Charlotte A Dodson

Magda Kosmopoulou

Mark W Richards

Butrus Atrash

Vassilios Bavetsias

Julian Blagg

Richard Bayliss
email: richard.bayliss@icr.ac.uk

Aura

Crystal structure of an

Keywords: Aurora-A kinase, MLN8054, DFG-in, DFG-up, DFG-out, drug design, inhibitor selectivity ePK, eukaryotic protein kinase, ESRF, European Synchrotron Radiation Facility, FAM, carboxyfluorescein, K i , dissociation constant

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Following the success of Glivec in the treatment of chronic myelogenous leukaemia, the development of small molecule kinase inhibitors has become an established goal in drug discovery [START_REF] Zhang | Targeting cancer with small molecule kinase inhibitors[END_REF]. A major obstacle is that the ATP binding pocket, the ubiquitous druggable binding site for inhibitors, is highly conserved across more than 500 human kinases. The rapid growth in the number and chemical variety of kinase inhibitors has been aided by the contribution of structural biology especially the high resolution snapshots of kinase/inhibitor complexes captured by X-ray crystallography [START_REF] Zhang | Targeting cancer with small molecule kinase inhibitors[END_REF][START_REF] Van Montfort | Structure-based design of molecular cancer therapeutics[END_REF]. These studies revealed the specific conformations recognized by the two main classes of kinase inhibitor. Type I inhibitors recognize the active, typically phosphorylated, form of the kinase, and Type II inhibitors recognize an inactive, typically dephosphorylated form of the enzyme. In Type I inhibitor co-crystal structures, the conformation of the activation loop, a motif containing the regulatory phosphorylation sites of kinases, adopts the same conformation as that observed in ADP/ATP-bound structures. This conformation is termed DFG-in after the highly conserved aspartic acid, phenylalanine, glycine motif at the N-terminus of the activation loop. In the DFG-in conformation, the aspartic acid sidechain points into the active site where it coordinates divalent cations and the phenylalanine sidechain points into the interior of the protein. By contrast, Type II compounds stabilize a DFG-out conformation of the activation loop, in which the phenylalanine sidechain points outwards and interacts with the compound and the aspartic acid sidechain is displaced away from the active site [START_REF] Schindler | Structural mechanism for STI-571 inhibition of abelson tyrosine kinase[END_REF][START_REF] Pargellis | Inhibition of p38 MAP kinase by utilizing a novel allosteric binding site[END_REF][START_REF] Wan | Mechanism of activation of the RAF-ERK signaling pathway by oncogenic mutations of B-RAF[END_REF]. Structure-based drug design can be used to develop Type II, DFG-out binding compounds based on Type I, DFG-in binding precursors [START_REF] Liu | Rational design of inhibitors that bind to inactive kinase conformations[END_REF]. The field is still a long way from the direct rational design of selective and potent kinase inhibitors with a controlled selectivity profile. Currently, multiple iterative design cycles are 3 required to optimise compounds towards the desired biological profile. However, structural biology has provided insight and direction to the design of selective inhibitors that avoid off-target kinases. The selectivity profile of kinase inhibitors is very difficult to explain, even with the help of a high-resolution crystal structure. For example, nine years after the crystal structure of cAbl bound to a Glivec variant showed that this inhibitor recognizes the DFG-out conformation, there is still debate about the structural and thermodynamic basis for its selectivity [START_REF] Schindler | Structural mechanism for STI-571 inhibition of abelson tyrosine kinase[END_REF][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][START_REF] Dar | Small molecule recognition of c-Src via the Imatinib-binding conformation[END_REF]. It is clear, however, that the cAbl/Glivec variant structure was the inspiration for a new approach to kinase inhibitor design that has yielded many valuable compounds [START_REF] Liu | Rational design of inhibitors that bind to inactive kinase conformations[END_REF].

Many mitotic kinases are promising cancer drug targets because they are overexpressed in tumours, and their inhibition generates an aberrant cell division that can selectively kill tumour cells [START_REF] Jackson | Targeted anti-mitotic therapies: can we improve on tubulin agents?[END_REF]. A great deal of attention has been focused on the three Aurora kinases (A, B and C), and a number of structurally diverse inhibitors have been developed that target this family [START_REF] Keen | Aurora-kinase inhibitors as anticancer agents[END_REF][START_REF] Pollard | Discovery and development of aurora kinase inhibitors as anticancer agents[END_REF][START_REF] Bavetsias | Hit generation and exploration: imidazo[4,5-b]pyridine derivatives as inhibitors of Aurora kinases[END_REF]. The Millennium Pharmaceuticals compound MLN8054 is an Aurora-family specific inhibitor that inhibits 7 out of 226 kinases more than 50% at a concentration of 1 µM [START_REF] Manfredi | Antitumor activity of MLN8054, an orally active small-molecule inhibitor of Aurora A kinase[END_REF] or has a K d < 100 nM for 6 out of 317 kinases [START_REF] Karaman | A quantitative analysis of kinase inhibitor selectivity[END_REF]. Unlike any of the other Aurora inhibitors, MLN8054 is based on a benzazepine scaffold and it has been of considerable interest to determine the binding mode of this compound and shed light on its specificity. MLN8054 is also the first inhibitor discovered to show increased selectivity towards Aurora-A both in a human colorectal tumour cell-line and in vitro [START_REF] Manfredi | Antitumor activity of MLN8054, an orally active small-molecule inhibitor of Aurora A kinase[END_REF][START_REF] Karaman | A quantitative analysis of kinase inhibitor selectivity[END_REF]. Aurora-A is amplified and/or overexpressed in many cancers and cancer cell lines including hepatocellular carcinoma [START_REF] Jeng | Overexpression and amplification of Aurora-A in hepatocellular carcinoma[END_REF], breast cancers [START_REF] Zhou | Tumour amplified kinase STK15/BTAK induces centrosome amplification, aneuploidy and transformation[END_REF] and neuroblastoma cell lines [START_REF] Zhou | Tumour amplified kinase STK15/BTAK induces centrosome amplification, aneuploidy and transformation[END_REF]. Polymorphisms in the Aurora-A gene confer increased breast and colon tumour susceptibility and somatic mutations in Aurora-A have been identified in colorectal adenocarcinoma, lung carcinoma and melanoma [START_REF] Ewart-Toland | Identification of Stk6/STK15 as a candidate low-penetrance tumor-susceptibility gene in mouse and human[END_REF][START_REF] Greenman | Patterns of somatic mutation in human cancer genomes[END_REF]. Inhibition of Aurora-A may be a useful therapeutic strategy in these cancers, and clinical trials of several Aurora inhibitors, including a modified version of