure. Flash column chromatography on silica eluting with 0-100% ethyl acetate in isohexane gradient afforded a pale yellow solid (29.7 g; 97%). Chiral separation of the racemic Letter reSeArCH mixture by supercritical fluid chromatography using a Chiralpak IA column with an isocratic mobile phase of 15% methanol (with 0.1% NH 4 OH) in carbon dioxide gave the desired product (second peak) as a single enantiomer. 1 tertButyl (4R,5R)5fluoro1(4amino1methyl1Hpyrazol5yl) azepan 4ylcarbamate: to a stirred solution of tertbutyl (4R,5R)5fluoro1(1methyl 4nitro1Hpyrazol5yl)azepan4ylcarbamate (15.0 g; 42 mmol) in ethanol (1,000 ml) and water (100 ml) was added ammonium chloride (11.34 g; 210 mmol) and iron powder (9.4 g; 168 mmol). The mixture was heated at 99 °C for 2.75 h, cooled, filtered through Celite and evaporated to approximately 100 ml. The concentrate was diluted with water (1,000 ml) and extracted with ethyl acetate (2 × 500 ml). The pooled extracts were washed with water (200 ml), dried (MgSO 4 ) and the solvent removed under reduced pressure to give tertbutyl (4R,5R)5 fluoro1(4amino1methyl1Hpyrazol5yl)azepan4ylcarbamate as a pale brown solid (12.6 g; 91%). 1 HNMR (400 MHz, CDCl 3 ) δ 7.13 (s, 1H), 6.35-6.20 (m, 1H) (14.4 g, 40.4 mmol), diisopropyleth ylamine (13.4 ml, 77 mmol), and PyBOP (26.1 g, 50 mmol) in dichloromethane (400 ml) was stirred at room temperature for 48 h. Saturated sodium hydrogen carbonate (600 ml) was added and stirring continued for 30 min. The mixture was filtered, the layers separated and the aqueous layer extracted with dichlorometh ane (500 ml). The pooled organics were dried (MgSO 4 ) and the solvent removed under reduced pressure. Flash column chromatography on silica eluting with 0-100% ethyl acetate in isohexane gradient afforded tertbutyl (4R,5R)1 (4(5tertbutoxycarbonylamino2(2,6difluorophenyl)thiazole4carboxamido)1 methyl1Hpyrazol5yl)5fluoroazepan4ylcarbamate as a pale yellow solid (2.5 g, 9.8%). Further elution with 0-10% methanol in ethyl acetate, then 10% methanol in dichloromethane, evaporation of relevant fractions under reduced pressure, and trituration of the residue with cold diethyl ether gave a further 7.61 g (30%) of product. 5AminoN(5((4R,5R)4amino5fluoroazepan1yl)1methyl1Hpyrazol 4yl)2(2,6difluorophenyl)thiazole4carboxamide (GDC0339): tertbutyl (4R,5R)1(4(5tertbutoxycarbonylamino2(2,6difluorophenyl)thiazole4 carboxamido)1methyl1Hpyrazol5yl)5fluoroazepan4ylcarbamate (10.11 g, 15.2 mmol) in 4 N HCl in dioxane (200 ml) and methanol (200 ml) was stirred at room temperature for 20 h. Evaporation under reduced pressure afforded a pale brown solid which was dissolved in 50% methanol in dichloromethane and added to a strong cation exchange (SCX) chromatography cartridge. After washing with dichloromethane and methanol, elution with 1 N ammonia in methanol and evaporation of the eluent under reduced pressure afforded 5aminoN(5((4R,5R)4amino5fluoroazepan 1yl)1methyl1Hpyrazol4yl)2(2,6difluorophenyl)thiazole4carboxamide as the free base (6.3 g, 89% yield). Purity by a 30min LC-MS at ultraviolet 254 nm: 98.00%; melting point: 185.73 °C; [α ] D 20 (in deg cm 3 g -1 dm -1 ) = + 6.312 (c = 0.00301 g cm -3 in methanol); 1 HNMR (400 MHz, DMSOd 6 ) δ 8.84 (s, 1H), 7.57-7.45 (m, 1H), 7.53 (s, 1H), 7.49 (s, 2H), 7.32-7.22 (m, 2H), 4.42 (dtd, J = 3.6, 8.3, 47.9 Hz, 1H), 3.64 (s, 3H), 3.24-3.02 (m, 5H), 2.18-2.01 (m, 1H), 2.01-1.73 (m, 4H), 1.65-1.52 (m, 1H); 13 C NMR (101 MHz, DMSOd 6 ) δ 32. 7, 33.6, 35.9, 46.4, 48.9, 55.5, 98.4, 111.4, 112.7, 112.9 113.1, 122.7, 131.6, 132.6, 133.9, 142.0, 158.7, 159.8, 162.5. 19 General methods: unless otherwise noted, reactions were conducted using commercially available reagents and solvents, which were used as received. Hydrogenation reactions were performed in a ThalesNano HCube reactor using 30mm 10% Pd/C catalyst cartridges. Silica gel chromatography was per formed using a Biotage Isolera Four flash purification system with Silicycle SiliaSep cartridges. 1 H NMR spectra were recorded on a Varian INOVA400 400 MHz spectrometer. Chemical shifts are reported in δ units (p.p.m.) relative to residual solvent peak. Coupling constants (J) are reported in hertz (Hz). LC-MS analyses were performed using a Waters 2795 Separations Module equipped with a Waters 2996 photodiode array detector, a Waters 2424 ELS detector, a Waters micromass ZQ single quadropole mass detector, and an XBridge C18 column (5 μ m, 4.6 mm × 50 mm).

tertButyl (3R)3[(2S)2{[(benzyloxy)carbonyl]amino}3phenylpropan amido]pyrrolidine1carboxylate: to a solution of Cbzlphenylalanine (0.9 g, 3.00 mmol) in dichloromethane (25 ml) were sequentially added HOBt monohy drate (0.46 g, 3.00 mmol) and EDAC (0.58 g, 3 mmol). To this mixture was added a solution of tertbutyl (3R)3aminopyrrolidine1carboxylate (0.56 g, 3.00 mmol). The reaction was stirred at ambient temperature for 18 h, then partitioned between dichloromethane and 5% aqueous citric acid. The layers were separated, and the organic phase was washed with aqueous saturated NaHCO 3 and brine, dried over MgSO 4 , filtered, and concentrated. The crude concentrate was purified by flash silica gel chromatography (25 g Silicycle column), eluting with ethyl acetate in hexanes (20-80%) to provide 1.27 g (91%) of the title compound.

(2S)N[(3R)1cyanopyrrolidin3yl]3phenyl2(3phenylpropanamido) propanamide (GNE3086): a solution of tertbutyl (3R)3[(2S)2{[(benzyloxy) carbonyl]amino}3phenylpropanamido]pyrrolidine1carboxylate (1.15 g, 2.46 mmol) in ethanol (50 ml) was passed through 10% Pd/C catalyst at a rate of 1 ml min -1 in the HCube reactor at 10 bar and 20 °C until the reaction was judged complete by LC-MS. The reaction mixture was concentrated under reduced pressure to obtain 0.725 g of crude tertbutyl (3R)3[(2S)2amino3 phenylpropanamido]pyrrolidine1carboxylate as an offwhite, oily, solid that was used without further purification. LC-MS: m/z = 334.18 [M+ H] + .

To a solution of tertbutyl (3R)3[(2S)2amino3phenylpropanamido] pyrrolidine1carboxylate (0.036 g, 0.108) in tetrahydrofuran (0.5 ml) were added 3phenylpropanoyl chloride (0.016 ml, 0.108 mmol) and DIPEA (0.02 ml, 0.108 mmol). The reaction was stirred at ambient temperature for 90 min and concentrated under reduced pressure to obtain crude tertbutyl (3R)3[(2S)3 phenyl2(3phenylpropanamido)propanamido]pyrrolidine1carboxylate that was used without further purification.

To the crude tertbutyl (3R)3[(2S)3phenyl2(3phenylpropanamido) propanamido]pyrrolidine1carboxylate in dichloromethane (1.0 ml), were added triethylsilane (1 ml), water (3 ml), and trifluoroacetic acid (0.5 ml). The reaction was stirred at ambient temperature for 90 min, concentrated under reduced pressure, and triturated with hexanes:diethyl ether (1:1) to obtain (2S)3phenyl 2(3phenylpropanamido)N[(3R)pyrrolidin3yl]propanamide as a trifluoro acetic acid salt.

To (2S)3phenyl2(3phenylpropanamido)N[(3R)pyrrolidin3yl]propan amide trifluoroacetic acid in acetonitrile (0.7 ml) were sequentially added DIPEA (0.07 ml, 0.4 mmol) and cyanogen bromide (2.0 M in MeCN, 0.05 ml, 0.1 mmol). The reaction was stirred at ambient temperature for 30 min and then diluted with dichloromethane. The diluted reaction was washed sequentially with water, 10% aqueous KHSO 4 , and brine. The organic layer was dried over MgSO 4 , filtered, and concentrated. The crude concentrate was purified by flash silica gel chroma tography (4 g Silicycle column), eluting with acetone/dichloromethane to obtain 25 mg (64%) of the title compound. 1 H NMR (400 MHz, CDCl 3 ) δ 7. 14-7.31 (m, 10 H), 5.93 (d, J = 6.6 Hz, 1 H), 5.88 (br. s., 1H), 4.52 (d, J = 7.1 Hz, 1 H), 4.28 (d, J = 6.1 Hz, 1 H), 3.49 (dd, J = 10.2, 6 Hz, 1 H), 3.33 (dd, J = 8.6, 5.1 Hz, 1 H), 3.17 (d, J = 9.5 Hz, 1 H), 3.03-3.08 (m, 2 H), 2.83-2.94 (m, 3 H), 2.50 (q, J = 7.6 Hz, 2 H), 1.94-1.98 (m, 1 H), 1.49-1.51 (m, 1 H). 13 C NMR (150 MHz, DMSOd 6 ) δ 30.9, 31.0, 36.7, 38.2, 48.3, 48.7, 53.7, 54.5, 117.2, 125.8, 126.3, 128.0, 128.1, 128.2, 129.2, 137.5, 141.3, 171.0, 171

.1. LC-MS: m/z = 391.2 [M + H] + .
Hit-to-lead selection cascade assays. Biochemical deubiquitinase assays. Biochemical deubiquitinase assays used ubiquitinRho110 as a substrate to enable kinetic monitoring of reactions for USP7, USP7 catalytic domain, USP5, and USP47. The deubiquitinase proteins and their concentrations that were used in biochemical reactions were as follows. USP7, fulllength Nterminal Histag, native C terminus: 0.18 nM (Genentech, Hs_USP7 21102); USP7 catalytic domain Nterminal Histag: 40 nM (Genentech, Hs_USP7.K208K554); USP5, fulllength: 0.5 nM (Boston Biochem catalogue number E322, lot 02010210); USP47, full length Nterminal Histag:0.8 nM (Genentech Hs_USP47.M1D1287). Substrate K m values for USP proteins were USP7 fulllength, 2 μ M; USP5 fulllength, 0.33 μ M; and USP47 fulllength, 0.175 μ M. The final assay conditions were as follows. The reaction buffer consisted of 50 mM Tris (pH 7.5), 0.01%(v/v) Triton X100, 2.5 mM dithiothreitol, and 0.1% (w/v) bovine gamma globulin (Sigma catalogue number G500925G). The final substrate ubiquitinRho110 (Boston Biochem catalogue number U555) concentration used for reactions was 1 μ M. Reactions were per formed for 1 h at room temperature, in black 20 μ lvolume polystyrene ProxiPlate 384 F Plus (PerkinElmer catalogue number 6008260). Test compounds, including a control USP7 inhibitor (Ubaldehyde, Boston Biochem catalogue number U201), were serially diluted in DMSO, in 384well clear Vbottom polypropylene plates (Greiner catalogue number 781280). Compounds in DMSO were diluted tenfold into reaction buffer, to achieve threefold the final desired concentration.

Letter reSeArCH

The substrate, ubiquitinRho110 (Boston Biochem catalogue number U555), was prepared at 3 μ M (threefold the final concentration) and 5 μ l was dispensed into the reaction plate. Five microlitres of the compounds (diluted in reaction buffer at threefold the final concentration) were transferred to the reaction plate. Five micro litres of DUB protein, which was diluted in reaction buffer at threefold the final concentration, was transferred to the reaction plate to initiate the reaction. After 1 h incubation at room temperature, the reaction was quenched by the addition of 5 μ l of 400 mM acetic acid, in the case of an endpoint reaction. The enzymatic product was measured by quantifying the fluorescence signal of cleaved Rhodamine110 using excitation at 485 nm and emission at 535 nm. When preincubation of DUB with compounds was required, the order of addition of reagents was modified to premix the compounds with DUB (with a 1 h incubation period), before the addi tion of the substrate and the initiation of the reaction period. Percentage inhibition values were calculated relative to a noenzyme control and an uninhibited enzyme control. Curve fitting and IC 50 calculations were performed using Genedata Screener software. Conversion between IC 50 and K i values was performed using the Cheng-Prusoff equation. Cellular ubiquitin-MDM2 assay. HCT116 colon cancer cells or SJSA1 osteo sarcoma cells were seeded at a density of 150,000 cells per well in 90 μ l RPMI 1640 medium, 10% FBS (or 0.5% FBS for low serum conditions), 1× GlutaMAX (Gibco) in 96well black clear bottom, tissueculturetreated microplates (Greiner, catalogue number 655090), and incubated for 2 h at 37 °C, 5% CO 2 in a tissue culture incubator. Compounds were prepared in a serial dilution in DMSO at 200× the final desired concentration in a 96well polypropylene Vbottom microplate (Greiner, catalogue number 651261), then diluted 1:20 in RPMI tissue culture medium and 10 μ l transferred to each well of the cell plate. Cell plates were incu bated overnight for 20 h, at 37 °C, 5% CO 2 . Twenty microlitres of a 120 μ M stock (in RPMI) of the proteasome inhibitor MG132 (Cayman Chemical, catalogue num ber 10012628) was added to each well. Cells were incubated for 1 h at 37 °C, 5% CO 2 . Quantification of ubiquitin-MDM2 was performed using a Ub/total MDM2 wholecell lysate kit (MSD, catalogue number K15168D2). Cells were lysed by adding 15 μ l of 5× MSD lysis buffer (containing additives: 10 mM NaF, 10 mM β glycero phosphate, 1.5 mM Na 3 VO 4 , protease inhibitor cocktail (Sigma, P8340)) to each well and incubated at 4 °C for 30 min with shaking. One hundred microlitres of lysate were transferred to each well of the MSD 96well plate and incubated at room temperature for 1 h while shaking (650 r.p.m.) in the dark. The MSD plates were washed three times in Trisbuffered saline (50 mM TrisCl, pH 7.5, 150 mM NaCl) using a Biotek EL405 plate washer. Three millilitres of detection antibody solution were prepared per plate (1 ml of block buffer A, 1.82 ml 1× Tris wash buffer, 150 μl 2% Blocker DM, 30 μ l 10% Blocker DR, 60 μ l 50× antitotal MDM2 antibody). Twentyfive microlitres of detection antibody solution were added per well and incubated for 1 h at room temperature (650 r.p.m.) in the dark. Plates were washed three times in Trisbuffered saline using a Biotek EL405 plate washer. MSD read buffer was prepared according to the manufacturer's instructions and 150 μ l added per well. Plates were read using a MSD Sector Reader. The final meas urement was the ratio of ubiquitinated MDM2 to total MDM2. The percentage increase in ubiquitin-MDM2 was calculated relative to DMSO controls using Genedata Screener software. Total MDM2 immunofluorescence. HCT116 cells were seeded at a density of 40,000 cells per well in 384well tissue culture plates (Greiner 781091) in 50 μl per well of RPMI, 10% FBS, 2 mM lglutamine, and incubated overnight. Test compounds were prepared in a 20point serial dilution (one to twofold) in DMSO using a Biomek FX in a 384well Labcyteapproved polypropylene plate (Labcyte P05525). Compounds were acoustically dispensed into the cell plates using a Labcyte Echo (final total volume transferred was 50 nl) (DMSO final concentration was 0.1% v/v). Cell plates were incubated at 37 °C, 5% CO 2 , for 24 h. Cells were fixed by addition of 15 μ l of 16% paraformaldehyde (Electron Microscopy Sciences 15710 S) directly to the 50 μ l cell culture medium in each well. Plates were incubated for 30 min at room temperature. The well contents were aspirated using a Biotek EL406 and 50 μ l per well of permeabilization/block buffer added (phosphatebuffered saline (PBS, pH 7.5), Triton X100 0.5% (v/v), BSA 0.5% (w/v), proclin 15 p.p.m.). Plates were incubated for 30 min then washed three times with 100 μ l per well of PBS. Twentyfive microlitres per well of antiMDM2 (rabbit polyclonal, AbCam ab58530 diluted 1:500 in PBS, BSA 0.5% (w/v), Triton X100 0.1% (v/v)) were dispensed into each well. Plates were incubated for 2 h at room temperature then washed four times with 100 μ l per well of PBS using a Biotek EL406. Twenty five microlitres per well of Alexafluor 555 conjugated antirabbit IgG (Life Technologies A31572, diluted 1:1,000 and Hoechst 33342 1 μ g ml -1 diluted in PBS, BSA 0.5% (w/v), Triton X100 0.1% (v/v)) were dispensed into each well. Plates were incubated for 2 h at room temperature then washed four times with 100 μ l per well of PBS using a Biotek EL406. Fluorescence images of the samples (channel 1: 38623_BGRFRN_ BGRFRN (DNA); channel 2: 54915_BGRFRN_ BGRFRN (MDM2)) were acquired using a Cellomics XTI Arrayscan with the Bioapplication 'Compartmental Analysis' . Channel 1 was used to define the nuclear region. Measurements were made of 'Mean_CircAvgIntCh2' , which was the Alexa Fluor 555 fluorescence intensity (MDM2) within the nuclear region measured on a percell basis and averaged over all the measured cells. Data analysis and EC 50 calculation were performed in GraphPad Prism using nonlinear fourparameter curve fitting. Cathepsin-B protease assay. CathepsinB proteolytic activity was quantified by an LC-MS, MRMbased detection method. In brief, varying concentrations of experi mental compound were incubated with 0.5 nM human liver cathepsinB (EMD Millipore, 219364) and benzyloxycarbonylArgArg7amino4 methylcoumarin (CbzRRAMC) fluorogenic substrate in buffer containing 10 mM MES pH 6.0 and 1 mM DTT. The reaction was incubated for 5 min at room temperature, followed by quenching with the addition of an equal volume of 2% formic acid in water. Free AMC liberated by cathepsinB was quantified on a Sciex 5500 QTRAP mass spectrometer (Sciex) equipped with a Biocius Rapidfire highthroughput LC system (Agilent). AMC product was captured on an Agilent C18 Rapidfire cartridge, desalted with a 0.1% formic acid wash, and then eluted with 80% acetonitrile, 0.1% formic acid. Measured MRM areaundercurve for AMC was plotted against compound concentration using GraphPad Prism and fitted for IC 50 using fourparameter fitting. MRM parameters for AMC were as follows: ESI positive mode, Q1 = 233.1, Q3 = 175.2, collision energy = 16. Caspase-3 protease assay. Caspase3 proteolytic activity was quantified by an LC-MS, MRMbased detection method. In brief, varying concentrations of exper imental compound were incubated with 0.025 nM of recombinant caspase3 (cloned and purified inhouse) and 1 μ M (ZDEVD) 2 Rho110 fluorogenic substrate in buffer containing 25 mM HEPES, pH 7.2, and 5 mM DTT. The reaction was incubated for 20 min at room temperature, followed by quench ing with the addition of an equal volume of 2% formic acid in water. Free ZDEVD liberated by caspase3 was quantified on a Sciex 5500 QTRAP mass spectrometer (Sciex) equipped with a Biocius Rapidfire highthroughput LC system (Agilent). ZDEVD product was captured on an Agilent C18 Rapidfire cartridge, desalted with a 0.1% formic acid wash, then eluted with 80% acetonitrile, 0.1% formic acid. Measured MRM areaundercurve for ZDEVD was plotted against compound concentration using GraphPad Prism and fitted for IC 50 using fourparameter fitting. MRM parameters for ZDEVD were as follows: ESI negative mode, Q 1 = 609.2, Q 3 = 440.2, collision energy = -35. USP7 aggregation analysis. Aggregation of fulllength USP7 was confirmed by dynamic light scattering using a Wyatt DynaPro Plate Reader. Dynamic light scattering data were acquired at 37 °C, with Dynamics V7 software, with a 10 s acquisition time, ten acquisitions per measurement in autoattenuation mode. Compounds were present at 100 μ M in a buffer containing 50 mM HEPES pH 7.2, 150 mM NaCl, 0.01% Triton X100, 1 mM TCEP, 0.1% DMSO. Fulllength USP7 was present at 1 mg ml -1 with all compounds except rottlerin. Aggregate is defined here as having a hydrodynamic radius greater than 10 nm. USP7 covalent modification and LC-MS analysis. To evaluate potential covalent modification of the proteins, fulllength USP7 was incubated with excess com pound at room temperature overnight and covalent modification was evaluated by LC-MS using standard methods. Cell culture and cell treatments. HCT116 parental, USP7null (HD R02028), and HCT116 p53null (HD 104001) cell lines were purchased from Horizon. All other cell lines were obtained from Genentech's repository and were cultured in standard conditions in RPMI medium containing 10% FBS (solid tumour cell lines) or 20% FBS (haematopoietic cell lines), 1% penicillin/streptomycin, and 1% lglutamine. All cell lines were mycoplasmatested and cell line identity was confirmed by short tandem repeat and single nucleotide polymorphism profiling as described 24 . For studies evaluating cellular effects of compound treatments, 2,500-5,000 cells were seeded in one well of a 96well plate (Corning 3904). The following day, the medium was changed from normal (10%) to low serum (0.5%) containing vehicle (DMSO) or compounds. Sixteen to 24 h later, FBS was added to each well to bring serum levels back to 10%. Cells were then allowed to grow for 2 additional days. All treatments were done in triplicate. For degradation rescue studies, the proteasome inhibitor bortezomib (Selleckchem) or the UAE inhibitor MLN7243 (Active Biochem) was added at 5 μ M 30-45 min before collection. Cycloheximide chase studies. MCF7 cells were treated for a total of 7 h with DMSO or 15 μ M of the indicated compounds. During the 7 h of compound treatment, 50 μ M cycloheximide was added for the indicated times before collection. Cells were subsequently collected and lysates processed for western blot analysis. Antibodies and reagents. Antibodies to the indicated proteins were purchased from the following vendors: USP7 (ab84098), USP5 (ab84695) (AbCam) or ( 4833 2664), phosphoS112Bad (5284), p21 (2947) (Cell Signaling Technology); K48 polyubiquitin (Genentech); USP47 (Bethyl A301190A); UCHL1 (Invitrogen 381000); HAHRP (clone HA7) and Flag (A8592) (Sigma); p53 (Thermo Scientific MS738P1); p21 (Millipore 05655), Bad (AF819) (R&D). Secondary antibodies were purchased from LICOR Biosciences or Jackson ImmunoResearch. Fulllength human USP7 was cloned into a pRK5 mammalian expression construct with a Cterminal Flagtag and muta tions were introduced by QuickChange sitedirected mutagenesis as instructed by the manufacturer (Agilent). Western blotting, quantification, immunoprecipitations, and deubiquitinase assays. Cells were treated with compounds as detailed above before lysis for western blot analysis. Primary antibodies were diluted 1:1,000 and incubated for 1 h at room temperature or 4 °C overnight. Secondary antibodies were diluted 1:10,000 and incubated for 30-60 min at room temperature. Blots were either imaged using a chemiluminescent reagent (Pierce) or were scanned and bands quantified using LICOR Odyssey instrumentation and software, respectively. Immunoprecipitations with ubiquitinspecific antibodies were performed as described 25 , and immuno precipitates and corresponding cell lysates were analysed by immunoblot analysis as previously described 25 using the antibodies detailed above. For in vitro deubi quitination of PIM2, ubiquitinated PIM2 was captured by treating MV411 cells for 45 min with the proteasome inhibitor MG132 (SelleckChem). Cells were washed, lysed in a 6 M urea lysis buffer, and antiK48 polyubiquitin immuno precipirates were washed and deubiquitinated with 250 nM wildtype or C223S fulllength recombinant USP7 following a similar protocol as described 25 .

Cell viability studies and analysis. IncuCyte live cell analysis. One day after cell seeding, 10% cell culture medium was changed to 0.5% serum medium containing 2 μ M CellEvent Caspase 3/7 reagent (Life Technologies C10423) and compounds. The plates were placed in an IncuCyte live cell imager and scanning was started within 15-20 min. Twentyfour hours later, 10% serum was added back to the cell cultures. Images were taken every 2 h for 68-72 h, using a 10× objective. Phase contrast was used to measure cell confluency/density and green fluorescence was used to measure caspase activity. The images were analysed using IncuCyte software (Basic Analysis parameters) and a ratio of caspase activity to cell density/ count was determined. For combination experiments, MCF7 cells were treated with 15 μ M USP7 inhibitors or 0.1 μ M doxorubicin alone or in combination. Similarly, U2OS cells were treated with 15 μ M USP7 inhibitors or 1 μ M cisplatin alone or in combination.

CellTiter-Glo assays with wild-type and USP7-null HCT-116 cells. Cells were plated and cultured as described for IncuCyte studies. Seventytwo hours after compounds were added, CellTiterGlo (Promega) reagent was added following the vendor's protocol. Three times more USP7null HCT116 cells were plated per well than wildtype HCT116 cells (7,500 versus 2,500 cells), given the slower proliferation of USP7null cells. Tumour cell-line panel viability. GNE6640 and GNE6641 were profiled for 3 days across 441 cell lines, and GNE6776, GNE6640, and GNE6641 were profiled for 5 days across a subset of 185 cell lines as previously described 26 . In brief, compounds were screened in ninepoint dose-response using a threefold dilution. Cells were seeded into 384well plates 24 h before compound addition. Cells were then incubated with compound for 72 h or 120 h before assaying viability (CellTiterGlo, Promega). Assays were performed in biological triplicate. Cells were incubated (37 °C, 5% CO 2 ) in RPMI1640, 2.5% FBS (72 h assay) or 5% FBS (120 h assay), and 2 mM glutamine throughout the assay. The reported IC 50 and mean viability metrics were as follows: IC 50 was the dose at which the estimated inhibition was 50% relative to untreated wells (that is, absolute IC 50 ). The mean viability was calculated as previously described 26 . Pharmacogenomics analysis. We determined genomic and nongenomic features that were associated with differences in compound sensitivity. Mean viability was calculated as the arithmetic average of the fitted viabilities at each tested dose, as described 26 . For this analysis, normalized mean viabilities of GNE 6640 and GNE6446 were determined by normalizing the mean viabilities of each compound by the mean viability of GNE6641. Cancer type, cell histology, loss offunction, and hotspot mutations were assessed for statistical association with changes in normalized mean viability. Lossoffunction protein coding mutations included (1) insertions, (2) deletions, and (3) substitutions resulting in predicted truncating, splice site, translational start site, or nonstop mutations present in more than 90% of reads sequenced (> 90% variant allele frequency). Hotspot missense mutations assessed here were previously reported as significantly recurrent mutations in a pancancer analysis 27 . Features present in at least three cell lines were assessed. Statistical significance was determined using a twosided Student's ttest. Q values were determined by correcting resulting P values for multiple hypothesis testing using the Benjamini-Hochberg procedure. Primary combination screen. A compound library comprising 589 compounds arrayed in ninepoint dose-response was screened in the absence or presence of fixed doses of GNE6776 (0 nm, 125 nM, 250 nM, 500 nM, 1,000 nM, and 2,000 nM) or GNE6640 (400 nM). In brief, 5,000 EOL1 cells were seeded into 384well plates, and compound was added 24 h later. Cell viability was deter mined 120 h after compound addition (CellTiterGlo). Curves were fitted, and both IC 50 and mean viability metrics were calculated. The IC 50 was the dose at which inhibition was 50% relative to untreated wells. The mean viability was the average of the fitted viabilities at each tested dose. Mean viability was equivalent to the area under the logdose/viability curve divided by the total number of tested doses. Mean viability values were used for the analysis described in Extended Data Fig. 6g. All data were fitted using Genedata Screener software. Primary combination screen analysis. Normalized mean viabilities were determined in the EOL1 cell line for 574 compounds that have known protein or mecha nistic targets, in the presence of DMSO or increasing concentrations of GNE 6776 (100 nM, 250 nM, 500 nM, 1,000 nM or 2,000 nM) or 400 nM of GNE6640. For each compound, we assessed the difference in mean viability between USP7 inhibitor treatment and the DMSO treatment. For targets targeted by three or more compounds, we calculated the enrichment of high mean viability difference for each concentration of USP7 inhibitor by using a Wilcoxon ranksum test. For visualization purposes, we combined the results of all concentrations by taking the mean of thelog 10 (transformed P values) for each target. Bliss analysis. PIM inhibitors were tested in a ninepoint dose-response matrix with GNE6676 in the same manner as described for the compound library screen. Inhibitors were screened in threefold dilution using a top concentration of 10 μ M for PIM inhibitors and 20 μ M for GNE6676. Bliss calculations were performed using Genedata Screener software and the synergy score was calculated as previously described 28 . Exome sequencing pipeline. Exome sequencing and analysis of the cell lines was performed using standard protocols and analysed by methods previously described 29 . FASTQ reads were aligned to the human reference genome (GRCh38) using GSNAP version '20131010' with the following parameters: M 2 n 10 B 2 i 1-pairmaxdna= 1,000-terminalthreshold= 1,000-gmapmode= none-clip overlap. Duplicate reads in the resulting BAM file were marked using PicardTools, and indels realigned using the GATK IndelRealigner tool. Variations were called using the Bioconductor package VariantTools version 1.9.4. with default options except for two exceptions: (1) no variants were called in repeat regions as defined by the annotation Dust, Satellite repeats, and Tandem repeats in EnsEMBl 77; and (2) the avgNborCount post filter was configured using all single nucleotide polymorphisms from dbSNP version 138. Deubiquitinase selectivity analysis. Recombinant deubiquitinase di-ubiquitin mass spectrometry cleavage assay. The MALDI-TOF DUB assay was performed using the indicated concentrations of recombinant deubiquitinases, diubiquitin substrates, and USP7 inhibitor compounds as described previously 4 . The inhibition efficiency for GNE6640 and GNE6776 against the UCHl family members was monitored on UbUbe2W (UbE2), an alternative substrate to diubiquitin. Endogenous deubiquitinase activity-based probe assay. HEK293T cells at 80% confluency were collected by rinsing the plate once with 10 ml PBS followed by scraping. Cells were cleared by spinning them for 3 min at 350g at 4 °C and cell pellets were flashfrozen in liquid nitrogen and stored at -80 °C until lysis. Frozen pellets were lysed by quickly rethawing them in buffer A (50 mM TrisHCl pH 7.5, 250 mM sucrose, 1× phosphatase STOP, 2.5 mM TCEP, 2 mM ATP, 50 μ M phenylmethylsulfonyl fluoride (PMSF), 120 mM NaCl, 5 mM MgCl 2 ) and the lysate cleared by centrifugation by spinning at 18,000g at 4 °C. The protein concentration was adjusted to 5 mg ml -1 , and 5 mg (1 ml) each of this cell lysate was incubated with indicated compounds or DMSO at the indicated concentrations for 20 min at 900 r.p.m., 25 °C in a Thermomixer (Eppendorf AG). After compound incubation, 300 ng of recombinant viral DUB was added and lysates were incubated with 6.6 μ g ml -1 of the indicated activitybased DUB probe, at 1,250 r.p.m. and 25 °C, for the indicated times. The reaction was terminated by adding a 20% SDS solution to a final concentration of 0.4% for at least 30 min at room temperature, rotating. Subsequently, the lysate was diluted ten times with buffer B (50 mM TrisHCl pH 7.5, 150 mM NaCl, 5 mM EDTA, protease inhibitor cocktail EDTAfree (Roche), 50 μ M PMSF, 0.5 % NP40) into 15 ml conical tubes. About 120 μ l slurry of preequilibrated antiHA affinity matrix (Roche) was added and HAtagged proteins were immunopurified with this matrix by rotating the samples overnight at 4 °C. The beads were then washed 1 ml of icecold buffer B (three times), buffer B without NP40 (once), and TEAB (15 mM triethylam moniabicarbonate, pH 8.5) (three times). Spins between washes were performed at 2,000g at 4 °C. To elute the immunopurified material, 330 μ l of buffer C (1 mg ml -1 HA peptide (Thermo Scientific), 15 mM TEAB, 0.02 % Rapigest (Waters)) was added and the samples were incubated at 37 °C for 30 min at 1,100 r.p.m. shaking in a Thermomixer (Eppendorf AG). The eluted material was cleared from the beads by spinning at 2,600g. The cleared material was stored at -80 °C until mass spectrometry preparation and analysis. For DUB Letter reSeArCH identifications, the eluted proteins were digested with trypsin using filteraided sample preparation. Eluents were added to Microcon30K filtration devices (Millipore) and briefly washed by 0.2 ml of 8 M urea in 200 mM TEAB, pH 8.5. Between each step, liquid was cleared by centrifugation at 14,000g, except if noted. Proteins were reduced by DTT for 20 min at 60 °C and subsequently alkylated by iodoacetamide for 15 min in the dark. The membrane was further washed by 8 M urea once and 200 mM TEAB three times. Trypsin (Promega) was added at 1:40 enzyme/substrate ratio. Devices were briefly centrifuged at 100g for 30 s and incubated overnight at 37 °C. Tryptic peptides were recovered by centrifu gation at 14,000g for 4 min. An additional 60 μ l of 200 mM TEAB, pH 8.5, was added to the devices and centrifuged as an additional elution step. This eluent was combined with the previous ones for further processing. Ten per cent of the eluents were dried in a SpeedVac and desalted with a C18 STAGE tip (Proxeon, Thermo Fisher) before being injected onto the mass spectrometer for LC-MS/MS acquisition. Deubiquitinase identification by mass spectrometry. Peptides were loaded onto an Acuity UPLC BEH130 C18 column (1.7 μ m, 12 Å, 100 μ m × 100 mm) at a flow rate of 1.5 μ l min -1 in solvent A (98% water/2% MeCN/0.1% formic acid) using a NanoAcquity UPLC system (Waters). Separation was achieved with a linear gradient of 2% solvent B (98% MeCN/2% water/0.1% formic acid) to 25% solvent B over 45 min. Eluted peptides were injected onto an Orbitrap Elite mass spectrometer (Thermo Fisher) using an Advance CaptiveSpray source (Bruker) at a voltage of 1.2 kV. Full MS scans were collected in the Orbitrap at 60,000 resolution, the top 15 most abundant ions were selected in a datadependent mode and frag mented with collisioninduced dissociation, and MS/MS were collected in the ion trap. MS/MS spectra were searched using Mascot (version 2.3.02) against a human proteome database (Uniprot, December 2011) with known contaminants along with all decoy sequences. Search parameters included trypsin cleavage allowing up to two missed cleavage events, a precursor ion tolerance of 50 p.p.m., and a fragment ion tolerance of 0.8 Da. Searches also allowed variable modifications of methionine oxidation (+ 15.9949 Da), and two cysteine modifications of either (+ 57.0215) for carbamidomethylation or (+ 192.0569 Da) for reacted DUB probe remnant. Peptide spectra matches were filtered with a false discovery rate of 5% on the peptide level and subsequently at 2% on the protein level. Each identified peptidespectrum match was quantified using areaundercurve, and the same quantification event was extended to other runs where such peptide was not identified by an MS/MS spectrum, based on exact precursor m/z and retentiontime matching. Statistical analysis of deubiquitinase activity data. Detected and quantified area undercurve measures from labelfree mass spectrometry were logarithmically (base 2) rescaled. Changes between inhibitor and controltreated lysate were compared by a linear mixedeffects model with fixed effects for treatments and random effects for peptide species and massspectrometry run. Computations were performed in R, version 3.3.0, using the R package lme4, with error degrees of freedom estimated by the Kenward-Roger method using the R package pbkrtest. For each protein, log 2 (fold change) in activity was normalized to that observed in a spikedin viral DUB or BSA control protein. Statistical significance of the observed 'inhibitorvscontrol' differences in log 2 (fold change) between each DUB and the control protein was then assessed by a tstatistic with Satterthwaiteestimated degrees of freedom, and corrected for multiplicity by the Benjamini-Hochberg false discovery rate method set at 0.10. Animal use and care. All animal work followed the recommendations of the Guide for Care and Use of Laboratory Animals with respect to restraint, husbandry, surgical procedures, feed and fluid regulation, and veterinary care. The animal care and use programme at Genentech is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International, which assures com pliance with accepted standards for the care and use of laboratory animals. Studies were tailored to minimize the number of animals used, yet allow sufficient numbers to address any variability in drug exposure or biomarker response. For efficacy studies where growth rate differences introduce some variability, n = 10 engrafted animals per treatment cohort followed recommended housing (five mice per cage) and provided sufficient statistical power for a wellbehaved xenograft model. For PD biomarker experiments, a lower number (n = 4) was sufficient to note a consistent response. Extra animals were engrafted to ensure sufficient animals had the desired range of tumour volumes at randomization, and cohorts were enrolled that had a balanced distribution of tumour volumes. Owing to the need to monitor potential adverse effects with first inlife assessment of novel compounds, no study was conducted under blinded conditions. DMPK analysis. In vitro DMPK studies were performed using standard protocols. GNE6776 was formulated as a suspension in 0.5% methylcellulose/0.2% Tween80 and was administered at 200 mg kg -1 (body weight) by oral gavage to female C.B-17 SCID mice, aged 12-16 weeks (Charles River Laboratories; n = 3 per time point). No randomization was used for DMPK studies. At 0.5, 1, 2, 4, 8 and 24 h postdose, blood samples were collected by terminal cardiac puncture into anticoagulant tubes (EDTA). Clarified plasma was then transferred to a fresh tube and snap frozen. GNE6776 plasma concentrations were determined by LC-MS/MS. In vivo pharmacodynamic response. For EOL1 AML xenograft studies, immu nodeficient C.B-17 SCID mice (Charles River Laboratories), aged 12-16 weeks, were inoculated subcutaneously on the right flank with five million cells in a 50:50 suspension of HBSS:Matrigel (BD Biosciences; 100 μ l). When tumour volumes reached between approximately 285 and 500 mm 3 , mice were distributed into volumematched cohorts (n = 4). For MCF7 breastcancer xenograft studies, immunodeficient nu/nu mice (Charles River Laboratories), aged 6-8 weeks, were implanted with 0.36 mg oestrogen pellets (Innovative Research of America) via trochar 1-3 days before tumour cell inoculation. Ten million MCF7Ser cells, an in vivooptimized MCF7 variant, were injected orthotopically into the 2/3 mammary fat pad of each mouse in a 50:50 suspension of HBSS:Matrigel (BD Biosciences) in a total volume of 100 μ l. When tumour volumes reached between approximately 285 and 450 mm 3 , mice were distributed into volumematched cohorts (n = 4). GNE6776 was formulated as a suspension in 0.5% methyl cellulose/0.2% Tween80 and administered at 200 mg kg -1 (body weight) by oral gavage at 0 and 4 h. 0.5% Methylcellulose/0.2% Tween80 control or GNE6776 treated samples were collected at 8 h after the first dose and excised tumours were flash frozen on dry ice. Tumours were lysed in RIPA buffer containing protease inhibitors (Roche) and 300 mM NaCl using a Qiagen TissueLyser. Samples were incubated on ice for 15 min and then centrifuged at 20,000g at 4 °C for 10 min. Protein levels in clarified lysates were quantified using a Pierce BCA assay kit and concentrations were normalized with sample buffer. Samples were run on gels, and proteins were transferred to membranes and western blotted as described above.

In vivo efficacy study. For EOL1 AML xenograft studies, immunodeficient C.B-17 SCID mice (Charles River Laboratories), aged 12-16 weeks, were inoculated subcutaneously on the right flank with five million cells in a 50:50 suspension of HBSS:Matrigel (BD Biosciences; 100 μ l). When tumours became established (150-300 mm 3 ), mice were distributed into tumourvolumematched cohorts (n = 7, mean tumour volume ~ 250 mm 3 ). GNE6776 was formulated as a suspension in 0.5% methylcellulose/0.2% Tween80 and was administered at 100 or 200 mg kg -1 (body weight) by oral gavage on a once or twice daily schedule. Tumour volume measurements, body weight and body condition data were collected two or three times per week. The maximum tumour volume limit of 2,000 mm 3 was not reached in any animal. USP7 enzymatic analysis. Michaelis-Menten kinetic measurements with full length USP7 were performed using 1 nM USP7 with a series of ubiquitin-AMC substrate titrations. The initial rate of substrate hydrolysis was determined using the Magellan software on a Tecan Safire2 plate reader, and kinetic parameters modelled using nonlinear regression analysis with GraphPad Prism software. Standard error was calculated from three technical replicates. For studies using the USP7 D305/E308 mutant, samples were reacted in a buffer consisting of 50 mM HEPES (pH 7.5), 100 mM NaCl, 2.5 mM dithiothreitol, and 0.1% (w/v) bovine gamma globulin (Sigma catalogue number G500925G). The starting substrate concentration of ubiquitinRho110 (Boston Biochem catalogue number U555) used for the Michaelis-Menten analysis was 100 μ M serial diluted to 781 nM. Reactions were performed for 1 h at room temperature with a final enzyme con centration of 100 nM (three independent experiments, see symbols in plots), in black 100μ l volume 96well half area plates (Corning catalogue number 3993). The enzymatic activity was calculated by fitting the data using the initial velocity with the linear V 0 values measured by analysing the fluorescence signal of cleaved Rho110 using excitation at 485 nm and emission at 535 nm. X-ray crystallography. Crystals were grown by the hangingdrop method by mixing the USP7 catalytic domain (residues 208-554) at 15 mg ml -1 with an equal volume of reservoir solution containing 100 mM TrisHCl, pH 7.0, and 20% PEG1000 (v/v). Costructures with compounds were obtained by soaking crystals with 1 mM of compound overnight. Crystals were cryoprotected with reservoir solution supplemented with 20% glycerol (v/v) and flashfrozen in liquid nitrogen. Data collection and refinement statistics are detailed in Extended Data Fig. 8e. Generation of isotopically labelled di-ubiquitins. In both K48 and K63linked diubiquitin, the distal ubiquitin has isotopically labelled 15 NThr residues and the proximal ubiquitin has 15 NLeu residues. These are marked with asterisks in Fig. 4a.

The selectively labelled ubiquitin monomers were generated and purified as follows: ubiquitin was cloned into a PET3a vector and transformed into the auxotrophic strains RF2 and ML2 (a gift from R. Gennis and T. Iwasaki) and expressed with the following modifications. Bacterial cultures were grown at 37 °C in M9 medium supplemented with NH 4 Cl (2 g l -1 ) and C 6 glucose (4 g l -1 ). Strain

Letter reSeArCH

RF2 was additionally supplemented with 6 mM [ 15 N]LThr (Cambridge Isoptopics catalogue number NLM142PK) and ML2 was supplemented with 6 mM [ 15 N] LLeu (Cambridge Isoptopics catalogue number NLM1421) and 6 mM unlabelled Ile and Tyr (SigmaAldrich catalogue number I2752 and catalogue number T3754). After reaching an absorbance of 0.6, the cells were induced with 1 mM IPTG and further grown for 6 h. Protein purification was performed at room temperature. Cells were collected and lysed in lysis buffer (50 mM HEPES 7.0). The cleared lysate was subjected to affinity chromatography using DEAE Sepharose Fast Flow (GE catalogue number 17070901). Ubiquitin was collected in the flow through and dialysed overnight into NaOAc, pH 4.5. Dialysed material was clarified by centrifugation at 35,000g and ubiquitin was subjected to ion exchange chromato graphy using a MonoS column (GE Healthcare, catalogue number 17516901). The following enzymes were obtained from Boston Biochem: UBE1 (catalogue number E305), UBE2K (catalogue number E2602) and UBE2N/UBE2V1 complex (catalogue number E2664).

K63 and K48linked diubiquitin chains were generated and purified as follows: in separate reactions incubating 250 nM E1 enzyme, 5 μ M UBE2K (K48 linked), or 5 μ M UBE2N/UBE2V1 complex (K63 linked) with equal molar ratios of 1 mM ubiquitin and 1 mM ubiquitin, 10 mM ATP, 50 mM HEPES (pH 8.0), 10 mM MgCl 2 in a 10 ml reaction at 37 °C. After 2 h, the reaction was acidified with 2 ml of 17.4 M glacial acetic acid. Obtained diubiquitins (K63 or K48linked) were purified by cation exchange using a MonoS column (GE Healthcare, catalogue number 17516901). All purified diubiquitin chains were buffer exchanged into 1× PBS buffer and proteins were flashfrozen in liquid nitrogen before storage at -80 °C. Generation of 5-TAMRA-peptide/tetra-ubiquitin conjugates. K63 and K48 linked tetraubiquitin chains were obtained from Boston Biochem, K63linked (catalogue number UC310B), K48linked (catalogue number UC210B). The 5TAMRA (5carboxytetramethylrhodamine) peptide was generated by CPC Scientific consisting of the sequence 5TAMRAYPYDVPDYAIREIVSRNKRR YQEDG 19 . K63 or K48 tetraubiquitin chains were conjugated to the peptide as follows. ( 1) Generation of tetraubiquitinMESNA: incubating 250 nM E1, 10 mM MgCl 2 , 10 mM MgATP, 1 mM tetraubiquitin, and 100 mM MESNA (SigmaAldrich, catalogue number 63705), in 20 mM Na 2 HPO 4 at pH 8.0 at 37 °C overnight. This was then dialysed into 0.4% TFA, and tetraubiquitinMESNA was lyophilized. ( 2) Lyophilized tetraubiquitin was dissolved in DMSO at a concen tration of 0.5 mM and 2 mg of peptide added until all components were dissolved; reaction volume 1 ml. The reaction was initiated by adding NHS 27.5 mM (final concentration), AgNO 3 3.3 mM (final concentration), and 22 μ l DIPEA, and incu bated at room temperature overnight. The reaction was diluted ten times with doubledistilled H 2 O and desalted into PBS (pH 7.5). Nonconjugated peptide was removed by sizeexclusion chromatography. In a second step, nonconjugated tetraubiquitin was removed by ion exchange chromatography as described above and buffer exchanged into 1× PBS (pH 7.5). The concentration of the purified final conjugate was determined by absorbance using an extinction coefficient for 5TAMRA at 80,000 cm -1 mol -1 .

TAMRA-peptide/tetra-ubiquitin conjugate depolymerization studies. For depolymerization assays, 100 nM USP7 (catalogue number E519, lot 09939314) or 100 nM USP7 catalytic domain Nterminal Histag (Genentech, Hs_USP7. K208K554) was diluted in 1× PBS buffer (pH 7.5) containing 5 mM DTT to generate 10× stock solutions in respect of the final concentration and preincubated at room temperature for 10 min. In a 90 μ l reaction, 9 μ g (2.7 μ M) of 5TAMRA peptide/tetraubiquitin (K63 or K48linked) was mixed with 9 μ l of diluted enzyme in 1× PBS buffer (pH 7.5). Aliquots of 10 μ l of the reaction were mixed with 4 μ l 2× SDS loading buffer at the time points indicated to stop the reaction. Samples (14 μ l) were subjected to SDS gelelectrophoresis using precast BioRad Criterion TGX 10-20% gels (catalogue number 5671114). Fluorescence was analysed using a FluorChem imager from Protein Simple according to the user's manual. Densitometry values were analysed using the software AlphaViewSA, ProteinSimple. Data and code availability. The 120 cell line exome dataset for Extended Data Fig. 5 and Supplementary Table 1 is available as part of a larger cell line exome study (S. Seshagiri et al., manuscript in preparation) from the European Genome phenome Archive under accession number EGAS00001002554. Atomic coordi nates and structures have been deposited in the PDB under accession numbers 5UQV (USP7/GNE6640) and 5UQX (USP7/GNE6776). The USP7 NMR assign ments in Figs 3a and4a and Extended Data Figs 8c, 9a and 10b are deposited in the Biological Magnetic Resonance Data Bank under accession number 26766. The authors declare that all other data supporting the findings of this study are available within the paper and its Supplementary Information files. Source data are provided with the paper as Supplementary Figs 1 and2 and Extended Data Fig. 4g, j linked source files, and Supplementary Tables 1 and2. R code written to run the linear mixed models and data analysis for endogenous deubiquitinase activity changes in Fig. 2b are available from the corresponding authors upon request (W.F.F. et al., manuscript in preparation). R code used in Extended Data Fig. 5 can be accessed at https://github.com/changmt/Kategaya_et_al_USP7.

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a.

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Extended Data Figure 1 | Screening cascades and hit-to-lead assay data for USP7 inhibitors. a, Highthroughput activitybased screening cascade to identify USP7 inhibitors. Screening stages are identified in bold print. Numbers of compounds at each stage are provided to the right of each box. Criteria for progression to the next stage are highlighted in italics to the left of each arrow. b, Fragment NMR screen diagram. Screening stages are identified in bold print. Numbers of compounds at each stage are provided to the right of each box. Criteria for progression to the next stage are highlighted in italics to the left of each arrow. Protein saturation transfer difference (STD) experiments were performed at 284 K. Primary USP7 catalytic domain binders were selected on the basis of the signaltonoise ratio of the respective compound with a cut off of greater than 5. All primary binders were remeasured as single compounds under otherwise identical conditions and confirmed binders selected having a signaltonoise ratio of greater than 10. Hits were further tested for specific binding to USP7 catalytic domain by measurement of 1 H/ 15 N TROSY spectra. Positive hits were defined as compounds that induced chemical shift perturbations. Perturbations were classified by the chemical shift patterns and selected compounds passed onto Xray soaking experiments. c, Table summarizing the hittolead assay results of the lead compounds identified by the highthroughput screening and NMR fragment screening campaigns. Compound series are grouped in columns and hittolead selection assay data are listed in the indicated rows. At least two experimental replicates were performed; averages are shown ± s.e.m. See Fig. 1b and text for more details. d, Endogenous MDM2 immunofluorescence studies. HCT116 human colorectal carcinoma cells were treated with a range of concentrations of the indicated USP7 inhibitors or DMSO vehicle for 24 h and endogenous MDM2 protein levels were detected by immunofluorescence imaging. Representative images show cells treated with 10 μ M of the indicated compounds or DMSO vehicle control. Scale bar, 20 μ m. The graph shows data from experimental duplicates of the quantified mean nuclear MDM2 protein levels per cell over a range of concentrations of GNE8735 and GNE2916. The halfmaximal effective concentration (EC 50 ) for the elevation in MDM2 caused by GNE8735 was 2.9 μ M. Image source data: Supplementary Fig. 1. e, Quantification of total and ubiquitinatedMDM2 (UbMDM2) in USP7 inhibitortreated cells. SJSA1 human osteosarcoma cells were treated with a range of concentrations of the indicated USP7 inhibitors or DMSO vehicle control for 24 h and the level of ubiquitinated MDM2 and total MDM2 were measured using a multiplexed mesoscale immunoassay. Graphs show experimental duplicates of the quantified level of either total MDM2 (right column, red), ubiquitinated MDM2 (central column, blue), or the percentage change in the ratio of the ubiquitinated MDM2 signal and the total MDM2 signal (left column, orange). All data are shown as percentage change in each value, relative to DMSO vehicle treated samples. The maximal extent of the increase in the ubiquitinated MDM2 to total MDM2 ratio varied between compounds; therefore to compare the potency of the increase in the ratio of ubiquitinatedMDM2 to total MDM2 between compounds, the top level was universally set to 100%. This normalization allowed calculation of the EC 50 of the percentage change in this ratio relative to DMSO (left column). Indole tricyclic compounds including GNE8735 increased total MDM2 levels and inhibited cathepsinB, indicating poor selectivity and induction of general cell stress by this chemical series (d, e). Indole tricyclics also precipitated caspase3, although they passed dynamic light scattering analysis (see also Extended Data Fig. 2a). The peptidomimetic compounds had weaker biochemical potency, poor selectivity, and covalently modified USP7 cysteine (Cys) residues other than the catalytic Cys (data not shown). Given these data, and because optimization of indole tricyclic and peptidomimetic compounds proved challenging, these series were discontinued. The tetrahydroacridine and fragment compounds were relatively potent, selective, and enhanced cellular MDM2 ubiquitination without significantly increasing total MDM2 (d, e). Tetrahydroacridine compounds passed cathepsinB inhibition assays, demonstrating additional protease selectivity. Neither tetrahydroacridine nor fragment compounds showed evidence of USP7 aggregation in dynamic light scattering or in NMR studies (data not shown; see Extended Data Fig. 2a). Tetrahydroacridine compounds, including GNE6831, covalently modified USP7, consistent with a previous report describing a similar series 30 (see also Extended Data Fig. 2b).

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Extended Data Figure 2 | Biophysical and cellular characterization of selected USP7 inhibitors. a, The dynamic light scattering autocorrelation functions are shown for 100 μ M rottlerin (red), fulllength USP7 with 100 μ M GNE8735 (blue), fulllength USP7 with 100 μ M GNE 2090 (brown), and fulllength USP7 with 0.1% DMSO vehicle control (black). The percentage aggregate by mass is shown in the legend. Two experimental replicates were performed. b, USP7 fulllength protein was incubated overnight with excess of GNE6831 and analysed by liquid chromatography-mass spectrometry. Unmodified and covalently modified USP7 are represented in the top and bottom panels, respectively. Three experimental replicates were performed. c, 1b and text for more details. e, Cell viability assays in AMO1 cells treated as indicated with the tetrahydroacridine compounds (top graph: purple lines, inactive controls; green line, active compound) and fragment compounds (bottom graph: red lines, inactive controls; blue line, active compound). Experimental triplicate data normalized to vehicle control are plotted as a function of compound concentration. f, Cell viability assays in KMS12PE cells treated as indicated with the tetrahydroacridine compounds (top graph: purple line, inactive control; green lines, active compounds) and fragment compounds (bottom graph: red line, inactive control; blue line, active compound). Experimental triplicate data normalized to vehicle control are plotted as a function of compound concentration. Tetrahydroacridine compounds GNE6831 and GNE2090 decreased viability of KMS12PE and AMO1 multiple myeloma cell lines but this activity was not differentiated from control compounds GNE0956, GNE2143, and GNE2148. By contrast, the fragment compound GNE2916 decreased multiple myeloma cell viability significantly more than control compounds GNE2917, GNE2931, and GNE9603. Thus work on tetrahydroacridine series was discontinued and the fragment series was further optimized. Graph source data for g and j can be accessed in the Supplementary Information. Gel source data: Supplementary Fig. 1.

Letter reSeArCH See Supplementary Table 1 for additional information.

Letter or cisplatin alone or in combination. f, Pie chart illustrating the distribution of compound classes in the Genentech Chemical Genomics Compound library, comprising 589 compounds. NHR, nuclear hormone receptor; GEF, guanine nucleotide exchange factor; DNA, DNAdamaging agent. Graph source data for a-e can be accessed in the Supplementary Information. g, Bar plot visualizing thelog 10 (transformed P value) from the Wilcoxon ranksum test evaluating the enrichment of a given compound target over all concentrations of USP7 inhibitors versus DMSO experiments in EOL1 cells. Only compound targets with three or more compounds in the screen were visualized; the distribution results are indicated by the individual data points from each compound. Higher values indicate synergy with USP7 inhibitors and were further evaluated by Bliss analysis (Fig. 2 and Extended Data Fig. 7). Compounds common to certain signalling pathways including PI3K/PIM, RTK/MAPK, epigenetic regulation, and DNA damage are colourcoded as indicated. See Supplementary Table 2 for additional information. 5 7 9 8 5 2 1 0 -2 0 -3 27 47 46 32 11 3 2 0 2 2 11 24 59 51 18 6 3 1 3 4 4 6 21 35 16 5 3 1 3 2 1 4 4 10 8 3 1 0 2 2 1 3 1 1 3 1 -1 0 2 -1 -2 -1 -4 -5 1 1 -2 0 2 -1 -2 0 -1 -1 1 1 -2 0 2 0 -1 1 -1 -1 0 1 -2 0 2 0 0 2 0 -1 0 1 -2 0

P I3 K -A P IM m T O R E R K F lt 3 G R C a s p a s e A L K J A K M E K 1 M E K 2 K D M 2 B K D M 5 A G u

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Extended Data Figure 7 | Mechanism of action studies with USP7 inhibitor and PIM inhibitor combinations. a, Schematic of the PI3K signalling pathway and regulation by PIM kinases. PIM and AKT kinases regulate Bad and TSC1/2 phosphorylation status. Phosphoproteins highlighted in yellow were profiled in cellular studies shown in Fig. 2d. b, Compound structure of GDC0339. c, PIM inhibitor viability curves at the indicated fixed doses of GNE6676 in EOL1 cells (technical duplicates). d, Bliss analysis of 9 × 9 dose-response matrix with PIM inhibitor GDC0339 and GNE6776 in EOL1 cells, representative of technical duplicates. Left: curvefitted viability values at each dose across the matrix. Zero represents no effect whereas 100 indicates complete loss of viability. Right: the difference in observed versus predicted values using the Bliss independence model. Positive values indicate a greater than predicted decrease in viability. Calculated synergy score = 4.84. e, Immunoblot analysis of cell lysates from the indicated cell lines treated with GNE6776 (2 μ M for 18 h), either alone or in combination with a UAE1 inhibitor MLN7243 (5 μ M for 45 min) or the proteasome inhibitor bortezomib (5 μ M for 45 min) as indicated. f, Immunoblot analysis of cell lysates from the indicated cell lines, either untreated or treated with the proteasome inhibitor bortezomib (5 μ M for 45 min). g, Immunoprecipitation of cellular lysates using an antiUSP7 antibody or an isotypematched control antibody followed by western blot analysis of lysates or immunoprecipitates with the indicated antibodies. h, Wildtype recombinant USP7, but not catalytically inactive USP7 C223S, deubiquitinates endogenous polyubiquitinated PIM2 that was immunoprecipitated from proteasome inhibitortreated MV411 cells. Data in e-h are representative of at least two experimental replicates. Gel source data: Supplementary Fig. 1.

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Extended Data Figure 8 | Enzymatic analysis and supporting structural biology data for USP7 inhibitors and USP7. a, Michaelis-Menten kinetic analysis of USP7 and a series of ubiquitinAMC substrate titrations with the indicated USP7 inhibitors. Initial rate of substrate hydrolysis was determined using the Magellan software on a Tecan Safire2 plate reader and kinetic parameters were modelled using nonlinear regression analysis with GraphPad Prism software. Averages with standard errors were calculated from three technical replicates and are representative of two (GNE6776) or three (GNE6640, GNE6641, and no inhibitor) biologically independent experiments. b, Affinity values of ubiquitin binding to USP7 catalytic domain in the absence and presence of USP7 inhibitors. The values were determined by titration of unlabelled ubiquitin to labelled USP7 catalytic domain and the NMR chemical shift changes were fitted as described in the Methods. The mean values and standard deviations are representative of three experimental replicates and were calculated by averaging the values obtained for eight independent chemical shift perturbations of the wellresolved cross peaks from Y339, S341, D342, G375, A381, G382, D412, and I419. c, Overlay of a region of the twodimensional 1 H/ 15 N TROSY spectrum of the USP7 catalytic domain (orange) highlighting changes induced by binding of ubiquitin in the absence (left, blue) and presence of GNE6640 (right, black). Individual peaks stemming from residues E371 and Q387 are highlighted. Data are representative of three experimental replicates. d, Comparison of the crystal structure of USP7 catalytic domain in complex with GNE 6640 (cyan) and GNE6776 (yellow). The catalytic domain is shown as an orange cartoon and the side chains of the residues in proximity to the inhibitor binding sites are shown as orange sticks. GNE6640 and GNE 6776 compound structures are indicated above. e, PDB accession numbers, data collection, and refinement statistics for GNE6640 and GNE6776 crystal structures with the USP7 catalytic domain.

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Extended Data Figure 10 | Analysis of differential USP7 binding to K48-and K63-linked poly-ubiquitin and depolymerization kinetics. a, Schematic diagrams of substratebound K48linked polyubiquitin chains (left) and K63linked chains (right), and proposed USP7 interactions. Dashed lines indicate residue side chains; filled circle indicates an isopeptide bond between the ubiquitin C terminus and a Lys residue side chain. The proximal ubiquitin subunits (ligated to the substrate) and their K48 or K63 side chains are indicated with 'a' subscripts, the next most distal ubiquitin subunits and side chains are indicated with 'b' subscripts, and so on. Preferential USP7 binding to free K48 side chains would direct USP7 to the distal ubiquitin subunit of K48 polyubiquitin and promote sequential exocleavage, whereas USP7 would bind all subunits of K63polyubiquitin and promote exo, endo, and basecleavage. b, The 1 H/ 15 N SOFAST spectrum of labelled ubiquitin at 100 μ M (cyan), superimposed with the spectrum of a 1:1 molar ratio of labelled ubiquitin in the presence of unlabelled USP7 catalytic domain (orange). Ubiquitin residues affected by the USP7 interaction, which results in exchange broadening of the residue cross peaks, are labelled with grey or blue text and correspond to the residues depicted in c. The K48 and K63 residues as well as the N and Cterminal residues are labelled in red type. The L43 and L50 residues labelled in green type do not broaden upon USP7 binding and serve as internal controls. The box highlights the region depicted in Fig. 4a

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Tumor samples were excluded from analysis if the protein yield or quality of tumor lysates was low.

No samples were excluded from analysis in the USP7 activity-based protein profiling screens or the combination compound library screen.

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Experiments were run at least in duplicate and standard deviation or SEM reported.

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Date: 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0 1 0 0 1 1 0 1 2 0 1 3 0 1 4 0 1 5 0 6 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0 1 0 0 1 1 0 1 2 0 1 3 0 1 4 0 1 5 0 1 6 0 1 7 0 1 8 0 f1 (ppm ) -2 0 0 0 0 0 0 -1 0 0 0 0 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0 1 0 0 1 1 0 1 2 0 1 3 0 1 4 0 1 5 0 1 6 0 1 7 0 8 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0 1 0 0 1 1 0 1 2 0 1 3 0 1 4 0 1 5 0 1 6 0 1 7 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0 1 0 0 1 1 0 1 2 0 1 3 0 1 4 0 1 5 0 1 6 0 1 7 0 8 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0 1 0 0 1 1 0 1 2 0 1 3 0 1 4 0 1 5 0 1 6 0 1 7 0 8 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0 1 0 0 1 1 0 1 2 0 1 3 0 1 4 0 1 5 0 6 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0 1 0 0 1 1 0 1 2 0 1 3 0 1 4 0 1 5 0 6 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0 1 0 0 1 1 0 1 2 0 1 3 0 1 4 0 1 5 0 1 6 0 1 7 3 0 4 0 5 0 6 0 7 0 8 0 9 0 1 0 0 1 1 0 1 2 0 1 3 0 1 4 0 1 5 0 1 6 0 1 7 0 f1 (ppm ) -2 0 0 0 0 0 0 -1 0 0 0 0 0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0 1 0 0 1 1 0 1 2 0 1 3 0 1 4 0 1 5 0 6 2013101144 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0 1 0 0 1 1 0 1 2 0 1 3 0 1 4 0 1 5 0 1 6 1 9 0 -1 8 5 -1 8 0 -1 7 5 -1 7 0 -1 6 5 -1 6 0 -1 5 5 -1 5 0 -1 4 5 -1 4 0 -1 3 5 -1 3 0 -1 2 5 -1 2 0 -1 1 5 -1 1 0 -1 0 8 0 -1 7 1 .7 9 -1 7 1 .7 6 -1 7 1 .7 5 -1 7 1 .7 2 -1 7 1 .7 0 -1 7 1 .6 8 -1 1 1 .2 4 -1 1 1 .2 2 -1 1 1

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INTRODUCTION

Deubiquitinating enzymes (DUBs) remove ubiquitin modifications from substrate proteins, thereby antagonizing the diverse post-translational ubiquitination signals that control protein stability, function, and localization to regulate various cellular processes [START_REF] Hershko | Immunochemical analysis of the turnover of ubiquitin-protein conjugates in intact cells. Relationship to the breakdown of abnormal proteins[END_REF][START_REF] Mukhopadhyay | Proteasome-independent functions of ubiquitin in endocytosis and signaling[END_REF][START_REF] Hoppe | Activation of a membrane-bound transcription factor by regulated ubiquitin/proteasome-dependent processing[END_REF][START_REF] Geng | Ubiquitin and proteasomes in transcription[END_REF][START_REF] Jackson | Regulation of DNA damage responses by ubiquitin and sumo[END_REF][START_REF] Teixeira | Ubiquitin ligases and cell cycle control[END_REF][START_REF] Hammond-Martel | Roles of ubiquitin signaling in transcription regulation[END_REF]. Proteolytic function of these specialized isopeptidases is tightly regulated, often through conformational activation mechanisms [START_REF] Khan | Molecular mechanisms for the conversion of zymogens to active proteolytic enzymes[END_REF]Yin et al., 2015;[START_REF] Li | Allosteric activation of ubiquitinspecific proteases by b-propeller proteins UAF1 and WDR20[END_REF]. Under normal conditions, DUBs are often kept inhibited until their function is essential to maintain cellular viability. In disease states, the regulatory mechanisms that keep DUBs in check no longer function properly, and have been reviewed extensively in recent articles (Clague et al., 2013;Heideker and Wertz, 2015;[START_REF] Jacq | Deubiquitylating enzymes and DNA damage response pathways[END_REF].

Of the �100 DUBs encoded by the human genome, ubiquitin-specific proteases (USPs) constitute the largest family, consisting of 58 members that share a common ''papain-like'' cysteine protease catalytic domain architecture. USP7, also known as Herpes virus-associated USP or HAUSP (Everett et al., 1997), is a USP-family DUB that acts on a wide range of cellular targets and plays a critical role in normal and disease biology [START_REF] Forand | Disruption of the phosphate transporter pit1 in hepatocytes improves glucose Structure 26, 72-84[END_REF][START_REF] Yim | Plk1-mediated stabilization of 53BP1 through USP7 regulates centrosome positioning to maintain bipolarity[END_REF][START_REF] Zhang | USP7 promotes cell proliferation through the stabilization of Ki-67 protein in non-small cell lung cancer cells[END_REF][START_REF] Tavana | HAUSP deubiquitinates and stabilizes n-myc in neuroblastoma[END_REF][START_REF] Yi | Stabilization of LSD1 by deubiquitinating enzyme USP7 promotes glioblastoma cell tumorigenesis and metastasis through suppression of the p53 signaling pathway[END_REF]. In particular, USP7 has emerged as a potential therapeutic target due to its regulation of p53-pathway components, including the oncogene MDM2 and tumor suppressors p53, FOXO4, and PTEN [START_REF] Reverdy | Discovery of specific inhibitors of human USP7/HAUSP deubiquitinating enzyme[END_REF]. USP7, like many DUBs, remains autoinhibited, and its deubiquitinating activity is regulated by a complex set of external and intramolecular factors (Sahtoe and Sixma, 2015). Indeed, USP7 is a multi-domain protein with accessory domains that tune the catalytic activity (Ma et al., 2010;Faesen et al., 2011;Holowaty et al., 2003). Specifically, USP7 has an N-terminal TRAF domain, the USP catalytic domain (USP7cd), five ubiquitin-like domains (UBLs 1-5), and a C-terminal segment that is poorly ordered in isolation. The TRAF domain and UBLs 1-3 help mediate target engagement, whereas the UBLs and C terminus modulate the catalytic competency of the USP7cd. Binding of the C-terminal segment to the USP7cd boosts the deubiquitinating activity of the catalytic domain, which has rather weak activity in the absence of the C-terminal tail (Ma et al., 2010;Faesen et al., 2011;[START_REF] Fernandez-Montalvan | Biochemical characterization of USP7 reveals post-translational modification sites and structural requirements for substrate processing and subcellular localization[END_REF][START_REF] Rouge | Molecular understanding of USP7 substrate recognition and C-terminal activation[END_REF].

Poor turnover by the isolated USP7cd has been attributed to the disrupted active site observed in the apo USP7cd crystal structure (Figure 1A) (Hu et al., 2002). By contrast, a ubiquitinconjugated USP7cd structure demonstrates the canonical cysteine protease active-site geometry (Figure 1A) (Hu et al., 2002). Thus, it has been postulated that ubiquitin binding ''realigns'' the USP7 active site for catalysis (Hu et al., 2002;Sahtoe and Sixma, 2015); however, any allosteric effect of ubiquitin binding on the active site still needs direct evidence for validation. An alternative explanation is that the USP7cd conformation observed in the ubiquitin-bound structure is a catalytically active but short-lived state visited by the isolated catalytic domain (Faesen et al., 2011). We sought to explore this conformational selection hypothesis and determine whether the conformational ensemble could be modulated to bias the active state over the competing inactive state and yield USP7cd variants with increased intrinsic activity. (B) Three distance metrics from a molecular dynamics trajectory show a transition from the inactive to active state starting from the inactive state with protonated catalytic H464 and deprotonated H294 and C223 (left). In the inactive state (PDB: 1NB8, gray cartoon), H294, likely protonated, participates in an electrostatic network involving W285, E298, and Y224, which is disrupted in the active state (PDB: 1NBF, green cartoon). (C) Crystal structures of USP2cd and USP7cd in the active and the inactive states. The densely packed phenylalanine cluster in USP2cd and corresponding residues in USP7cd are depicted in space-filling representation. In the active USP7cd, core packing by smaller hydrophobics (mostly leucines) is suboptimal, leaving a small, open groove, whereas in the inactive state a denser packing of these residues negates the groove.

Toward this aim, we employed a combination of rational design and directed sampling to identify residues within USP7cd that play a key role in the transition between inactive and active states. Using these positions for conformational remodeling, we designed three sets of USP7cd mutations with significantly improved intrinsic deubiquitinating activity in the absence of C-terminal tail activation. In fact, a subset of our designs mimics the native activation by the regulatory C-terminal segment, despite the fact that our design approach was blind to the location and binding mode of this regulatory element on USP7cd. Full kinetic analyses and X-ray crystallographic data confirm that the increases in activity are achieved through two main mechanisms: destabilization of electrostatic interactions that favor the inactive state and stabilization of the active state through improved packing of the USP7cd core.

RESULTS

Distal Electrostatics Affect Organization of the Active Site

To reveal the catalytic domain structural features that differ between the active and the inactive states, we quantitatively compared the catalytic domain crystal structures. The overall folds of the two states are remarkably similar with the Ca rootmean-square deviation (RMSD) difference of <1.0 A ˚(Table S1), but significant rearrangements occur in the active site (Figure 1A) (Hu et al., 2002). Rapid pKa calculations based on local geometry predict that two histidines, out of a total of 13, are differentially protonated in the active versus the inactive states (Table S2). One of the two histidines, H464, is the catalytic histidine, which was expected to have alternative protonation states due to the significant changes in the active-site geometry. The second histidine, H294, is located on a loop region distal to the active site called the switching loop (Figure 1B). In the inactive apo state, positively -charged H294 makes a p-cation interaction with W285 and a salt bridge with E298, forming an electrostatic network in the switching loop (Figure 1B). W285 also makes an aromatic carbon-oxygen hydrogen bond [START_REF] Horowitz | Carbon-oxygen hydrogen bonding in biological structure and function[END_REF] with Y224 that is located at the N terminus of the a1 helix (Ye et al., 2009), neighboring the catalytic cysteine C223. To accommodate this conformation of the switching loop, C223 adopts backbone f/c angles that lie outside the Ramachandran favored regions (Figure S1). Given the highly similar overall fold in the active and inactive states, we reasoned that the backbone of C223 is likely held in this unfavorable conformation by the strong electrostatic interactions within the switching loop described above and not by other structural features shared by both states. By contrast, neutral H294 in the active state cannot form a salt bridge, leading to the disruption of the electrostatic network; as a result, Y224 is no longer sequestered. In the absence of stabilizing electrostatics of these nearby residues, C223, being located at the N-terminal a1 helix, improves the unfavorable f/c angles by helical refolding, which brings it into the proximity of the catalytic histidine, leading to the formation of the active site.

To test the hypothesis that H294 deprotonation enables the formation of the active state, we set up parallel molecular dynamics simulations with several combinations of protonation states for H294 and the catalytic residues H464 and C223 (Table S3 and Figure S2). The transition from the inactive to the active state was only captured when H294 was neutral with a deprotonated C223 and charged H464. The transition from inactive to active state happens rapidly and can be observed in relatively short molecular dynamics simulations (100 ns), as illustrated by the decreased distance between catalytic residues C223 and H464 (see Movie S1). Concomitant with this transition, we observed destabilization of the H294mediated electrostatic interactions seen in the inactive state, quantified by the H294-E298 and H294-W285 distances (Figure 1B). These simulations clearly validated the coupling between the switching loop electrostatic network and the active-site geometry.

Identification of Suboptimal Core Packing in the Active State

To further probe the USP sequence-structure-activity relationship, we compared USP7cd with the catalytic domain of USP2 (USP2cd), a DUB catalytic domain with robust, high activity. USP2 and USP7 catalytic domains, with �24% sequence similarity, share a remarkably conserved backbone conformation with a Ca RMSD of �1.0 A ˚ [START_REF] Renatus | Structural basis of ubiquitin recognition by the deubiquitinating protease USP2[END_REF]. However, USP2cd has a phenylalanine (Phe) cluster formed by side chains from the switching loop and switching helix (Figure 1C) while the corresponding residues in USP7cd (L260, L279, L288, L299) are leucine residues with the exception of Y224 and V302. In the active state, smaller leucine side chains are suboptimally packed, leaving an open groove in the hydrophobic core. By contrast, the switching loop is in a closed conformation in the inactive state, resulting in a more densely packed leucine cluster (Figure 1C). Based on this comparative analysis, we hypothesized that the suboptimal packing in wild-type USP7cd core is a destabilizing factor for the active state, and bulkier hydrophobic side chains may improve the local packing and stabilize the active state.

Rational Design to Increase Deubiquitinating Activity

Having identified two structural features that appear to favor the inactive state of USP7cd-the electrostatic network that helps sequester the catalytic cysteine and the suboptimal packing of the core in the active state-we sought to rationally design USP7cd mutants that would shift the protein toward the active state. First, while the molecular dynamics simulations indicate that H294 deprotonation may aid in organizing the catalytic triad, the experimental validation of this hypothesis is challenging due to the difficulty of modifying the protonation state of a single histidine in a protein that has multiple histidine residues. Therefore, to biochemically probe the coupling between switching loop electrostatics and active-site geometry, we replaced H294 with a negatively charged glutamic acid to disrupt the E298-H294-W285-Y224 electrostatic network and push the conformational equilibrium toward the active-like conformational ensembles. Second, to test the hypothesis that the suboptimal packing in wild-type USP7cd core is a destabilizing factor for the active state and that bulkier hydrophobic side chains may improve the local packing and stabilize the active state, combinations of leucine-to-phenylalanine mutations were introduced into the hydrophobic core of USP7cd (Y224F/L260F/L279F/L288F/ L299F/V302F). smaller hydrophobic side chain. Across USP7 homologs, position 299 is primarily either leucine or phenylalanine (Figure S4). Rationalizing the replacement of the leucine with a smaller hy-drophobic residue is challenging; fortunately, the experimental structural studies shed light on this interesting computational design, as discussed later. Finally, V302K is a mutation located on the switching helix close to the electrostatic network residues E298 and W285. Having a lysine residue at this position may satisfy the local electrostatics in the absence of the protonated H294. All three RosettaDesign mutations that preferentially stabilized the active state (Y224F, L299A, and V302K) were cloned into the USP7cd to evaluate their deubiquitinase activity.

Designs Affect Intrinsic Activity, but Not Specificity, of USP7 For initial evaluation of our designed variants, we performed parallel small-scale expressions of a panel of USP7cd variants using a semi-automated in vitro translation and purification system. The deubiquitinating activity of this series of variants was assessed at a single substrate concentration using a quenched ubiquitin-AMC substrate where the fluorescent dye, 7-amino-4-methylcoumarin (AMC), is released from ubiquitin upon hydrolysis (Figure S5) [START_REF] Dang | Kinetic and mechanistic studies on the hydrolysis of ubiquitin C-terminal 7-amido-4-methylcoumarin by deubiquitinating enzymes[END_REF]. This preliminary screen showed an overall increase in the catalytic activity in all mutants relative to the wild-type USP7cd, with the H294E single-point mutation demonstrating the most dramatic enhancement in catalytic turnover. The core-repacking leucine-to-phenylalanine mutants displayed only minor increases in activity as compared with wild-type USP7cd. Moreover, the boost in activity for a quadruple repacking mutant, Y224F/L260F/L299F/V302F, was almost the same as that measured for the single Y224F mutant alone, suggesting that the tyrosine-to-phenylalanine mutation predominantly drives the modest effect. In light of these results, we narrowed the list of designs for detailed kinetic characterization down to four: H294E, Y224F, which was also predicted by directed sampling, and the two other Rosetta variants, L299A and V302K.

Steady-state kinetic analysis revealed that all of these variants but Y224F increase the catalytic efficiency relative to wild-type USP7cd. The electrostatics-altering design, H294E, is 4-fold catalytically more efficient than the wild-type USP7cd, with a 2.4-fold increase in k cat and a 1.7-fold decrease in K M (Figure 3 and Table S4). The impact of the Rosetta mutants is remarkably different. While the Y224F USP7cd variant shows activity roughly equivalent to that of wild-type USP7cd, L299A and V302K enhance USP7cd catalysis (k cat ) by 6-and 4-fold, respectively. Both L299A and V302K exhibit reduced substrate binding (K M ) by �2-fold, resulting in overall enhancements of USP7cd intrinsic catalytic efficiency (k cat /K M ) of 3-and 2-fold, respectively, for these two Rosetta mutants.

To assess whether the functional impact of our designs extends to USP7 that is already activated by intramolecular accessory elements, we cloned the single mutations into a construct containing the catalytic domain, the ubiquitin-like domains 4 and 5, and the C-terminal segment (USP7cd-45)-a construct previously reported to have the same activity on Ub-AMC as the full-length enzyme (Faesen et al., 2011). In this context, H294E still slightly improves catalytic efficiency by 1.4-fold relative to wild-type USP7cd-45 (Figure 3 and Table S4), mainly due to a 1.7-fold increase in k cat . Strikingly, the Rosetta variants L299A and V302K display the same catalytic efficiency in both the USP7cd and USP7cd-45 frameworks, suggesting that the also obtained crystals for apo versions of the V302K and H294E USP7cd constructs; however, the latter did not diffract below �15 A ˚. All other crystals diffracted to a resolution range of 1.4-2.0 A ˚and structures were solved by molecular replacement using the USP7cd from the wild-type Ub�USP7cd crystal structure (PDB: 1NBF/A) or the apo wild-type USP7cd crystal structure (PDB: 1NB8/A) as the search model. The resulting refined structures all possess the same canonical USP7cd fold, with a backbone RMSD of <1.0 A ˚; however, local structural rearrangements that appear to dictate the differential impact of each mutant are described below.

The Ub�L299A co-crystal structure (1.4 A ˚) has one complex in the asymmetric unit, which aligns well with the wild-type ubiquitin-conjugated structure (PDB: 1NBF/A), displaying 0.4 A ˚RMSD over 268 Ca atoms. Like the wild-type complex, the L299A active site is clearly formed, with the C223 sulfur only 3.8 A ˚apart from the delta nitrogen of H464. Interestingly, the switching loop exists in a different ''closed'' conformation that is distinct from both the active and inactive states of the wild-type USP7cd.

This conformation is intermediate between the wild-type active and inactive switching loop structures, as quantified by intermolecular Ca-Ca distances (Figure S6). Unexpectedly, the Ub�L299A crystal structure reveals that in the absence of the extra isopropyl group at position 299, residue F283 adopts a different rotamer conformation, allowing it to reach across the hydrophobic groove in the USP7cd core to pack against V302, buttressed by the aromatic residue, W285 (Figure 5). As a result, the L299A mutation improves upon the suboptimal packing in the wild-type active state by a mechanism that was not apparent from our initial modeling efforts.

The asymmetric unit of the 1.9-A ˚-resolution Ub�H294E cocrystal structure contains two ubiquitin-conjugated USP7cd molecules (chains A and B) that possess almost identical backbone structure (0.23 A ˚RMSD superposition over 267 Ca atoms). Similar to the L299A design, both copies of Ub�H294E display active-like catalytic triad geometry (Figure 5). Although the two molecules have slightly different crystal contacts around the switching loop region, the conformation of the 49, 76.56, 88.39 99.55, 99.55, 83.77 116.33, 43.54, 84.76 62.05, 80.47, 86.56 75.62, 68.28, 77 switching loop backbone is the same for the two and resembles that of the wild-type active structure (Table S5). The positions of key residues H294E, D289, and W285 are also the same in chains A and B (Figure S7). One notable difference between the two structures is that the F283 side chain is once again flipped out in chain B, reaching over to pack against residue V302, whereas chain A shows the same F283 rotamer as the wild-type inactive state. Thus, upon H294E mutation, F283 appears to sample alternative conformations without directly affecting the active switching loop conformation. As observed in the Ub�L299A structure, the orientation of F283 in Ub�H294E chain B improves the suboptimal packing by partially filling the groove in the hydrophobic core (Figure 5). In addition, E294 forms a salt bridge with residue R74 of the ubiquitin tail, whereas the wild-type H294 side chain has no specific interactions with ubiquitin. Finally, our crystallization of the V302K variant yielded several apo and ubiquitin-conjugated structures. The initial Ub�USP7cd crystal structure contains one complex in the asymmetric unit that surprisingly exhibits an inactive-like catalytic triad geometry despite being covalently conjugated to ubiquitin at the active-site cysteine. The switching loop conformation also resembles that of the wild-type inactive state. To our added surprise, a malonic acid molecule from the crystallization buffer is bound to the cleft where the C-terminal tail of ubiquitin typically sits, resulting in a ubiquitin binding mode that is slightly altered compared with the wild-type co-crystal structure. A follow-up co-crystal structure utilizing different crystallization conditions yielded a complex structure in which buffer components are not associated with the ubiquitin binding site; however, even in this structure, the USP7 active site is not properly formed. Apo V302K structures also resemble the inactive wild-type state; the USP7cd conformation is nearly identical with or without ubiquitin conjugation (Figure 5).

In all Ub�USP7cd complexes, including the wild-type, residue E298 typically interacts with Q49 and R72 of the ubiquitin tail. In the malonic acid-bound Ub�USP7cd structure of the V302K variant, the change in conformation of the ubiquitin tail replaces R72 with R74 in these interactions. In this structure, residue K302 does not interact with ubiquitin but instead extends into the USP7cd hydrophobic core where the non-branched aliphatic portion of the side chain allows interactions with, and repacking of, F283, W285, and L299; it makes no specific charge interactions. In the second co-crystal structure of V302K, K302 forms electrostatic interactions with ubiquitin residues Q49 and E51, the latter being unique to this structure (Figure 6). This additional contact contributes to a network of electrostatic interactions across the Ub�USP7cd interface, involving residues E51-K302-Q49-E298-R72. Such an electrostatic network, which involves several residues across a protein-protein interface, may increase the residence time of bound ubiquitin and contribute the apparent increase in the catalytic efficiency of V320K.

DISCUSSION

USP7cd displays poor catalytic activity in the absence of key regulatory elements, preferentially adopting a conformational state with incompetent active-site geometry (Ma et al., 2010;Faesen et al., 2011;[START_REF] Rouge | Molecular understanding of USP7 substrate recognition and C-terminal activation[END_REF]. Through computational modulation of conformational states, we have identified features within USP7cd that are important for its activation and have successfully (with and without a bound malonic acid) complex crystal structures. Wild-type USP7cd is colored green, designs teal, and ubiquitin yellow. The side chains that surround the hydrophobic grooves F283, L299, and V302 are represented by spheres. The ubiquitin residues that make charge interactions with USP7cd and key USP7cd residues, including the K302 side chain in V302K complex structures, are shown as sticks.

engineered mutants of USP7cd with improved intrinsic activity. Importantly, the relatively rapid calculations described here yielded successful designs in a directed and streamlined computational approach that did not require several rounds of random mutagenesis to find impactful substitutions. To highlight this efficiency, we chose to report all of the mutations that we designed and tested biochemically in this study-not merely a subset with improved activity. Full kinetic analyses and X-ray crystal structures confirm that our designs promote activation through two distinct mechanisms: disruption of electrostatic interactions in the distal ''switching loop'' that stabilize the inactive state and repacking of the USP7cd hydrophobic core that favors the active conformation.

Three of our designs, L299A, V302K, and H294E, cause significant local structural rearrangements affecting intrinsic activity. Comparative pKa predictions on wild-type active and inactive states pointed to the significance of a distal histidine, H294, protonation state in the active site organization. Molecular dynamics simulations sampling alternative protonation states at H294/ H464/C223 uncovered the atomistic details of this crosstalk; in the inactive apo state, positively charged H294 stabilizes an electrostatic network with W285, E298, and Y224. This network of strong electrostatic interactions balances the unfavorable f/c angles of C223. By contrast, neutral H294 in the active state cannot make charge interactions, leading to the disruption of the electrostatic network. As a result, the C223 unfavorable backbone angles are improved by helical refolding; hence, the active site is formed. In addition, based on the structural differences between USP7cd and USP2cd, local packing in the wild-type USP7cd is not ideal in the active state. Our designed mutations, L299A and to a certain extent H294E, stabilize an alternative sidechain rotamer for F283 within the catalytic domain that improves core packing in the USP7cd active state. Moreover, the switching loop adopts an intermediate ''closed'' conformation in L299A while V302K simply fills in the groove via the larger lysine side chain, with both serving to repack the core and thereby increase USP7cd intrinsic activity. These findings provide experimental evidence that conformational selection can affect USP7 activation. A more in-depth analysis of how conformational selection and induced fit mechanisms affect USP7 activation would greatly benefit from apo crystal structures of our designs. Unfortunately, none of the apo proteins for these mutants could be induced to crystallize despite considerable efforts to this end. Intriguingly, we found that the catalytic triad is not organized in ubiquitin-conjugated crystal structures of the V302K protein despite its elevated kinetic activity on both Ub-AMC and tetra-Ub substrates. The fact that ubiquitin is not able to induce an active conformation in this more catalytically efficient variant lends credence to the hypothesis that ubiquitin binding cannot be the sole mechanism of organizing the catalytic triad for enzymatic activity. Indeed, in the absence of ubiquitin, we observed increased reactivity of several USP7cd designs with a small-molecule active-site probe, suggesting that the designed mutations are sufficient to pre-organize the active site in the absence of ubiquitin binding. Mutations predicted to destabilize the inactive state (H294E and Y224F) were most reactive with the small-molecule inhibitor, with Y224F proving substantially more reactive than wild-type USP7cd despite showing no increase in activity with ubiquitin-based substrates. By contrast, one of the most active variants against ubiquitin chains substrates (L229A) displayed poor reactivity with the probe, suggesting that the ''intermediate'' conformation adopted by the switching loop is L299A is compatible with native substrates but not with the peptidomimetic inhibitor. Taken together, these observations of differential intrinsic activity with and without ubiquitin binding likely reflect a combination of induced fit and conformational selection at play in the USP7 activation mechanism.

Interestingly, detailed kinetic analyses of our designs suggested that the core-repacking mutations compete with native activation by the C-terminal tail of USP7. Visualization of USP7 C-terminal tail binding, provided by the recently published Ub�USP7cd�C-terminal tail ternary complex, reveals that our core-repacking strategy does indeed closely mimic the native activation mechanism observed in full-length USP7 [START_REF] Rouge | Molecular understanding of USP7 substrate recognition and C-terminal activation[END_REF]. Specifically, the C-terminal segment fills the hydrophobic groove created by the suboptimal core packing observed in the USP7cd active conformation, whereas our designs collapse this groove to improve packing of the isolated catalytic domain. The overall structures of the USP7 catalytic domain and ubiquitin are nearly identical between the wild-type Ub�USP7cd complex and the Ub�USP7cd�C-terminal tail ternary complex, with Ca RMSD of <0.4 A ˚. The USP7 C-terminal tail from the ternary complex can dock into the wild-type ubiquitin-conjugated USP7cd structure with no clashes (Figure S8). In particular, the hydrophobic residues I1098 and I1100 of C-terminal tail are essential to the native activation mechanism, supporting the role of local packing in the USP7cd core. These critical hydrophobic sites are occupied by F283 and W285 in the switching loop of the L299A design. While these side-chain changes improve local packing and boost the intrinsic activity, the aromatic side chains are not evolutionarily optimized for repacking the hydrophobic groove in the absence of accessory domains; hence, full-length USP7 activity levels are not achieved in the USP7cd designs. In addition, our core-repacking designs effectively compete with C-terminal tail binding in the context of multi-domain USP7 constructs (Figure 7); thus, no boost to activity is seen for these mutants in going from the USP7cd to USP7cd-45 constructs. By contrast, our design that destabilizes the inactive state via disruption of switching loop electrostatic network is compatible with the native activation mechanism and realizes a higher level of catalysis than wildtype in the context of the C-terminal tail.

It is curious that a small hydrophobic segment at the C-terminal tail is able to activate the catalytic domain through rather weak and non-specific van der Waals contacts without triggering major conformational changes in the ubiquitin-bound state captured in crystals. Our rapid design approach uncovers why the catalytic domain is so stable in the inactive state and provides the conformational basis for the intriguing activating ability of C-terminal segment. The suboptimal packing in the catalytic core destabilizes the active state and the switching loop electrostatic network further stabilizes the inactive state unless the C-terminal segment is bound to the core to stabilize the active conformation via hydrophobic interactions. In the absence of the C-terminal segment, our designs modulate these two structural features of the catalytic domain, allowing subtle yet functionally significant conformational changes within the catalytic domain and enabling the regulation of deubiquitinating activity of USP7. In the native setting, the C-terminal segment of full-length USP7 binds to this small groove, which we have identified as suboptimally packed, and stabilizes the activationcompetent conformational state of the catalytic domain. Moreover, an allosteric activator of USP7, GMP synthase, appears to rely on this intramolecular tail-groove interaction to exert its effect on USP7 activity from its distal binding site on UBLs 1-3 (Faesen et al., 2011). It would be interesting to investigate whether small molecules could also be designed to take advantage of this allosteric switch behavior to inhibit USP7 activity, as these molecules would have significant therapeutic potential.

Activation through subtle changes in conformational ensembles appears to be a broader theme in DUBs. Domains within DUBs or external cofactors can activate DUB catalytic domains through a variety of mechanisms; yet, often with only very subtle impacts on conformational states. Charge interactions between USP46 and WDR48, a common activator of a series of USPs, cause only limited conformational changes in ubiquitin co-crystal structures, but these unexpected interactions far from the active site remarkably enhance the catalytic efficiency (Yin et al., 2015). Another recent study reveals slightly altered conformations for the two loops of USP12 upon allosteric interactions with WDR48 [START_REF] Li | Allosteric activation of ubiquitinspecific proteases by b-propeller proteins UAF1 and WDR20[END_REF]. These small changes in conformational ensembles have profound impacts on function. Subtle fine-tuning of USP catalytic domain conformational space by intramolecular and external factors emerges as a common theme for regulating this class of enzymes to achieve specific cellular outcomes. Our selective modulation of co-existing conformational states in USP7cd yielded designs mimicking aspects of this theme in the absence of native regulation.

Precise activity level is crucial for USP7 and other DUBs following Goldilocks principles whereby too much or too little activity can be detrimental for the downstream biological effect [START_REF] Hao | USP7 acts as a molecular rheostat to promote wash-dependent endosomal protein recycling and is mutated in a human neurodevelopmental disorder[END_REF]. Our results show that engineering hydrophobic packing is an effective way to modulate protein dynamics in USP7cd to fine-tune the activity. Local packing has been re-ported as a stabilizing factor for certain conformational ensembles in other systems, e.g., hydrophobic sliding in viral proteases and hydrophobic regulatory spines in kinases [START_REF] Foulkes-Murzycki | Hydrophobic sliding: a possible mechanism for drug resistance in human immunodeficiency virus type 1 protease[END_REF][START_REF] Shaw | Kinases and pseudokinases: lessons from RAF[END_REF]. Typically, hydrophobic interactions are not considered as functionally significant due to their non-specific character. However, facile exchange of van der Waals contacts that facilitate motion within protein cores are necessary for function. Because DUB catalytic domains, despite the sequence variation, are structurally conserved, quantitative characterization of the intramolecular hydrophobic interactions across DUBs will be indispensable for understanding their impact on stabilizing certain conformational populations and possibly revealing mechanisms of activation. We anticipate that local packing will be critical, especially for allosteric regulatory mechanisms that yield only subtle conformational changes but cause functionally significant activation levels [START_REF] Li | Allosteric activation of ubiquitinspecific proteases by b-propeller proteins UAF1 and WDR20[END_REF].

An important lesson on modulating protein activity by design, relearned here from our efforts to repack the USP7cd hydrophobic core with phenylalanines inspired from USP2cd, is that protein folds are highly complex, interdependent entities, and structural epistasis dictates the apparent impact of site-directed mutagenesis. Swapping residues between homologs with differential functional features does not always yield a protein with intended intermediate properties [START_REF] Seeliger | C-Src binds to the cancer drug imatinib with an inactive Abl/c-Kit conformation and a distributed thermodynamic penalty[END_REF]. While swapping solvent-exposed residues can be effective in engineering molecular interactions across protein-protein interfaces [START_REF] Taherbhoy | BMI1-RING1B is an autoinhibited RING E3 ubiquitin ligase[END_REF], engineering the core of a protein to stabilize a certain conformation is more complicated (Zhang et al., 2013;[START_REF] Wilson | Kinase dynamics. Using ancient protein kinases to unravel a modern cancer drug's mechanism[END_REF]. Protein engineering methods, including directed evolution, typically require highly specialized human experts for effective execution. Conversely, continuous advancements in the accuracy, throughput, and usability of computational methods that account for sequence variation and protein dynamics [START_REF] Sastry | Protein and ligand preparation: parameters, protocols, and influence on virtual screening enrichments[END_REF][START_REF] Case | Amber 12[END_REF][START_REF] Smith | Backrub-like backbone simulation recapitulates natural protein conformational variability and improves mutant sidechain prediction[END_REF][START_REF] Leaver-Fay | Rosetta3: an object-oriented software suite for the simulation and design of macromolecules[END_REF] eliminate the dependence on deep expertise on any single highly specialized domain and render our approach to systematically modulate the competing conformational states broadly applicable.

Modulating conformational states for desired aspects of function has many potential applications. Interrogating the biology of biomedically important enzymes such as DUBs, where members of the family adopt fairly similar globular structures and nearly identical active sites with distinct functions in distinct pathways, has been challenging. Taking a rapid design approach, proteinbased reagents could be generated to create homogenized populations biased toward a desired function, which could then be utilized to probe unexplored biology. In addition, the ability to control and understand the subtleties of a drug target's conformational landscape would assist in drug discovery processes. Historically, well-documented conformational states of kinases have been tremendously helpful in designing therapeutics and interpreting the biological impacts of small-molecule inhibitors targeting distinct states of kinases. By contrast, DUBs, which are relatively newer drug targets, appear to have much more nuanced conformational landscapes. Through our rapid and efficient design approach, characterizing the subtleties of the conformational states that are relevant for a desired aspect of function and being able to lock the enzyme at a state of interest, will add value in efforts to understand and target functions of biomedically important proteins.

STAR+METHODS

Protein Purification

The USP7 variants carrying several mutations were generated with the QuikChange Lighting Multi Site-Directed Mutagenesis Kit (Agilent). QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent) was used to generate the single point mutations. An initial series of designs were expressed and purified using the ExiProgen TM protein synthesizer (Bioneer, Daejon, Korea) with EC-Tagfree Protein Synthesis Kit according to manufacturer's protocol. For all other analyses, plasmids encoding N-terminal His-tagged wildtype and mutants of USP7cd and USP7cd-45 were expressed in BL21 (DE3) Gold cells; cultures were grown in LB media supplemented with carbecillin until OD600nm reached 0.7 and were induced with 0.5mM IPTG at 16 � C overnight. Cells were harvested by centrifugation, and stored at -80 � C. Cells were lysed in the lysis buffer (25 mM Tris (pH 8.0), 150mM NaCl, 5% glycerol, 0.5mM tris(2-carboxyethyl)phosphine (TCEP)) with added benzonase, lysozyme, cOmplete, EDTA-free protease inhibitor tablets (Roche Life Sciences). Clarified lysate (homogenized, passed through a microfluidizer, spun down at 13,000 rpm for 40 minutes, filtered through a 0.2uM membrane) was loaded onto a 5 mL Ni-nitrilotriacetic acid (Ni-NTA) affinity column (Qiagen) pre-equilibrated with lysis buffer, washed with lysis buffer plus 45mM imidazole and eluted with lysis buffer plus 300mM imidazole. The six-histidine tags were removed using thrombin (Calbiochem, 1u / mg of fusion protein) and 4mM CaCl2 (for USP7cd) or TEV protease (for USP7cd-45) while dialyzing against the lysis buffer overnight at 4 � C. The cleaved material was loaded onto a fresh 3 mL Ni-NTA column prequilibrated with lysis buffer. Column was then washed with lysis buffer plus 45mM imidazole and finally eluted with lysis buffer plus 300mM imidazole. Fractions containing protein were concentrated down to 4 mL and loaded onto a Superdex 75 column (GE Healthcare) with 25 mM Tris pH 8.0, 150mM NaCl and 0.5mM TCEP. Peak fractions were pooled and concentrated.

Synthesis of Peptidomimetic Inhibitor

General Methods for synthesis of benzyl N-[(1S)-1-{[(3R)-1-cyanopyrrolidin-3-yl]carbamoyl}-3-phenylpropyl]carbamate (G'3093(SMDC-751644)). Unless otherwise noted, reactions were conducted under air atmosphere using commercially available reagents and solvents, which were used as received. Silica gel chromatography was performed using a Biotage Isolera Four flash purification system with Silicycle SiliaSep cartridges. 1 H NMR spectra were recorded on a Varian INOVA-400 400 MHz spectrometer. Chemical shifts are reported in d units (ppm) relative to residual solvent peak. Coupling constants (J) are reported in hertz (Hz). LC-MS analyses were carried out using a Waters 2795 separations module equipped with a Waters 2996 photodiode array detector, a Waters 2424 ELS detector, a Waters micromass ZQ single quadropole mass detector, and an XBridge C18 column (5 mm, 4.6 x 50 mm).

Step 1 Tert-butyl (3R)-3-[(2S)-2-{[(benzyloxy)carbonyl]amino}-4-phenylbutanamido]pyrrolidine-1-carboxylate. To a solution of Cbz-L-homophenylalanine (125 mg, 0.4 mmol) in dichloromethane (2 mL) were sequentially added HOBt monohydrate (61 mg, 0.4 mmol) and EDAC (77 mg, 0.4 mmol). Next was added a solution of tert-butyl (3R)-3-aminopyrrolidine-1-carboxylate (74 mg, 0.4 mmol) in dichloromethane (1 mL). The reaction was stirred at ambient temperature for 2 h, then partitioned between CH 2 Cl 2 and 5% aqueous citric acid. The layers were separated, and the organic phase was washed with NaHCO 3 (sat. aq.) and brine, dried over MgSO 4 , filtered, and concentrated. The crude concentrate was purified by flash silica gel chromatography (12 g Silicycle column), eluting with EtOAc in hexanes (20%-85%) to provide �175 mg (95%) of the title compound.

Step 2 Benzyl N-[(1S)-3-phenyl-1-{[(3R)-pyrrolidin-3-yl]carbamoyl}propyl]carbamate trifluoroacetic acid. To a solution of tert-butyl (3R)-3-[(2S)-2-{[(benzyloxy)carbonyl]amino}-4-phenylbutanamido]pyrrolidine-1-carboxylate (48 mg, 0.1 mmol) in dichloromethane (1.5 mL) were sequentially added four drops of triethylsilane, three drops of water, and trifluoroacetic acid (0.7 mL). The reaction was stirred at ambient temperature for 1.5 h then concentrated to �0.5 mL. The concentrate was triturated with 1:1 diethyl ether/ hexanes twice at 0 � C. The liquid was decanted, and the residue dried under vacuum. The crude product was used without further purification in the next reaction.

  Afin d'élucider les différents mécanismes régulateurs contrôlant son activité, nous avons tout d'abord validé l'importance de la région localisée au C-terminal d'USP7 dans l'activation de cette enzyme. Utilisant diverses méthodes (ingénierie des protéines, enzymologie et cristallographie), Lionel Rougé-« Study of the intramolecular activation mechanism of the deubiquitinase USP7 » -BioSPC -2019 Lionel Rougé-« Study of the intramolecular activation mechanism of the deubiquitinase USP7 » -BioSPC -2019 6 nous avons ainsi démontré que cette séquence permet de stabiliser la « switching loop » dans une conformation active lorsque l'ubiquitine est présente. Nous sommes également parvenus à obtenir deux autres structures (USP7CD-UBL123 et USP7CD-UBL45-CTP en complexe avec l'ubiquitine) suggérant un possible agencement des UBLs à proximité du domaine catalytique et nous permettant ainsi de proposer un modèle d'activation pour USP7. Ce modèle a été testé par électron microscopie et suggère que les échantillons analysés requièrent de l'optimisation. Notre étude révèle un mécanisme de régulation intramoléculaire d'USP7, à ce jour unique dans la famille des DUBs. Compte tenu du rôle majeur joué par USP7 dans de nombreux procédés cellulaires et par conséquent de son intérêt thérapeutique, ces résultats ont le potentiel d'aider au développement d'inhibiteurs spécifiques.

  Lionel Rougé-« Study of the intramolecular activation mechanism of the deubiquitinase USP7 » -BioSPC -2019 Lionel Rougé-« Study of the intramolecular activation mechanism of the deubiquitinase USP7 » -BioSPC -2019 7 Title: Study of the intramolecular activation mechanism of the deubiquitinase USP7

A

  . Introduction ....................................................................................................................................................... 1. The Ubiquitin -Proteasome System ............................................................................................................. a) The Proteostasis Network ......................................................................................................................... b) The Ubiquitin Code .................................................................................................................................. 2. Deubiquitinases and their function ................................................................................................................ a) Deubiquitinases role in the UPS ............................................................................................................... b) Specificity of DUBs ................................................................................................................................. c) DUBs and their regulation ........................................................................................................................ d) Involvement in diseases ............................................................................................................................ 3. The Ubiquitin Specific Protease 7 ................................................................................................................. a) USP7 is a key regulator of the p53/MDM2 axis ....................................................................................... b) Other functions of USP7 ........................................................................................................................... c) Structural features and regulation of USP7 .............................................................................................. d) USP7 cleavage specificity ........................................................................................................................ e)

Lionel

  Rougé-« Study of the intramolecular activation mechanism of the deubiquitinase USP7 » -BioSPC -2019 Lionel Rougé-« Study of the intramolecular activation mechanism of the deubiquitinase USP7 » -BioSPC -2019 15 of ubiquitin (Ub)-mediated protein degradation, and in 2016, to Yoshinori Ohsumi for his work on autophagy.

  Lionel Rougé-« Study of the intramolecular activation mechanism of the deubiquitinase USP7 » -BioSPC -2019 Lionel Rougé-« Study of the intramolecular activation mechanism of the deubiquitinase USP7 » -BioSPC -2019 16

Lionel 17 Figure 2 :

 172 Figure 2: Overview of ubiquitin biology in the UPS

Lionel

  Rougé-« Study of the intramolecular activation mechanism of the deubiquitinase USP7 » -BioSPC -2019 Lionel Rougé-« Study of the intramolecular activation mechanism of the deubiquitinase USP7 » -BioSPC -2019 19 2009

Figure 3 : 2 .

 32 Figure 3: Ubiquitin chains and their biological functions The ability of ubiquitin to form various chains determine different functional outcomes for protein substrates (adapted from Y. Ye & Rape, 2009, with permission).

Figure 4 :

 4 Figure 4: Schematic representation of cysteine protease catalytic mechanism Catalytic triad is typically composed of an aspartic acid, a histidine and a cysteine. Asp orients His which activates catalytic Cys thiol group leading to formation of a thioether covalent bond with substrate. This transition state is stabilized by movement of substrate oxyanion of intermediate into the oxyanion hole. After bond cleavage, product is released and acyl enzyme

Figure 5 :

 5 Figure 5: Domain diversity and organization of DUB family members Domain organization of members of the UCH, Josephin, OTU and JAMMs families (a) and of the USP family (b).MINDYs and ZUFSP are not represented. From Komander, Clague, & Urbé, 2009, with permission.

Lionel

  Rougé-« Study of the intramolecular activation mechanism of the deubiquitinase USP7 » -BioSPC -2019 Lionel Rougé-« Study of the intramolecular activation mechanism of the deubiquitinase USP7 » -BioSPC -2019 25

Figure 6 :

 6 Figure 6: Examples of linkage specificity of different DUBs and their associated E2 or E3 enzymes

Lionel

  Rougé-« Study of the intramolecular activation mechanism of the deubiquitinase USP7 » -BioSPC -2019 Lionel Rougé-« Study of the intramolecular activation mechanism of the deubiquitinase USP7 » -BioSPC -2019 26 2007

Figure 7 :

 7 Figure 7: Examples of intramolecular and external factors regulating DUB activities (from Sahtoe & Sixma, 2015, with permission) a) Phosphorylation of DUBA at Ser177 has been shown to be necessary for enzyme activity. Phosphorylation helps coordinating a number of interactions between the DUB and C terminal tail of ubiquitin (colored in yellow) promoting substrate recognition and stabilizing active site b) WD-repeat protein WDR48 acts as a positive modulator of USP1, USP12 and USP46. The crystal structure of the ternary WDR48: USP46: ubiquitin complex reveals that the scaffold protein interacts with both the DUB and ubiquitin respectively through a small surface of its bpropeller and a SUMO-like domain encircling the exposed surface of ubiquitin. c) In apo form, UCH-L1 catalytic triad can be found misaligned with nucleophilic cysteine Cys90 7.7Å away from His161 (represented in white sticks). However, ubiquitin binding induces a series of conformational changes leading to the flipping of His161, now positioned in close proximity of the catalytic cysteine (3.9Å) consistent with a catalytically competent active site.

Lionel

  Rougé-« Study of the intramolecular activation mechanism of the deubiquitinase USP7 » -BioSPC -2019 Lionel Rougé-« Study of the intramolecular activation mechanism of the deubiquitinase USP7 » -BioSPC -2019

Figure 8 :

 8 Figure 8: Role of USP30 in mitophagy One aspect of mitochondria quality control is continuous intermixing of mitochondria. Damaged mitochondria can acquire damage components by fusing with healthy mitochondria. If mitochondria are damaged beyond repair, it can be completely eliminated through degradation in lysosomes, process known as mitophagy. This process starts by ubiquitination of damaged mitochondria by the ligase Parkin after its activation by the kinase PINK1. Loss of PARKIN/PINK1 function prevents proper sequestration of these toxic mitochondria leading to a contamination of the healthy pool. USP30 antagonizes the action of PARKIN by removing ubiquitin chains from damaged mitochondria making it a good target for the development of therapeutics in the development of Parkinson's and other disease where mitochondria homeostasis plays a major role.

Figure 9 :

 9 Figure 9: USP7 role in p53 homeostasis For details, please refer to text.

Lionel

  Rougé-« Study of the intramolecular activation mechanism of the deubiquitinase USP7 » -BioSPC -2019 Lionel Rougé-« Study of the intramolecular activation mechanism of the deubiquitinase USP7 » -BioSPC -2019

Lionel

  Rougé-« Study of the intramolecular activation mechanism of the deubiquitinase USP7 » -BioSPC -2019 Lionel Rougé-« Study of the intramolecular activation mechanism of the deubiquitinase USP7 » -BioSPC -2019 34 solved over the years (except for the C terminal region) but no full-length structure or model were available thus preventing proper understanding of USP7 activation upon substrate binding.

Figure 10 :

 10 Figure 10: Schematic representation of USP7 domain organization

Figure 11 :

 11 Figure 11: Binding mode of USP7 TRAF-like to a canonical Pro-Ala-X-X-Ser sequence a) Crystal structure of the TRAF-like domain (shown in cartoon and colored in cyan) in complex with p53 peptide (Pro359-Ser363 shown in sticks). b) Overlay of all co-structures of TRAF domain (shown in surface) bound to different peptides (shown as sticks)

Figure 12 :

 12 Figure 12: USP7 catalytic domain in both apo and bound conformations (based of PDB 1NB8 and 1NBF, please refer to text for legend).

Lionel

  Rougé-« Study of the intramolecular activation mechanism of the deubiquitinase USP7 » -BioSPC -2019 Lionel Rougé-« Study of the intramolecular activation mechanism of the deubiquitinase USP7 » -BioSPC -2019 37 protein-protein modules function. In 2011, Faesen et al., solved the first structure of these five

Figure 13 :

 13 Figure 13: Structural organization of UBL domains in presence or absence of substrates a) Overlay of X-ray structures of UBL12345 apo (2YLM, domains colored based on legend from Figure 10) and bound to DNMT1 (4YOC, UBLs colored in gray, DNMT1 represented in surface). UBL1-2 superimpose pretty well (RMSD of 0.408 Å for 188 Cα atoms) and diverge starting at UBL3. C-terminal residues are 40 Å away from each other. b) Alignment of two structures of UBL123 bound to ICP0 [Gly618-Arg626] peptide from two different space groups solved byPfoh et al. 4WPI (also called "extended" conformation) is colored based on legend from Figure10and the ICP0 peptide is represented in sticks(colored in

  BL21(DE3) Gold competent cells (Stratagene) and protein expression was induced by the addition of 0.5 mM IPTG. Cells were harvested by centrifugation and resuspended in 25 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.5 mM TCEP (Buffer A), lysed using a microfluidizer and clarified by centrifugation (15,000 x g for 45 min). Supernatant was loaded onto a 5 mL GST sepharose column (GE Healthcare) pre-equilibrated with Buffer A. The column was washed with 50 mL of Buffer A and eluted with 30 mL of Buffer A containing 10 mM reduced glutathione (Sigma). GST tag was removed using thrombin (Calbiochem). Cleaved material was loaded onto a HiLoad 16/60 Superdex 75 prep grade column (GE Healthcare) pre-equilibrated with Buffer A. The peak fractions were pooled and protein concentration was determined by absorbance at 280 nm.

Figure 14 :

 14 Figure 14: Purification of USP7CD-UBL123

  filtration using a HiLoad 16/60 Superdex 75 column (GE Healthcare) pre-equilibrated with 25 mM Tris-HCl pH 8.0, 150 mM NaCl and 0.5 mM TCEP. The peak fractions were pooled and protein concentration was determined by absorbance at 280 nm.

Figure 15 :

 15 Figure 15: Purification summary of USP7CD-CTP

Figure 16 :

 16 Figure 16: Purification summary of USP7 [Lys208-Asn1102]

(intermediate 3 )

 3 . This intermediate (3 mL at 3 mg/mL) was subsequently mixed with 450 mg of bromoethylamine (MP Biomedicals) and 600 μL of 2 N NaOH. The reaction was incubated at room temperature for 30 minutes and quenched by loading it onto a HiLoad 16/60 Superdex 75 column (GE Healthcare) pre-equilibrated with 25 mM Tris-HCl pH 7.5 and 150 mM NaCl.

Figure 17 :

 17 Figure 17: Schematic representation of preparation of Ub-BEA using intein chemistry (fromBorodovsky et al., 2002, with permission).

Figure 18 :

 18 Figure 18: Example of purification and LC-MS analysis of USP7 covalently bound to Ub-BEA a) Purification of USP7CD-CTP + ubiquitin-BEA complex over MonoQ column b) Top panel shows LC-MS of purified apo USP7 [Lys208-Asn1102]. Expected molecular weight is 104808 Da. Observed molecular weight is + 3 Da which is within experimental errors of the instrument. Bottom panel shows LC-MS of USP7-ubiquitin covalent complex; USP7 molecular weight shifted by 8 536 Da, characteristic of Ub-BEA covalent binding.

Figure 19 :

 19 Figure 19: Purification summary of DNMT1 [Arg600-Ala1600] a) Three step purification of DNMT1: 1) Affinity purification (Ni-NTA), 2) tag removal followed by reverse Ni-NTA purification (removal of uncleaved material and HisTag-TEV protease) and finally 3) size exclusion chromatography (S200 16/60). b) LC-MS of purified DNMT1 sample after tag removal. Expected molecular weight is 113787 Da. Observed molecular weight is + 3 Da which is within experimental errors of the instrument.

Figure 20 :

 20 Figure 20: Purification of USP7 [Lys208-Asn1102] bound to Ub + DNMT1 complex Purified DNMT1 and USP7 [Lys208-Asn1102] + Ub were independently loaded onto the Superose 6 (respective UV traces at 280nm are shown in red and pink). Elution profile for USP7 [Lys208-Asn1102] bound to Ub + DNMT1 complex is shown in blue. Complex with estimated molecular weight of 230kDa (assuming 1:1 stoichiometry) elutes earlier compared to independent proteins. Small shoulder eluting later represents excess DNMT1.

Figure 21 :

 21 Figure 21: Principle of Multi-angle static light scattering (MALS) and data analysis of USP7CD-UBL45-CTP a) MALS requires precise alignment of the beam direction, laser polarization and detector position in order to provide accurate measurements (from wyatt.com). b) 100μL of a solution of 2 mg/mL of purified USP7CD-UBL45-CTP was injected. Data analysis indicates sample behaves as a 72 kDa (estimated molecular weight: 75 kDa).

Figure 22 :

 22 Figure 22: Example of kinetic measurement of Ub-AMC hydrolysis and Km determination a) Schematic representation of Ub-AMC enzymatic assay b) Measurement of AMC release (relative fluorescence) in function of time. Serial dilutions of Ub-AMC are visualized. Error bars represent standard deviation from three technical replicates in each curve.

  52

Figure 23 :

 23 Figure 23: Crystallization phase diagram and dehydration methods a) Phase diagram of a protein crystallization experiment(Pichlo, Montada, Schacherl, & Baumann, 2016, with permission).

  Lionel Rougé-« Study of the intramolecular activation mechanism of the deubiquitinase USP7 » -BioSPC -2019 Lionel Rougé-« Study of the intramolecular activation mechanism of the deubiquitinase USP7 » -BioSPC -2019 53 Crystals of the USP7CD-CTP-ubiquitin complex were obtained at 19 ºC using the hangingdrop vapor-diffusion method by mixing equal volumes of protein at 10 mg/mL with well solution containing 0.05 M calcium chloride, 0.1 M 2-(N-morpholino) ethanesulfonic acid (pH 6.0) and 45% PEG 200. Crystals of the USP7CD-UBL45-CTP-ubiquitin complex were obtained at 4 ºC by the hanging-drop vapor diffusion method by mixing equal volumes of protein at 10 mg/mL with well solution containing 0.2 M K chloride, 0.05 M HEPES pH 7.5 and 35% (v/v) pentaerythritol propoxylate. Diffraction images for both crystals were collected at the Advanced Photon Source beamline ID22. Finally, crystals of USP7CD-UBL123 were obtained at 19 °C by the hanging-drop vapor diffusion method by mixing equal volumes of USP7CD-UBL123 at 15 mg/mL with a well solution containing 0.2 M tri-potassium citrate pH 8.3 and 20% PEG 3350. Crystals typically appeared after a couple of days and initial diffraction was poor. Diffraction images were collected at the Diamond Light Source beamline I02.

Figure 24 :

 24 Figure 24: Crystallization of different USP7 constructs in presence or absence of ubiquitin a) Crystals of the USP7CD-CTP ubiquitin complex b) Crystals of the USP7CD-UBL45-CTP ubiquitin complex c) Crystals of the USP7CD-UBL123 after overnight dehydration in 50% PEG 3350 d) In house diffraction pattern for dehydrated crystals of USP7CD-UBL123. Reflections can be seen out 4 Å.

Figure 26 :

 26 Figure 26: Overview of negative stain and cryo-EM sample preparation ( fromOrlova & Saibil, 2011, with permission) a) For negative stain sample preparation, 4 uL of a solution at 0.01 mg/mL is placed onto a CF2/1 300 grid from Electron Microscopy Sciences (EMS) previously cleaned using a GloQube glow discharge (Quorum Technologies) system. After 30 seconds incubation, sample is blotted with Whatman paper, dipped three times into 30uL of distilled water and stained twice into 30uL of a filtered solution of 2% uranyl acetate. At the last step, grid is incubated with the stain for 30 seconds before final blotting and air dried. b) For cryo EM sample preparation, hydration of macromolecules is necessary to maintain their structural integrity. Rapid freezing into liquid ethane(-182 ºC) prevents sample dehydration and reduces effects of electron beam damages. This procedure is performed using a VitroBot (ThermoFischer Scientific) instrument.

Figure 27 :

 27 Figure 27: Basics of electron microscopy a) Schematic representation of an electron microscope (from Orlova & Saibil, 2011, with permission). b) Interaction of the electron beam with the sample (from "Part 3: Amplitude and phase contrast 2016: G Jensen, youtube.com)

Figure 28 :

 28 Figure 28: Representation of EM workflow (from "Fundamentals of cryo-EM", with permission from Claudio Ciferri)

Figure 29 :

 29 Figure 29: Description of the constrast transfer function a) TEM diagram with the contrast transfer function (Wikipedia, 2018) b) Representation of the CTF diagram (fromJiang & Chiu, 2001, with permission)

Figure 30 :

 30 Figure 30: Overview of the iterative process of 3D reconstruction (from Orlova et al., 2011 with permission).

Figure 31 :

 31 Figure 31: Cryo-EM and data analysis at Genentech a) Description of the two microscopes operational at Genentech b) cisTEM is a complete image processing pipeline for single-particle cryo-EM based on the following programs: Unblur and Summovie for movie alignment[START_REF] Grant | Measuring the optimal exposure for single particle cryo-EM using a 2.6 Å reconstruction of rotavirus VP6[END_REF]

  Figure33).

Figure 33 :

 33 Figure 33: Plot of percent initial velocity relative to USP7CD-CTP for Ub-AMC cleavage using a C-terminal activation peptide titration series. The mutant peptides Ile1098A and Ile100A are included as negative controls.

Figure 34 :

 34 Figure 34: Overview of the USP7CD-CTP structure a) 2Fo -Fc electron density for the C-terminal peptide contoured at 1 sigma (electron density is colored in blue, modeled C-terminal peptide in red). The catalytic domain is represented as a cartoon and colored according to Figure 10; ubiquitin is shown in surface representation and colored in pale cyan. b) Close up of C-terminal peptide binding in the activation cleft. Hydrogen bond interactions involving the C-terminal peptide are shown as yellow dashes.

Lionel

  Rougé-« Study of the intramolecular activation mechanism of the deubiquitinase USP7 » -BioSPC -2019 Lionel Rougé-« Study of the intramolecular activation mechanism of the deubiquitinase USP7 » -BioSPC -2019 74 Large crystals of USP7CD-UBL123 were obtained within 48 hours; unfortunately, these crystals failed to diffract to high resolution. Several techniques were used to alter crystal packing, reduce solvent content and improve diffraction:

Figure 35 :

 35 Figure 35: Structural representation of USP7 CD-UBL123 a) Residues 211 to 881 are represented in cartoon. Domains are colored according to Figure 10. Cys223 is depicted as a yellow sphere and switching loop is colored in cyan. Disordered loops in 1NB8 structure are shown in red. "Compact" structure of UBL123 reported by Pfoh et al., (PDB code 4WPH, shown in maroon) was overlaid to this structure and is shown in box. b) Symmetry related molecule (shown in surface, colored based on Figure 10) makes extensive interactions with USP7 CD-UBL123. Most of these interactions are found between the catalytic and UBL12 domains. 90 degrees rotation (right figure) shows location of binding groove where ICP0 peptide has been reported to bind (shown in orange sticks).

Lionel

  Rougé-« Study of the intramolecular activation mechanism of the deubiquitinase USP7 » -BioSPC -2019 Lionel Rougé-« Study of the intramolecular activation mechanism of the deubiquitinase USP7 » -BioSPC -2019 78 catalytic and UBL45 domains, and this created some ambiguity in the connectivity of the different domains.

Figure 36 :

 36 Figure 36: Structural representation of USP7 CD-UBL45-CTP a) Overview of "dimer of dimer" organization in the crystal packing; monomer is represented in cartoon and colored based on Figure 10. Ubiquitin is colored in light cyan and switching loop in teal. NCS dimer is depicted in ribbon. Symmetry-related dimer is represented in surface and colored based on Figure 10. b) Only one dimer is visualized with one predicted monomer shown in cartoon and the other one in surface. Linker is not visible but close proximity between C-terminus of catalytic domain and N-terminus of UBL4 suggests catalytic domain is stabilized by C-terminal peptide from NCS dimer.

Lionel

  Rougé-« Study of the intramolecular activation mechanism of the deubiquitinase USP7 » -BioSPC -2019 Lionel Rougé-« Study of the intramolecular activation mechanism of the deubiquitinase USP7 » -BioSPC -2019 80 a) Molecular model of full length USP7

Figure 37 :

 37 Figure 37: Model building of full length USP7 a) Our structure of USP7 CD-UBL123 was superimposed with structure of TRAF-CD and either USP7 UBL12345 (2YLM) or USP7 UBL12345 complexed with DNMT1 (4YOC). Distances between Asn1084 (last visible residues of UBL5) from these last two models and catalytic Cys233 are shown in yellow. Domains are colored based on Figure 10 except for PDB 2YLM which is colored in teal. This model suggests that when USP7 is not bound to substrate (2YLM), C terminal peptide is too far to bind activation cleft hence maintaining catalytic domain in an inactive conformation.

Figure 38 :

 38 Figure 38: Model of full length USP7 bound to DNMT1 DNMT1 and ubiquitin (both shown in surface and colored in wheat in light teal respectively) have been added to Figure 37b "active" model. Lysine residues from DNMT1 have been colored in blue.

Figure 39 :

 39 Figure 39: Negative stain micrographs and 2D classes of USP7 [Lys208-Asn1102] bound to ubiquitin a) Examples of negative stain micrographs of USP7 [Lys208-Asn1102] bound to ubiquitin (left panel), DNMT1 (middle) and the resulting complex (right panel). Note that DNMT1 was used as a positive control since multiple crystal structures were previously solved (including apo protein, PDB code 4WXX [Ser351-Trp1600], Z.-M.Zhang, Liu, et al., 2015) suggesting that recombinant

  Lionel Rougé-« Study of the intramolecular activation mechanism of the deubiquitinase USP7 » -BioSPC -2019 Lionel Rougé-« Study of the intramolecular activation mechanism of the deubiquitinase USP7 » -BioSPC -2019 85 After several rounds of classification, a few 2D classes seemed to suggest the presence of a boomerang shaped object, reminiscent of the USP7CD-UBL123 structure and of the full length

Figure 41 :

 41 Figure 41: Study of the hydrophobic core packing of USP7 a) Sequence logo representation for the sequence variation in the catalytic hydrophobic core

Figure 43 :

 43 Figure 43: Binding mode of hydroxy-piperidine core series a) Chemical structures and IC50 of compounds used for structural studies. Core hydroxypiperidine is shown using brackets. b) Overlay of apo USP7 catalytic domain with four structures in complex with inhibitors. Structures are colored according to panel a). Apo USP7 in shown in gray. c) Overlay of PDB 5NGE and apo USP7. C terminal tail of ubiquitin is show in the cyan. Upon compound binding, Phe409 is pushed away side chain adopts a different rotamer opening a hydrophobic pocket that is occupied by residue Leu73 in ubiquitin. Side chains for Phe409 and catalytic triad residues are shown in sticks.

LionelFigure S4 :Figure S5 :Figure S6 :Figure S8 :Figure S9 :

 S4S5S6S8S9 Figure S1: Structural organization of the different di-ubiquitin linkages Linear (PDB code 2W9N), Lys6 (PDB code 2XK5), Lys11 (PDB code 3NOB), Lys27 (PDB code 5UJN), Lys29 (PDB code 24S22), Lys33 (PDB code 24XYZ), Lys48 (PDB code 5EMZ), and Lys63 (PDB code 3H7P) di-ubiquitin chains are represented in cartoon with the proximal ubiquitin colored in cyan and the distal in wheat. Lysines side chains forming isopeptide bonds with Cterminal residue of distal ubiquitin is shown in red. Top four linkages corresponds to so-called "compact" chains whereas bottom ones more elongated. All structures were generated by X-ray crystallography except for Lys27 di-ubiquitin (solved by NMR).

Figure 1 .

 1 Figure 1. C-Terminal Peptide of USP7 Restores Full Ubiquitin Cleavage Activity to the Catalytic Domain of USP7 and Regulates Enzyme Activity

(

  A) Schematic of the USP7 domain structure and boundaries. (B) Sequence alignment of the C-terminal 19 residues of USP7 orthologs. (C) Enzyme kinetics for USP7CD-CTP constructs of varying linker lengths. See also Table

Figure 2 .

 2 Figure 2. Structure of USP7CD-UBL45 Covalently Bound to Ubiquitin (A) Cartoon and surface representation of the complex between USP7CD-UBL45 covalently bound to ubiquitin showing trans activation of the C-terminal peptide from an NCS-related molecule. One chain is represented as a light blue molecular surface and the other chain as a violet ribbon; ubiquitin is wheat colored. The catalytic cysteine is colored green and the side chains of Ile1100 and Ile1098 from one protomer are shown as spheres. Only one dimer from the dimer of dimers present in the asymmetric unit is shown. (B) Close up of C-terminal peptide binding in the activation cleft. The catalytic domain is represented as a cartoon colored dark green. Hydrogen bond interactions involving the C-terminal peptide are shown as black dashes. See also Figure S2 for details of the C-terminal peptide interaction in the USP7CD-CTP structure. (C) 2jF o j jF c j electron density for the C-terminal peptide contoured at 1 s.

Figure 3 .

 3 Figure 3. Structure of the USP7CD-UBL123 (A) Cartoon diagram for USP7CD-123 with the catalytic domain colored orange and UBL1, UBL2, and UBL3 colored cyan, slate, and pink respectively. (B) Close up of the catalytic triad showing the residues are in the inactive state. (C) 2jF o j jF c j electron density for the C-terminal a10 helix contoured at 1 s.

Figure 4 .

 4 Figure 4. A Model for Full-Length USP7 in the Inactive Conformation and the Proposed Model for Full-Length USP7 in the Active Conformation (A) Schematic of the structures and their domain boundaries that were used to generate the proposed model for full-length USP7. (B) Structure of the UBL12345 domain (PDB: 4YOC) with UBL123 colored according to (A). (C) Model of USP7CD-UBL12345 generated by the superposition of the UBL123 domain of PDB: 4YOC on to the UBL123 domains of the USP7CD-UBL123 structure. (D) Proposed mechanism of cis activation of full-length USP7 by the C-terminal region. See also Figure S3A for a molecular model of full-length USP7 in complex with DNMT1 and Figure S3C for a molecular model of full-length USP7 consistent with trans activation by the C-terminal peptide. (E) Schematic model depicting activation of the catalytic domain by the C-terminal peptide in the presence of ubiquitin.

Figure 5 .

 5 Figure 5. MDM2 Binding by Full-Length USP7 (A) SPR sensorgrams showing binding to immobilized full-length MDM2 by (A) USP7-full length or (B) NTD-CD. (C) Superposition of sensorgrams for MDM2 binding by USP7-full length (red) USP7CD-UBL12345 (blue), negative protein control (green). (D) Superposition of sensorgrams for MDM2 binding by USP7-full length (red) and USP7DCTP (blue) constructs. Residuals are shown in insets for (A) and (B). (E) Binding constants for USP7, MDM2 interactions. MDM2 was immobilized at a target surface density of 1,000 RU. Analyte concentrations are 1,000 to 26 nM in 1.5-fold dilutions for USP7-FL and USP7DCTP or 2000-336 nM for USP7CD-UBL12345. For experiments in (C) and (D), USP7 was applied at a fixed concentration of 500 nM, and sensorgrams for association and dissociation phases are compared. Data shown are representative of multiple experiments.

Figure 1 |

 1 Figure 1 | Identification and characterization of USP7 inhibitors. a, Schematic of USP7regulated signalling. b, USP7 inhibitor hitto lead selection cascade. See text for more details. HTS, highthroughput screening; IF, immunofluorescence; MSD, mesoscale discovery; DLS, dynamic light scattering. c, Immunoblot analysis of MDM2 turnover on USP7 inhibition, with actin loading controls. See also Extended Data Fig. 3b. Actinnormalized MDM2 levels are plotted from three biological replicates. DMSO, dimethylsulfoxide; IP, immunoprecipitation; WB, western blot. d, Analysis of endogenous MDM2 polyubiquitinated with K48linked chains. Denatured lysates from cells, treated with the indicated compounds, were immunoprecipitated with K48polyubiquitin linkagespecific antibody, and immunocomplexes or cell lysates were immunoblotted as indicated. e, Immunoblot analysis of p53pathway proteins in isogenic cell lines treated with USP7 inhibitors or inactive controls. d, e, Representative of three experimental replicates. Gel source data: Supplementary Fig.1.

0 InhibitionFigure 2 |Figure 3 |

 023 Figure 2 | Selectivity of USP7 inhibitors and synergy with PIM kinase inhibition. a, Activity heatmap of recombinant deubiquitinases (DUB) incubated with USP7 inhibitors, representative of experimental duplicates. Diubiquitin cleavage activity was detected by matrixassisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry. b, Volcano plots of endogenous deubiquitinase activity changes in HEK293T cells. Data from experimental triplicates (left) or quadruplicates (right) are plotted versus Benjamini-Hochberg false discovery rate adjusted P values. Numbers indicate detected deubiquitinases relative to the 20% false discovery rate line. c, Combination treatments with PIM inhibitor GDC0570 and GNE6776 in EOL1 cells. Left: inhibition scores: 0, no effect; 100, no viable cells. Right: Bliss analysis of observed versus predicted viability. Positive values: greater than predicted viability decrease. Synergy score = 4.11. d, Immunoblot analysis of EOL1 wholecell lysates treated as indicated. Asterisks * and ** indicate cleaved and uncleaved PARP, respectively. e, Analysis of endogenous PIM2 polyubiquitinated with K48linked chains, as in Fig. 1d. d, e, Representative of three experimental replicates. Gel source data: Supplementary Fig. 1.

  shift (p.p.m.)1 H chemical shift (p.p.m.)

Figure 4 |

 4 Figure4| USP7 preferentially binds and cleaves ubiquitin moieties with free K48 side chains. a, Top row: schematics of distal (Dist.)15 NThr or proximal (Prox.)15 NLeulabelled diubiquitins (see also Extended Data Fig.10b, c). Middle/bottom rows: two dimensional bandselective optimized flipangle short transient (SOFAST) spectra overlays and onedimensional traces of labelled diubiquitin. Left/middle:15 NThr or15 NLeu peaks from K48linked diubiquitin without (green) and with (orange) USP7. Right/middle:15 NThr or15 NLeu peaks from K63linked diubiquitin without (purple) and with (orange) USP7. Bottom row:15 NThr or15 NLeu peaks from K48linked diubiquitin without (green) and with (orange) indicated USP7 mutants. b, Time course of TAMRA/peptideconjugated tetraubiquitin cleavage by fulllength USP7 (top); corresponding densitometry plots (bottom). a, b, Representative of four experimental replicates. Gel source data: Supplementary Fig. 1.

  Figure4| USP7 preferentially binds and cleaves ubiquitin moieties with free K48 side chains. a, Top row: schematics of distal (Dist.)15 NThr or proximal (Prox.)15 NLeulabelled diubiquitins (see also Extended Data Fig.10b, c). Middle/bottom rows: two dimensional bandselective optimized flipangle short transient (SOFAST) spectra overlays and onedimensional traces of labelled diubiquitin. Left/middle:15 NThr or15 NLeu peaks from K48linked diubiquitin without (green) and with (orange) USP7. Right/middle:15 NThr or15 NLeu peaks from K63linked diubiquitin without (purple) and with (orange) USP7. Bottom row:15 NThr or15 NLeu peaks from K48linked diubiquitin without (green) and with (orange) indicated USP7 mutants. b, Time course of TAMRA/peptideconjugated tetraubiquitin cleavage by fulllength USP7 (top); corresponding densitometry plots (bottom). a, b, Representative of four experimental replicates. Gel source data: Supplementary Fig. 1.

received 8

 8 February; accepted 16 August 2017. Published online 18 October 2017.

1 H

 1 NMR (400 MHz, CDCl 3 ) δ 8.06 (d, J = 5.1 Hz, 1H),7.21-7.11 (m, 2H), 7.06-6.97 (m, 2H), 6.64 (d, J = 5.4 Hz, 1H), 4.48 (s, 2H), 3.86 (s, 3H), 2.31 (q, J = 7.6 Hz, 2H),1.11-0.88 (m, 3H). LC-MS (ESI m/z): 264.1 (M + H + ).

1 H

 1 NMR (400 MHz, DMSOd 6 ) δ 8.01 (d, J = 8.7 Hz, 1H), 7.82 (d, J = 1.7 Hz, 1H), 7.62-7.59 (m, 2H), 6.67 (s, 1H), 4.90-4.72 (m, 2H), 3.79 (s, 3H), 3.73 (s, 3H), 2.98-2.90 (m, 4H), 1.85 (m, 4H).13 C NMR (150 MHz, DMSOd 6 ) δ21.8, 21.8,

  1 HNMR (400 MHz, CDCl 3 ) δ 8.03 (s, 1H), 3.85-3.77 (m, 1H), 3.77 (s, 3H), 3.65-3.55 (m, 1H), 3.45-3.15 (m, 4H), 2.85-2.70 (m, 1H), 2.25-2.10 (m, 2H), 2.05-1.85 (m, 2H). LC-MS (ESI m/z): 282.1 [M + H + ].

  1 HNMR (400 MHz, CDCl 3 ) δ 8.03 (s, 1H), 4.60-4.40 (m, 1H), 3.77 (s, 3H), 3.45-3.10 (m, 5H), 2.35-1.90 (m, 3H), 1.80-1.65 (m, 1H), 1.60 (br s, 2H). LC-MS (ESI m/z): 258.3 [M + H + ].

  1 HNMR (400 MHz, CDCl 3 ) δ 10.34 (s, 1H), 8.75 (s, 1H), 7.89 (s, 1H), 7.40-7.25 (m, 1H), 7.15-7.00 (m, 2H), 4.95-4.85 (m, 1H), 4.85-4.65 (m, 1H), 4.15-4.00 (m, 1H), 3.77 (s, 3H), 3.45-3.30 (m, 2H), 3.25-3.00 (m, 2H), 2.35-2.10 (m, 3H), 1.95-1.75 (m, 1H), 1.55 (s, 9H), 1.43 (s, 9H).

  ) (Cell Signaling Technology); MDM2 (Santa Cruz sc965 or EMD Millipore 07575); tubulin (LICOR 92642211); tubulinHRP (5346), actinHRP (12620), GAPdH HRP (8884), caspase3 (9662), PARP (9441), total S6 (2217), phosphoS235/236S6 Letter reSeArCH (2945), Mcl1 (4572), PIM2 (2730), cleavedcaspase3 (

1 |

 1 See next page for caption.

Extended Data Figure 2 |

 2 See next page for caption.

3 |

 3  wild-type USP7 null USP7 null6640Deubiquitinase inhibition and cellular activity of optimized fragment compounds. a, Table summarizing deubiquitinase biochemical assay data and ubiquitin-MDM2 assay data from optimized fragment compounds and inactive controls. Averages are shown ± s.e.m, where applicable, with the number of biologically independent experiments indicated below. n = 1 experimental replicate, if not specified. Fragment compound structures are shown to the right. b, Representative western blot analysis of USP7, p53, and p21 levels from the cycloheximide (CHX)chase study of MDM2 turnover shown in Fig. 1c, performed in experimental triplicate. c. Analysis of endogenous MDM2 polyubiquitinated with K48linked chains. Top: denatured lysates from MCF7 cells treated for 8 h with the indicated compounds were immunoprecipitated with a K48 polyubiquitin linkagespecific antibody and immunocomplexes were western blotted with an antiMDM2 antibody. Western blot analysis of wholecell lysates for the indicated proteins are shown below. Representative of two experimental replicates. d, Cell viability of wildtype and USP7null HCT116 colon adenocarcinoma cells, treated as indicated, and analysed with a CellTiterGlo assay. Experimental triplicates normalized to vehicle control are plotted as a function of compound concentration. Top: averages ± s.e.m. Twosided ttests were used to calculate P values between wildtype HCT116 and USP7null cells treated with GNE6640: 7.5 μ M P = 0.01, 10 μ M P = 0.041, 12.5 μ M P = 0.009, 15 μ M P = 0.011, 20 μ M P = 0.017. Bottom: experimental triplicate data are shown. e. Cell viability of wildtype and USP7null HCT116 colon adenocarcinoma cells, treated and analysed as in d with the indicated doses of GNE6776. Top: averages are shown ± s.e.m. Twosided ttests were used to calculate P values between wildtype HCT116 and USP7null cells: 1 μ M P = 0.023, 2.5 μ M P = 0.003, 5 μ M P = 0.001, 7.5 μ M P = 0.003, 10 μ M P = 0.007, 12.5 μ M P = 0.001, 15 μ M P = 0.007, 17.5 μ M P = 0.001, 20 μ M P = 0.008. Bottom: experimental triplicate data are shown. Gel source data: Supplementary Fig.1.

4 | 4 |

 44 See next page for caption. © 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved. Selectivity of USP7 inhibitors, in vitro and in vivo drug metabolism and pharmacokinetic (DMPK) profiling, and xenograft growth inhibition studies with GNE-6776. a, GNE6776 dosedependent cleavage inhibition by USP7 of the indicated diubiquitin chains as measured by MALDI-TOF. Data from experimental triplicates are shown. b, Percentage inhibition of the indicated deubiquitinases for cleaving diubiquitins after treatment with 100 μ M of the indicated USP7 inhibitor compounds, representative of one experimental duplicate. Deubiquitinase concentrations and diubiquitin substrates are as in Fig. 2a. c. Supporting western blots for Fig. 2b, left. HEK293T cell lysates were treated with the indicated USP7 inhibitors (0.1% DMSO control = 0 μ M compound) and endogenous deubiquitinases were reacted with the HA-ubiquitin-vinylsulfone activitybased probe (HA-Ub-VS).Reacted cell lysates were immunoblotted with the indicated antibodies. * Unreacted deubiquitinases; * * probereacted deubiquitinases; arrowhead points to a band identified by antiHA immunoblotting that runs at the expected molecular mass of USP7 and is diminished in lysates treated with GNE6640. Representative of one experimental duplicate. d, Supporting western blots for Fig.2b, right. HEK293T cell lysates were treated with the indicated USP7 inhibitors, endogenous deubiquitinases were reacted with the HA-Ub-VS activitybased probe, and reacted cell lysates were immunoblotted with the indicated antibodies.

5 |

 5 Bioinformatics analysis of USP7 inhibitor cell viability screens. a, Schematic of the cellular viability assay workflow and bioinformatics analysis. The six tumour cell line indications included leukaemias, lymphomas, lung carcinomas, and breast, colon, and prostate adenocarcinomas. b, Histogram of IC 50 values of GNE6640, GNE6446, and GNE6641 in 181 cell lines. Mean viability is calculated as the arithmetic average of the fitted viabilities at each tested dose of GNE6640 or GNE6446 normalized to the mean viability of GNE6641. See also Supplementary Table1. c, Univariate analysis of features associated with viability differences. The x axis represents the fold change (log 2 ) in normalized mean viability between cell lines present or absent for a given feature. The y axis represents the nominal P value (-log 10 scale). Features with q values less than 0.05 and absolute log 2 (fold change) greater than 0.1 are coloured in red. Features with only absolute log 2 (fold change) greater than 0.1 are coloured in gold. Other analysed features that did not reach significance are indicated in grey. P values were determined using the two sided Student's ttest, and q values were determined by correcting resulting P values for multiple hypothesis testing using the Benjamini-Hochberg approach. The size of each point corresponds to the number of cell lines present with the feature. d, e, Boxplots of selected features and their respective associations with normalized mean viability. The respective P and q values are indicated below and were calculated as outlined in the Methods. 'TP53 LOF' indicates lossoffunction mutations and previously identified hotspot mutations inTP53 (ref. 27). The number of samples, minima, 25th percentile, centre, 75th percentile, and maxima for each boxplot are as follows: GNE6640 TP53 LOF TRUE (20, -0.261, -0.119, -0.071, 0.017, 0.304), GNE6640 TP53 LOF FALSE (156, -0.623,0.186, -0.084, -0.008, 0.294), GNE6640 TP53 WT TRUE(80, -0.623, -0.182, -0.085, -0.014, 0.293), GNE6640 TP53 WT FALSE (96, -0.425, -0.190, -0.083, 0.012, 0.304), GNE6776 TP53 LOF TRUE (20, -0.166, -0.082, -0.003, 0.029, 0.105), GNE6776 TP53 LOF FALSE(156, -0.434, -0.087, -0.032, 0.015, 0.225), GNE6776 TP53 WT TRUE (80, -0.434, -0.098, -0.036, 0.008, 0.225), GNE6776 TP53 WT FALSE(96, -0.304, -0.077, -0.021, 0.023, 0.113), GNE6640 AMl TRUE(8, -0.623, -0.394, -0.340, -0.271, -0.222), GNE6640 AMl FALSE(168, -0.488, -0.173, -0.079, 0.001, 0.304), GNE6640 TP53 Y220 TRUE(3, 0.070, 0.091, 0.112, 0.142, 0.171), GNE6640 TP53 Y220 FALSE(173, -0.623, -0.184, -0.085, -0.010, 0.304), GNE6640 TP53 R175 TRUE(5, -0.425, -0.261, -0.155, -0.119, 0.028), GNE6640 TP53 R175 FALSE(171, -0.623, -0.183, -0.082, -0.004, 0.304), GNE6776 AMl TRUE(8, -0.434, -0.216, -0.061, -0.036, -0.009), GNE6776 AMl FALSE(168, -0.363, -0.085, -0.030, 0.021, 0.225), GNE6776 TP53 Y220 TRUE(3, -0.033, 0.010, 0.052, 0.070, 0.088), GNE6776 TP53 Y220 FALSE(173, -0.434, -0.087, -0.031, 0.018, 0.225), GNE6776 TP53 R175 TRUE(5, -0.304, -0.081, -0.040, -0.034, -0.012), GNE6776 TP53 R175 FALSE(171, -0.434, -0.087, -0.031, 0.019, 0.225).

6 |

 6 Extended Data Figure | See next page for caption. Live cell imaging of USP7 inhibitor-treated cells and combination studies with chemotherapeutic and targeted agents. a-e, Graphs showing cell confluence as a function of time (top rows) and normalized caspase activity (bottom rows) in cells treated with the indicated USP7 inhibitors. Data are representative of three biologically independent experiments and each data point represents the average ± s.e.m. of 12 technical replicates. a, TP53 wildtype or TP53null HCT116 colon adenocarcinoma cells. b, TP53 wildtype MCF7 or TP53 null MDAMB157 breast adenocarcinoma cells. c, TP53 wildtype U2OS cells or TP53null SaOS osteosarcoma cells. At least three experimental replicates were performed for all experiments. d, Graphs showing cell confluence as a function of time (top rows) and normalized caspase activity (bottom rows) in MCF7 breast adenocarcinoma cells treated with GNE6640 or doxorubicin alone or in combination. e, Graphs showing cell confluence as a function of time (top rows) and normalized caspase activity (bottom rows) in U2OS osteosarcoma cells treated with GNE6640

Extended Data Figure 7 |

 7 See next page for caption.

8 |

 8 See next page for caption.

9 |

 9 Analysis of the functional significance of the interactions between ubiquitin-K48 and USP7-D305, USP7-E308 residues. a, Titration curves showing the effect of unlabelled wildtype ubiquitin (left) and ubiquitin K48A (right) addition to [ 2 H/ 15 N/ 13 C] USP7 catalytic domain. The weighted combined 1 H/ 15 N chemical shift change is plotted against the ubiquitin concentration for five wellresolved peaks stemming from E371, Q387, A381, D342, and Y339 residues in the 1 H/ 15 N TROSY spectrum and fitted as described in the Supplementary Information. The standard error was calculated on the basis of the average values over the five fits. Single data points are representative of three experimental replicates. b, Timecourse analysis of peptideconjugated tetraubiquitin chains reacted with the USP7 catalytic domain D305A/E308A mutant. Data are representative of three experimental replicates. c, Michaelis-Menten analysis of USP7 catalytic domain D305A/E308A mutant showing the results of three experimental replicates. d, Evaluation of endogenous MDM2 ubiquitination status upon expression of wildtype, C223A, or D305A/E308A fulllength USP7. Top: denatured lysates from MCF7 cells transfected with the indicated USP7 expression constructs were immunoprecipitated with a K48 linkage specific antibody and immunocomplexes were blotted with an MDM2 antibody. Western blot analysis of wholecell lysates for the indicated proteins are shown below. Data represent three experimental replicates. Gel source data: Supplementary Fig. 1. Letter reSeArCH Extended Data Figure 10 | See next page for caption.

  Figure 1c

Figure 1 .

 1 Figure 1. Comparative Analysis of Inactive and Active USP7 States

(

  A) Active and inactive states of USP7cd were previously captured in crystal structures; the catalytic triad geometry is disrupted in the apo crystal structure (PDB: 1NB8) while ubiquitin-aldehyde conjugated USP7cd structures reveal canonical cysteine protease active-site geometry (PDB: 1NBF).

Figure 3 .

 3 Figure 3. Kinetic Analysis of Designs

Figure 5 .

 5 Figure 5. Switching Loop Interactions and Core Packing of USP7 Variants Detailed views of the (A) Ub�USP7cd WT, (B) apo USP7cd WT, (C) Ub�USP7cd L299A, (D) Ub�USP7cd H294E protomer A, (E) Ub�USP7cd H294E protomer B, (F) Ub�USP7cd V302K with bound malonic acid, (G) Ub�USP7cd V302K, and (H) apo USP7cd V302K protomer A; and (I) protomer B. Key residues are shown as sticks or spheres (i.e., core-packing residues).

Figure 6 .

 6 Figure 6. Molecular Interactions across the Ub�USP7cd Interface Detailed view of ubiquitin-USP7cd interactions in the (A) wild-type, (WT) (B and C) H294E (chains A and B), (D) L299A,and (E and F) V302K (with and without a bound malonic acid) complex crystal structures. Wild-type USP7cd is colored green, designs teal, and ubiquitin yellow. The side chains that surround the hydrophobic grooves F283, L299, and V302 are represented by spheres. The ubiquitin residues that make charge interactions with USP7cd and key USP7cd residues, including the K302 side chain in V302K complex structures, are shown as sticks.

Figure 7 .

 7 Figure 7. Switching Loop Conformations in Designs Compete with C-Terminal Tail Activation The potential clashes between the USP7cd C-terminal tail and the switching loop are assessed by comparing the recently published ternary C-terminal tail-bound Ub�USP7cd structure with the crystal structures of (A) the wild-type (WT), (B) the H294E/B, (C) the L299A, and (D) the V302K complexes. The clashes are shown in red. C-terminal tail overlaid onto the Ub�USP7cd complex structures of the designs reveals clashes at predominantly two hydrophobic sites I1098 and I1100 that are critical for activation by the C-terminal tail. The complexes are in surface representation with ubiquitin colored yellow, with the wild-type and mutant USP7cd colored green and teal, respectively. The switching loop residues 283-298 are highlighted in purple.

Step 3 Structure 26 ,

 326 Benzyl N-[(1S)-1-{[(3R)-1-cyanopyrrolidin-3-yl]carbamoyl}-3-phenylpropyl]carbamate (G'3093/SMDC-751644). To a solution of benzyl N-[(1S)-3-phenyl-1-{[(3R)-pyrrolidin-3-yl]carbamoyl}propyl]carbamate trifluoroacetic acid (�0.1 mmol) in acetonitrile (1.5 mL) were sequentially added DIPEA (43 mL, 0.15 mmol) and cyanogen bromide (2.0 M in CH 3 CN, 50 mL, 0.1 mmol). The reaction mixture was stirred at ambient temperature for 30 min, then diluted with CH 2 Cl 2 . The diluted reaction was washed sequentially with 72-84.e1-e7, January 2, 2018 e5 at 0, 1, 4, and 7 hr time points and added to 5uL of SDS loading buffer to stop the reaction. All samples were loaded onto 26-well 4-12% Bis-Tris NuPAGE gels and stained with SYPRO Ruby stain (Life Technologies). Gels were imaged on Typhoon (GE Helathcare) imager and cracked gels are aligned digitally for clarity.QUANTIFICATION AND STATISTICAL ANALYSISData are represented as the mean +/-standard deviation unless otherwise indicated. For kinetic analysis, each Michaelis-Menten curve reflects n = 3 technical replicates. GraphPad Prism was used to fit each curve and determine k cat and K m . As shown in FigureS5, reported k cat and K m values reflect the mean of 1-3 independent kinetic curves.DATA AND SOFTWARE AVAILABILITYCoordinates and structure factors have been deposited in the Protein Data Bank under accession codes 5KYB, 5KYC, 5KYD, 5KYE, 5KYF.

  excitation wavelength of 380 nm and monitoring emission at 460 nm with a 435nm cutoff filter. Initial rate of substrate hydrolysis was determined using the Magellan software and kinetic parameters (Km and kcat) were modeled using nonlinear regression analysis with GraphPad Prism software. Standard error was calculated from three technical replicates.

1 mM DTT, 0.05% Tween 20, and 0.1 mg/mL bovine serum albumin. As Ub-AMC is provided in 100% DMSO stock, the final DMSO concentration in each reaction well was kept at 2.5%. Assays were monitored in Corning 96-well half area microplate with the total reaction volume of 40 µL.

Kinetic data was collected in time intervals of 30 seconds over 1 hour using Tecan Safire2 plate

Lionel Rougé-« Study of the intramolecular activation mechanism of the deubiquitinase USP7 » -BioSPC -2019 Lionel Rougé-« Study of the intramolecular activation mechanism of the deubiquitinase USP7 » -BioSPC -2019 50 reader with an

Table 1 :

 1 Crystallographic data collection and refinement

	Data Collection	USP7CD-UBL45-CTP-Ub	USP7CD-CTP-Ub	USP7CD-UBL123
	PDB-ID	5JTV	5JTJ	5J7T
	Wavelength (Å)	1.0	1.0	0.979

Table 2 :

 2 Enzyme kinetics for different USP7 constructs.

Table 3 :

 3

Enzyme kinetics for USP7CD-CTP constructs of varying linker lengths. ND stands for "Not Detectable".

Table 4 :

 4 Alanine scanning of C-terminal residues. Residues were mutated to Ala except for A1097

  Switching loop and residues Ala299 and Phe283 are colored in blue. Switching loop from inactive and active USP7 are colored in cyan and green, respectively.

	Lionel Rougé-« Study of the intramolecular activation mechanism of the deubiquitinase USP7 » -BioSPC -2019
	As described earlier, USP7 has been a target of interest in the pharmaceutical industry for
	many years. Many companies and academic groups have been working towards the development
	of inhibitors for the treatment of several cancers as shown in Figure 42 (Zhou et al., 2018).
	Figure 42: Chemical structures of specific and non-specific USP7 inhibitors.
	A number of USP7 inhibitors have been identified by companies such as Hybrigenics
	(https://www.hybrigenics.com/),	Progenra	(http://progenra.com),	Forma
	(http://www.formatherapeutics.com/), Almac (https://www.almacgroup.com/) or Genentech. Most
	of IC50 reported for these inhibitors varies between 4 μM (i.e. Progenra) and 0.045 μM (Almac)
		for 58 human USP sequences (left) or in USP7 sequences
	across species (right) (from Ozen et al.,). Numbering is based on USP7 sequence.
	b) Comparison of enzymatic values (Km, Kcat and Km/Kcat) between WT and Leu299Ala USP7
	constructs; assays were ran using both catalytic and USP7CD-UBL45-CTP constructs (adapted
	from Ozen et al.,)
	c) Overall representation of USP7CD [Leu299Ala] + Ub-BEA crystal structure at 1.4Å
	(Rwork/Rfree: 17.5% / 18.5%). Catalytic domain and ubiquitin are colored as described earlier.
	Lionel Rougé-« Study of the intramolecular activation mechanism of the deubiquitinase USP7 » -BioSPC -2019	91

Lionel Rougé-« Study of the intramolecular activation mechanism of the deubiquitinase USP7 » -BioSPC -2019 Lionel Rougé-« Study of the intramolecular activation mechanism of the deubiquitinase USP7 » -BioSPC -2019 90

Table 1 .

 1 Enzyme Kinetic Constants for Point Mutations in USP7CD-CTP

	C-Terminal			k kat /K M
	Mutations K M (mM)	k cat (s 1 )	(M 1 s 1 10 5 ) % activity
	Wild-type	3.4 ± 0.4	0.5 ± 0.1	1.5 ± 0.3	52
	N1102(Del)	3.0 ± 0.9	0.16 ± 0.03	0.5 ± 0.2	17
	N1102A	4 ± 1	0.13 ± 0.03	0.3 ± 0.1	10
	H1101A	2.7 ± 0.7	0.27 ± 0.07	1.0 ± 0.4	34
	I1100A	>79	>0.06	ND	ND
	K1099A	4 ± 2	0.11 ± 0.03	0.3 ± 0.2	10
	I1098A	>79	>0.06	ND	ND
	A1097M	2.74 ± 0.06 0.205 ± 0.006 0.75 ± 0.03	26
	A1097Y	2.37 ± 0.04	0.27 ± 0.01	1.14 ± 0.05	39
	K1096A	6.1 ± 0.4	0.253 ± 0.005 0.41 ± 0.03	14
	E1095A	4.1 ± 0.1	0.78 ± 0.03	1.9 ± 0.09	66
	L1094A	5.05 ± 0.07	0.89 ± 0.04	1.76 ± 0.08	61

Table 2 .

 2 Crystallographic Data Collection and Refinement

		USP7CD-UBL45-Ub	USP7CD-CTP-Ub	USP7CD-UBL123
	Data Collection
	PDB ID	5JTV	5JTJ	5J7T
	Wavelength (A ˚)	1.0	1.0	0.979
	Resolution range (A ˚)a	46.95-

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The ubiquitin system regulates essential cellular processes in eukaryotes. Ubiquitin is ligated to substrate proteins as monomers or chains and the topology of ubiquitin modifications regulates substrate interactions with specific proteins. Thus ubiquitination directs a variety of substrate fates including proteasomal degradation 1 . Deubiquitinase enzymes cleave ubiquitin from substrates and are implicated in disease 2 ; for example, ubiquitin- specific protease-7 (USP7) regulates stability of the p53 tumour suppressor and other proteins critical for tumour cell survival 3 . However, developing selective deubiquitinase inhibitors has been challenging 4 and no co-crystal structures have been solved with small-molecule inhibitors. Here, using nuclear magnetic resonance-based screening and structure-based design, we describe the development of selective USP7 inhibitors GNE-6640 and GNE- 6776. These compounds induce tumour cell death and enhance cytotoxicity with chemotherapeutic agents and targeted compounds, including PIM kinase inhibitors. Structural studies reveal that GNE- 6640 and GNE-6776 non-covalently target USP7 12 Å distant from the catalytic cysteine. The compounds attenuate ubiquitin binding and thus inhibit USP7 deubiquitinase activity. GNE-6640 and GNE- 6776 interact with acidic residues that mediate hydrogen-bond interactions with the ubiquitin Lys48 side chain 5 , suggesting that USP7 preferentially interacts with and cleaves ubiquitin moieties that have free Lys48 side chains. We investigated this idea by engineering di-ubiquitin chains containing differential proximal and distal isotopic labels and measuring USP7 binding by nuclear magnetic resonance. This preferential binding protracted the depolymerization kinetics of Lys48-linked ubiquitin chains relative to Lys63-linked chains. In summary, engineering compounds that inhibit USP7 activity by attenuating ubiquitin binding suggests opportunities for developing other deubiquitinase inhibitors and may be a strategy more broadly applicable to inhibiting proteins that require ubiquitin binding for full functional activity.

  , high resolution mass spectrometry (HRMS) m/z 331.1633. (M + H + , C 20 H 19 ON 4 requires 331.1653).

  . HRMS m/z 349.1659. (M + H + , C 20 H 21 O 2 N 4 requires 349.1650).

	4(6Amino4ethyl5(4(methoxymethyl)phenyl)pyridin3yl)phenol (GNE 2118): 1 H NMR (400 MHz, DMSOd 6 ) δ 9.49 (s, 1H), 7.69 (s, 1H), 7.51 (d, J = 8.1 Hz,
	2H), 7.31 (d, J = 8.0 Hz, 2H), 7.17 (d, J = 8.0 Hz, 2H), 6.86 (d, J = 8 Hz, 2H), 4.99
	(s, 2H), 4.55 (s, 2H),

  156.1, 156.2, HRMS m/z 331.1750 (M + H + , C 21 H 22 O 2 N 2 requires 331.1754).

  .0. HRMS m/z 345.1710. (M + H + , C 21 H 21 ON 4 = 345.1650).

  . HRMS m/z 334.1550 (M + H + , C 20 H 20 O 2 N 3 requires 334.1700).

	4[6Amino4ethyl5(4hydroxyphenyl)3pyridyl]benzamide (GNE2916):

1

H NMR (400 MHz, DMSOd 6 ) δ 9.53 (s, 1H), 7.98 (d, J = 6.1 Hz,1H), 7.95-7.87 (m, 2H),

7.72 (s, 1H), 7.43-7.31 (m, 3H), 7.14-6.95 (m, 2H), 6.93-6.74 (m, 2H), 5.04 (s, 2H

  . HRMS m/z 349.1655. (M + H + , C 20 H 21 O 2 N 4 requires 349.1659). Synthesis of GNE-6831 is representative of the syntheses for GNE-0956, GNE-2090, GNE-2143, and GNE-2148.

  . HRMS m/z 470.1033 (M + H + , C 24 H 22 O 3 N 3 Cl 2 , requires 470.1029). 9ChloroN(5chloro2,4dimethoxyphenyl)N((3methyloxetan3yl)methyl) 5,6,7,8tetrahydroacridine3carboxamide (GNE0956): 1 H NMR (400 MHz, DMSOd 6 ) 1H NMR (400 MHz, DMSOd 6 ) δ 7.96 (d, J = 8.2 Hz, 1H), 7.71 (d, J = 1.4 Hz, 1H), 7.56 7.48 (m, 1H), 7.51 (s, 1H), 6.57 (s, 1H), 4.21 (d, J = 5.9 Hz, 2H), 2H), 3.74 (d, J = 13.5 Hz, 6H), 3.37 (s, 2H), 2.99 (s, 2H), 2.91 (s, 2H),1.88-1.77 (m, 3H), 1.42 (s, 3H).

  . HRMS m/z 515.1499 (M + H+ , C 27 H 27 O 4 N 2 Cl 2 requires 515.1400).

	9ChloroN(5chloro2,4dimethoxyphenyl)N(2,3dihydroxypropyl)5,6,7, 8tetrahydroacridine3carboxamide (GNE2090): 1 H NMR (400 MHz, DMSOd 6 ) δ 7.96 (dd, J = 8.6, 3.4 Hz, 1H), 7.77-7.68 (m,1H), 7.66-7.47 (m, 2H), 6.56
	(d, J = 14.2 Hz, 1H), 4.99 (d, J = 5.2 Hz, 1H), 4.54 (dt, J = 15.4, 5.8 Hz, 2H), 4.21
	(dd, J = 13.6, 4.3 Hz, 1H), 3.93 (s, 1H), 3.80-3.63 (m, 5H), 3.46-3.33 (m, 2H), 2.99
	(s, 2H), 2.91 (s, 2H), 1.85 (s, 1H).

  . LC-MS (ESI) m/z 505.2 [M + H + ].

	9ChloroN(2hydroxyethyl)Nmethyl5,6,7,8tetrahydroacridine3carbox amide (GNE2143): 1 H NMR (400 MHz, DMSOd 6 ) δ 8.16 (d, J = 8.2 Hz, 1H), 7.95
	(s, 1H), 7.62 (d, J = 8.6 Hz, 1H), 4.79 (s, 1H), 3.66 (s, 1H), 3.57 (s, 1H), 3.48 (s, 1H),
	3.27 (s, 2H), 3.10-2.94 (m, 7H), 1.90 (td, J = 3.8, 1.8 Hz, 4H).

  . HRMS m/z 319.1208 (M + H + , C 17 H 20 O 2 N 2 Cl requires 319.1500). 9ChloroNmethylN(2morpholino2oxoethyl)5,6,7,8tetrahydroacridine 3carboxamide (GNE2148): 1 H NMR (400 MHz, DMSOd 6 ) δ 8.18 (dd, J = 22.2, 8.6 Hz, 2H), 7.93 (d, J = 1.6 Hz, 1H), 7.81 (s, 1H), 7.65 (dd, J = 8.6, 1.6 Hz, 1H), 7.54 (d, J = 8.7 Hz, 1H),4.40 (s, 2H), 4.18 (s, 2H), 4H), 3.15 (s, 2H), 3.02 (t, 20.9 Hz, 4H), 4H).

  .1. HRMS m/z 343.0900, 345.0882 (M + H+ , C 18 H 22 N 2 Br requires 343.0850, 345.0911).

	(1S)1(5bromo1Hindol2yl)N(2phenylethyl)ethanamine (GNE8735): H NMR (400 MHz, DMSOd 6 ) δ 10.83 (s, 1H), 7.53 (s, 1H), 7.24-7.12 (m, 6H), 1
	7.05-7.03 (m, 1H), 3.88 (d, J = 4 Hz, 1H), 2.81-2.60 (m, 6H), 2.10-1.75 (m, 2H),
	1.75-1.60 (m, 2H), 1.50-1.30 (m, 2H).

13

C NMR (150 MHz, DMSOd 6 ) δ

23.4,

  . HRMS m/z 385.1126, 383.1114 (M + H + , C 21 H 24 N 2 Br requires 383.1117, 385.1130).

  HNMR (400 MHz, CDCl 3 ) δ 8.04 (s, 1H),5.05 (m, 1H), 4.80-4.55 (m, 1H), 4.15-4.05 (m, 1H), 3.79 (s, 3H), 3.45-3.10 (m, 4H), 2.35-2.05 (m, 3H), 1.95-1.80 (m, 1H), 1.47 (s, 9H). LC-MS (ESI m/z): 358.3 [M + H + ].

3086 GNE-2090 GNE-6831 GNE-2916

	Primary Screen UbA10 TR-FRET inhibition 40 -150%	1.37 million library compounds		b.	Primary Screen STD NMR with 5-compound mixtures	4871 library fragments
	normalized F615 -170 -80%
	primary structural filters						S/N >5
		IC50 confirmation UbA10 TR-FRET USP7 diUb FRET Ub-Rho110	3064 initial hits				primary confirmation STD NMR with single compounds	810 preliminary hits
	confirmed IC50 in ≥2 assays secondary confirmation di-Ub cleavage assay Hill slope < 2 in UbA10 TR-FRET no Rho-110 interference	preliminary actives 1315			titration with labeled USP7 secondary confirmation S/N >10	confirmed binders 478
								chemical shift
	hit-to-lead candidates IC50 < 50µM	101 confirmed actives			X-ray crystallography perturbation clusters	candidates 83 crystallography
							crystal structure solved
								hit-to-lead candidates
		Indole tricyclic	Peptidomimetic	Tetrahydroacridine	Fragment
	Compound GNE-Full Length GNE-0300 GNE-8735 USP7 IC 50 (µM) 1.03 0.47±0.39 8.80±5.20	4.64±1.38	0.75±1.30	2.63±0.43
	USP7 Catalytic Domain IC50 (µM)	1.40	0.50±0.31	13.80	41.7±30.6	>200	1.40±0.18
	Full Length USP5 IC 50 (µM)	160	>200	>100		>200	N/D	>200
	Full Length USP47 IC 50 (µM)	140	>200	7.0		63.3	>200	>200
	Total MDM2	none	increase	none		none	none	none
	increase (IF)	>20µM	2.9µM	>20µM	>20µM	>20µM	>20µM
	Ub-MDM2 MSD (µM)	not done	>50.00	9.65±3.18	3.55±1.91	3.20±2.69	7.60±0.57
	Cathepsin-B Inhibition (µM)	> 40.00	6.12	not done	not done	> 40.00	not done
	Caspase-3 Inhibition	apparent precipitator	apparent precipitator	not done	not done	not done	not done
	Dynamic Light Scattering	pass	pass	pass		pass	pass	not done
	Covalent Modification	not done	not done	yes	not done	yes	not done

  Table of active and inactive tetrahydroacridine compounds with structures. Hittolead selection assay data are listed in the indicated rows. Averages are shown ± s.e.m., where applicable, with the number of biologically independent experiments indicated below. n = 1 experimental replicate, if not specified. See Fig. 1b and text for more details. d, Table of active and inactive fragment compounds with structures. Hittolead selection assay data are listed in the indicated rows. Averages are shown ± s.e.m., where applicable, with the number of biologically independent experiments indicated below. n = 1 experimental replicate, if not specified. See Fig.

GNE-6640 GNE-6776 MW/LogD7.4/ tPSA 330

.38/4.40/87.0 348.40/3.30/3.4/101 LM CLhep (h/r/m/d/c) 14/46/75/24/33 4.1/15/37/14/12 Hep CLhep (h/r/m/d/c) 16/45/68/24/39 9.4/39/44/18/33 PPB % (h/r/m) 99.6/98.7/99.0 97.3/93.4/93.4 MDCK Papp ratio (B to A, A to B) 0.83 (12.17, 14.7) 0.75 (12.06, 16.10) j.

  * Unreacted deubiquitinases; * * probereacted deubiquitinases. Representative of one experimental duplicate. e, In vitro pharmacokinetic assessment of USP7 inhibitors. Calculated drug properties are indicated: molecular mass (MW), lipophilicity at pH7.4 (logD 7.4 ), total polar surface area (tPSA), stability in hepatic microsomes (LM CL hep ), or hepatocytes (Hep CL hep ) from human/rat/mouse/dog/cynomolgus monkey (h/r/m/d/c) species, percentage plasma protein binding (PPB %), and permeability across an MDCK cell monolayer from basolateral to apical (B to A) or apical to basolateral (A to B) directions. f, EOL1 cell line viability in response to GNE6776 as measured in a 5day CellTiterGlo assay performed in experimental triplicates. g, In vivo pharmacokinetic analysis of GNE6776. Mice (three per group) were dosed PO with 100 mg kg -1 (body weight) or 200 mg kg -1 (body weight) of GNE6776. Plasma concentrations of GNE 6776 were measured at the indicated time points and the mean ± s.d. (left) or individual data points (right) plotted as a function of time. Exposure metrics relating to the free fraction EC 50 for EOL1 cells are also indicated, where target exposure = (EOL1 IC 50 )/(1percentage plasma protein binding) = 1.54 μ M/0.066 = 23.33 μ M. h, Western blot analysis of MCF7 Ser xenografted tumours. Mice bearing MCF7Ser xenograft tumours were were dosed by mouth with vehicle or 200 mg kg -1 (body weight) GNE 6776 at 0 and 4 h; 8 h after the initial treatment, tumours were excised and the indicated proteins were examined by immunoblotting tumour lysates. Data show three biological replicates. i, Western blot analysis of EOL1 xenografted tumours. Mice bearing EOL1 xenograft tumours were dosed by mouth with vehicle or 200 mg kg -1 (body weight) GNE6776 at 0 and 4 h; 8 h after the initial treatment, tumours were excised and the indicated proteins were examined by immunoblotting tumour lysates. Data show three biological replicates. j, EOL1 xenograft growth inhibition study of mice administered vehicle or the indicated doses of GNE6776 by mouth with n = 7 mice per group. The mean ± s.e.m. (top) or individual data points (bottom) are plotted as a function of time. The P values were calculated using Dunnett's multiple comparison test. Asterisks indicate significant growth inhibition relative to vehicletreated mice. Day 4 100 mg kg -1 (body weight) P = 0.0163, day 4 200 mg kg -1 (body weight) P = 0.0138, day 6 200 mg kg -1 (body weight) P = 0.0344.

  2

			Percent Inhibition			Bliss Excess
		20						20
	GNE-6776 (µM)	6.67 2.22 0.74 0.25 0.082 0.027 0.009						6.67 2.22 0.74 0.25 0.082 0.027 0.009
		0.003						0.003
		0						0
		0	0.002	0.005 0.014	0.04	0.12	0.37	1.1 3.3 10.0	0	0.002	0.005 0.014	0.04	0.12	0.37	1.1 3.3 10.0
			[GDC-0339] (µM)		[GDC-0339] (µM)

Table 1 :

 1 .2 1 Summary of cell line TP53 mutation status and viability in response to treatment with USP7 inhibitors.

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Supplementary

Table 1 .

 1 Crystallographic Data Reduction and Refinement Statistics for the Various Datasets

		USP7 L299A + Ub	USP7 H294E + Ub	USP7 V302K + Ub (mal)	USP7 V302K + Ub	Apo USP7 V302K
	PDB ID	5KYF	5KYE	5KYC	5KYD	5KYB
	Data Collection
	Space group	P2 1 2 1 2 1	P4 3	C 121	P2 1 2 1 2 1	P 1 2 1 1
	Cell dimensions
	a, b, c (A ˚)	62.

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Lionel Rougé-« Study of the intramolecular activation mechanism of the deubiquitinase USP7 » -BioSPC -2019 Lionel Rougé-« Study of the intramolecular activation mechanism of the deubiquitinase USP7 » -BioSPC -2019

Structure 24, 1335-1345, August 2, 2016 1341

Structure 24, 1335-1345, August 2, 2016

Structure 24, 1335-1345, August 2, 2016 1343

© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

Structure 26, 72-84, January 2, 2018 73

Structure 26, 72-84, January 2, 2018 75

Michaelis-Menten parameters (A) k cat /K M , (B) k cat , and (C) K M for wild-type and four designs in the backgrounds of USP7cd and USP7cd-45. Error bars represent SDs from at least three replicates (see FigureS5and TableS4).

Structure 26, 72-84, January 2, 2018 77

Structure 26, 72-84, January 2, 2018 79

Structure 26, 72-84, January 2, 2018 81

e4 Structure 26, 72-84.e1-e7, January 2, 2018

e6 Structure 26, 72-84.e1-e7, January 2, 2018

Structure 26, 72-84.e1-e7, January 2, 2018 e7

Acknowledgements

I would like to thank my managers within the Structural Biology department (Dr. Alexis Rohou, Dr. Claudio Ciferri, Dr. Christopher Koth and Dr. Sarah Hymowitz) for their continued support and encouragement throughout this entire process. Beyond what they have done in the last 12 months, I really value their enthusiasm and support of my growth as a scientist throughout the ACKNOWLEDGMENTS Portions of this research were carried out at the Advanced Light Source, supported by the Director, Office of Science, Office of Basic Energy Sciences of the US Department of Energy, under contract no. DE-AC02-05CH11231. We thank Diamond Light Source for access to beamline I02, which contributed to the results presented here, the Bio-Molecular Engineering group for generating many of the DNA constructs and for help with protein expression, Sarah Hymowitz and James Kiefer for critical reading of the manuscript.

Acknowledgements We thank W. Fairbrother, L. Frick, S. Fong, E. Helgason,

ACKNOWLEDGMENTS

We thank the Genentech Structural Biology Expression Group for their support in protein production, Elizabeth Helgason for assistance with the in vitro expression system, and Allyn Schoeffler, Jacob Corn, Jeremy Murray, and Till Maurer for discussions. Many thanks to the Roche Pharma Technical Development (U.S.) department for their generosity allowing the authors to be trained and run molecular dynamics simulations in their computer cluster in support of this project. At the time of submission, A.O., L.R., C.B., N.J.S., and E.C.D. were all employees of Genentech. A.O. and B.R.H. are now employees of Pfizer and ProLynx, respectively. Received: May 26, 2017 Revised: August 31, 2017 Accepted: November 15, 2017 Published: December 14, 2017

SUPPLEMENTAL INFORMATION

Supplemental Information includes Supplemental Experimental Procedures, four figures, and three tables and can be found with this article online at http://dx.doi.org/10.1016/j.str.2016.05.020.

AUTHOR CONTRIBUTIONS

Conceptualization, I.E.W., J.A.E., and J.M.; Methodology, L.R., T.M., T.W.B., R.T., J.A.E., and J.M.; Formal Analysis, L.R., T.W.B., M.K., R.T., P.D.L., T.M., and J.M.; Investigation, L.R., T.W.B., R.T., T.M., and J.M.; Visualization, T.M., T.W.B., J.A.E., and J.M.; Supervision: J.A.E., I.E.W., and J.M.; Writing -Original, L.R., J.A.E., T.W.B., M.K., and J.M.; Writing -Review, L.R., T.W.B., M.K., R.T., I.E.W., T.M., P.D.L., J.A.E., and J.M.

CONFLICT OF INTEREST

The authors declare competing financial interests: all authors are employees of Genentech.

Supplementary Information is available in the online version of the paper. nature24006 2 5 3 0 3 5 4 0 4 5 5 0 5 5 6 0 6 5 7 0 7 5 8 0 8 5 9 0 9 5 1 0 0 1 0 5 1 1 0 1 1 5 1 2 0 1 2 5 1 3 0 1 3 5 1 4 0 1 4 5 1 5

SUPPLEMENTARY INFORMATION

Directed Sampling to Selectively Stabilize the Active Conformation

In addition to our rational design efforts to affect the switching loop and helix conformations, we also utilized a computational approach (RosettaDesign) to design more efficient packing in this region. This protein design software explores mutations at user-specified positions that will lead to stabilization of a target conformation (Kuhlman and Baker, 2000). Based on our rational design, the hydrophobic core (leucine cluster) and H294 in the switching loop were good candidates for side-chain optimization. Yet, since the overall domain fold in the active and inactive states is very similar, a mutation that stabilizes the active state may also result in stabilization of the inactive state. To identify and filter out such non-selective stabilization, we set up independent design simulations to sequence-optimize the active and inactive states. Separate design simulations were started from active and inactive USP7cd crystal structures, with optimization of all selected residue positions occurring simultaneously.

The probability of a particular mutation appearing in a resulting low-energy design is depicted by the height of the amino acid in the logo plot of the RosettaDesign consensus sequences (Figures 2A, 2B, and S3A). While some mutations were favored in both active and inactive designs (e.g., L288D, or V302E), other mutations were specific to one of the distinct states. From these analyses, three mutations were predicted to preferentially stabilize the active state: Y224F, L299A, and V302K (Figure S3B). The first mutation, Y224F, was also among our rationally designed mutations. Y224 is a part of the switching loop electrostatic network in the USP7cd inactive state and is a position that participates in the core Phe cluster in USP2cd. We hypothesized that a tyrosine-to-phenylalanine mutation, with the missing hydroxyl group, would destabilize the electrostatic network but would be hydrophobic and large enough to possibly improve local packing in the USP7cd active state. Sequence analysis of the USP family reveals that phenylalanine is found at this position in more than 20 other USPs (Figure S4). The second mutation, L299A, appears contradictory to the rationale of the repacking mutations as it replaces the leucine with an even C-terminal tail can no longer activate the catalytic domain when the L299A or V302K mutations are present. Taken together, the differences in kinetic behaviors suggest that the rationally designed H294E mutant and the Rosetta variants may function through two distinct activation mechanisms.

Given their effects on USP7 activity, we next sought to evaluate whether these mutations might also alter USP7 specificity. To this end, we performed gel-based cleavage assays on all tetra-ubiquitin chain types. As shown in Figure 4, wild-type USP7cd-45 reduces most of the lysine-linked tetra-ubiquitin chains to monomeric ubiquitin over 1-4 hr, whereas it only processes K29-linked chains to dimers in that time frame and The activity of the designs on tetrameric ubiquitin chain substrates was assessed by gel cleavage assays for the wild-type (WT) USP7cd-45 and USP7cd constructs and the four USP7cd single mutants Y224F, H294E, L299A, and V302K.

does not cleave M1-linked tetra-ubiquitin chains. Wild-type USP7cd shows similar specificity, albeit significantly slower processing due to its relatively poor catalytic turnover. H294E, L299A, and V302K variants of USP7cd again appear generally more active than the wild-type USP7cd on the lysine-linked tetra-ubiquitin chains, as best illustrated by the relative abundance of monomeric ubiquitin released over time. In particular, L299A most readily cleaves the native isopeptide bonds of the ubiquitin chains in comparison with wild-type and the other USP7cd variants. Importantly, the designs did not result in non-specific cleavage of M1linked tetra-ubiquitin chains. In short, while the designs improve USP7cd activity on the chain types that can be cleaved by wild-type USP7 constructs, they do not appear to alter substrate specificity.

Finally, as a measure of the intrinsic activity of our designs in the absence of ubiquitin, we monitored the reactivity of the USP7cd variants with a small, peptidomimetic inhibitor bearing an electrophilic cyanopyrrolidine warhead [START_REF] Deaton | Novel and potent cyclic cyanamide-based cathepsin K inhibitors[END_REF] capable of forming a covalent bond with the catalytic cysteine (see STAR Methods; Kategaya et al., 2017). Incubation of the USP7cd variants with this inhibitor resulted in the formation of covalent USP7cd-inhibitor adducts, which we monitored over time by liquid chromatography-mass spectrometry (Figure S5C). Consistent with the activity measurements that utilized ubiquitin substrates, H294E and V302K displayed greater reactivity with the small-molecule probe than with wild-type USP7cd. Interestingly, Y224F also exhibited increased intrinsic activity over wild-type, whereas L299A showed very weak reactivity (albeit greater than the catalytically dead mutant, C223S, negative control), suggesting that ubiquitin binding may have more influence over the apparent activity of these two variants.

Crystal Structures Reveal Improved Local Packing in the USP7cd Core

To better understand the activation mechanisms of our designs, we crystallized the H294E, L299A, and V302K mutants of the USP7 catalytic domain conjugated to ubiquitin (Table 1). We Detailed methods are provided in the online version of this paper and include the following:

CONTACT FOR REAGENT AND RESOURCE SHARING

Inquiries for further information may be directed to and fulfilled by corresponding author Erin C Dueber. To request reagents please submit a form to Genentech at https://www.gene.com/scientists/mta.

EXPERIMENTAL MODEL AND SUBJECT DETAILS

Recombinant proteins generated in this paper were purified from E. coli BL21 (DE3) gold transformed with the appropriate plasmids and were grown in LB media with 50mg/mL carbenecillin.

METHOD DETAILS Molecular Dynamics Simulations and Modeling

Crystal structures were prepared with the Protein Preparation workflow from Schrodinger Suite 2014-2. [START_REF] Sastry | Protein and ligand preparation: parameters, protocols, and influence on virtual screening enrichments[END_REF] As a part of preparation, hydrogens were added, side chains with missing atoms were built. pKa predictions were performed using PROPKA at pH 7.0. [START_REF] Olsson | PROPKA3: consistent treatment of internal and surface residues in empirical pKa predictions[END_REF] The hydrogen bonding network was also optimized by flipping the terminal chi angle of Asn, Gln, and His residues and sampling hydroxyl/thiol polar hydrogens. The exhaustive sampling option with the inclusion of water orientational sampling was used. The structure was then minimized in vacuum with restraints on heavy atoms using the Impact Refinement module with the OPLS2005 force field [START_REF] Banks | Integrated modeling program, applied chemical theory (IMPACT)[END_REF] and terminated when the root-mean square deviation (RMSD) reached a maximum cutoff of 0.3 A ˚allowing the hydrogen atoms to be freely minimized, while allowing heavy-atom movement to relax strained bonds, angles, and clashes. The AMBER simulation package (version 12) with the ff99SB force field was used in all further minimization, equilibration, and production stages. [START_REF] Case | Amber 12[END_REF] All structures were solvated explicitly in a truncated octahedron box using the TIP3P water mode with a 10 A ˚buffer of solvent between the solute's furthest dimensions in each direction to avoid interaction with self-images upon tumbling. [START_REF] Jorgensen | Comparison of simple potential functions for simulating liquid water[END_REF] Each system was neutralized by adding Cl-counterions using a Coulombic potential on a 1 A ˚grid and added extra Na+ and Cl-ions to keep the simulation box at 150 nM salt concentration. First, the system was minimized in six stages with a harmonic restraint on the solute slowly decreasing from 40 to 0 kcal/mol/A ˚2, each stage over 100 steps of steepest descent followed by 100 steps conjugate gradient algorithm with an electrostatic cutoff of 9 A ˚and a solvent dielectric constant of 78.5. Following the energy minimization, the system was equilibrated for 20 ps at NPT ensemble with 10 kcal/mol/A ˚2 restraint on solute. Subsequently the system temperature was increased from 150 to 300 K over 50 psec at 1 fs integration steps. Bonds involving hydrogens were constrained by the SHAKE algorithm with a relative geometric tolerance of 10E-5 A ˚. [START_REF] Ryckaert | Numerical-integration of cartesian equations of motion of a system with constraints-moleculardynamics of n-alkanes[END_REF] A typical system density increased from 0.90 g/cm 3 to 1.02 g/cm 3 during equilibration of the solvated system. Production data of 100 ns were collected at NVT ensemble at 3 fsec time steps and recorded every 15 psec. The particle mesh Ewald (PME) method [START_REF] Essmann | A smooth particle mesh Ewald method[END_REF] was used to calculate the full electrostatic energy of a periodic box, bypassing pair list creation and nonbonded force and energy evaluation by calling special PME functions to calculate the Lennard-Jones and electrostatic interactions with a cutoff distance of 9 A ˚. A time step of 3 fs was employed in the Leapfrog integrator. Coordinates and energies were written every 15 ps. 224,260,279,288,294,299,302 were allowed to vary during the simulations (see resfile below). Two types of design were performed: rotamer only, in which the amino acids were allowed to adopt romateric positions, and backrub, in which the backbone flexibility was taken into account. [START_REF] Smith | Backrub-like backbone simulation recapitulates natural protein conformational variability and improves mutant sidechain prediction[END_REF] Sequences were extracted from all 5,120 designs (256 x 20 designs), narrowed down to the seven positions allowed to vary during design simulations and used to generate a sequence logo. dilute aqueous KHSO 4 , dilute aq. NaHCO 3 , and brine. The organic layer was dried over MgSO 4 , filtered, and concentrated. The crude concentrate was purified by flash silica gel chromatography (4 g Silicycle column), eluting with acetone/CH 2 Cl 2 . Pure, product-containing fractions were combined and concentrated to afford 17 mg (42% over two steps) of the title compound. 1 H NMR (400 MHz, CHLOROFORM-d) d 7.31 -7.40 (m, 5 H), 7.25 -7.30 (m, 2 H), 7.19 (m, 1 H), 7.13 (d, J=7.1 Hz, 2 H), 6.41 (br. s., 1 H), 5.21 (m, 1 H), 5.11 (s, 2 H), 4.39 (br. s., 1 H), 4.06 (q, J=7.0 Hz, 1 H), 3.56 (dd,J=10.0,6.0 Hz,1 H),2 H),3.14 (d,J=7.7 Hz,1 H),2.66 (t,J=7.7 Hz,2 H),2 H),1.93 (m,1 H),1.81 (br. s.,1 H). LC-MS: m/z = 407 [M+H] + .

Monitoring Reactivity of Inhibitor with Usp7

To initiate reaction, a two-fold excess of compound was incubated with Usp7 mutants in PBS buffer with 0.5mM TCEP. At 0,10,20,30, and 40 minutes, 1.0mg protein was injected over a 6224 TOF LC/MS (Agilent). Mass spectrometry data were analyzed using MassHunger Qualitative Analysis B.06.00 (Agilent). Peaks corresponding to the correct mass for Usp7 and Usp7 + inhibitor were integrated and plotted in GraphPad Prism as a function of time.

Synthesis of Ubiquitin-bromo-ethyl-amine

Ubiquitin-bromo-ethyl-amine (Ub-BEA) was produced according to published procedures. (Borodovsky et al., 2002;Wilkinson et al., 2005) The ubiquitin thiolester intermediate (3 mL at 3 mg/mL) was mixed with 450 mg of bromoethylamine (MP Biomedicals) and 600 mL of 2N NaOH. Reaction was incubated at room temperature for 30 minutes and stopped by loading it onto a HiLoad 16/60 Superdex 75 prep grade column (GE Healthcare) pre-equilibrated with 25 mM Tris-HCl pH 7.5, 300 mM NaCl and 0.5 mM TCEP; 10 mgs of purified USP7 CD point mutant were immediately added to purified Ub-BEA and the reaction was incubated for 72h at 4 � C. The resultant complexes were dialyzed against 25 mM Tris-HCl pH 8.0, 75 mM NaCl and 0.5 mM TCEP (Buffer A) before loading onto a Mono Q 10/100 GL column (GE Healthcare) pre-equilibrated with 40 mL of Buffer A. The column was washed with Buffer A followed by elution of the complex with a gradient of 0-0.75 M NaCl in the same buffer. Peak fractions were pooled and complex formation was confirmed by analysis on SDS PAGE gels (BioRad). The complexes were concentrated to a final concentration of 10 mg/mL.

Crystallization and Structure Determination

Crystals of USP7cd L299A-Ub complex were obtained at 19 � C by hanging drop diffusion method by mixing equal volumes of protein at 10 mg/mL with a well solution containing 0.1M HEPES pH 7.5 and 25% w/v PEG 3350 (Hampton Research). Crystals of USP7cd H294E-Ub complex were obtained at 19 � C by hanging drop diffusion method by mixing equal volumes of protein at 10 mg/mL with a well solution containing 0.1M HEPES pH 7.5, 0.2M Ammonium acetate and 25% w/v PEG 3350 (Hampton Research). Crystals of USP7cd V302K-Ub (Malonate bound) complex were obtained at 19 � C by hanging drop diffusion method by mixing equal volumes of protein at 10 mg/mL with a well solution containing 8% tacsimate pH 4.0 and 20% w/v PEG3350 (Hampton Research). Crystals of USP7 V302K-Ub complex were obtained at 19 � C by hanging drop diffusion method by mixing equal volumes of protein at 10 mg/mL with a well solution containing 0.2M ammonium fluoride and 20% w/v PEG3350 (Hampton Research). Crystals of apo USP7 V302K were obtained at 19 � C by hanging drop diffusion method by mixing equal volumes of protein at 10 mg/mL with a well solution containing 1% Tryptone, 0.05M HEPES Na pH 7.0, 20% w/v PEG3350 (Hampton Research). Crystals were transferred into a cryo-protectant solution containing their respective crystallization buffers supplemented with 25% glycerol and frozen in liquid nitrogen. Diffraction images were collected at the Advanced Light Source beamline 5.0.2 and at the Stanford Synchrotron Radiation Lightsource beamline 12-2. The images were indexed, integrated and scaled with iMosflm [START_REF] Battye | iMOSFLM: a new graphical interface for diffraction-image processing with MOSFLM[END_REF] and the structures were solved by molecular replacement using PHASER (McCoy, 2007). Ub�USP7cd L299A and Ub�USP7cd H294E datasets were solved using PDB 1NBF as the search models whereas both Ub�USP7cd V302K datasets were solved using PDB 1NB8 and 1UBQ as models. Apo USP7cd V302K was solved using PDB 1NB8 as search model. The final models were obtained after iterative cycles of manual rebuilding in COOT (Emsley and Cowtan, 2004) and refinement in PHENIX (Adams et al., 2010). For data collection and refinement statistics, see Table 1.

Ubiquitin-AMC Cleavage Assays

Activity on Ub-AMC (Boston Biochem) was assayed at 30 � C in PBS with 1 mM DTT, 0.05% Tween 20, and 0.1 mg/mL bovine serum albumin. As Ub-AMC is provided in 100% DMSO stock, the final DMSO concentration in each reaction well was kept at 2.5%. Assays were monitored in black 384-well microplate (ProxiPlate-384. Plus F; PerkinElmer) with the total reaction volume of 20 mL. Kinetic data was collected in time intervals of 30 seconds over 1 hour using a SpectraMax M5e plate reader (Molecular Devices) an excitation wavelength of 360 nm and monitoring emission at 460 nm with a 435-nm cutoff filter. We determined the reaction rates using quadratic regression in Matlab, and converted fluorescence signal to mmol AMC using an AMC standard curve (Cayman Chemical). Rates (in nmol/s) as a function of Ub-AMC concentration were fitted into the Michaelis-Menten equation to solve for K m and k cat using GraphPad Prism.

Tetra-ubiquitin Cleavage Assays

All tetrameric ubiquitin chains were purchased from Boston Biochem. Cleavage reactions were carried out at 37 � C in PBS with 1 mM DTT, 0.05% Tween 20, 0.1 mg/mL bovine serum albumin, and 2.5% DMSO with constant enzyme concentration of 200nM USP7cd or USP7cd-45 and constant substrate concentration of 3 uM tetra-ubiquitin in 23uL total reaction volume. 5uL samples were taken

Supplemental Information

Selectively Modulating Conformational States of USP7 Catalytic Domain for Activation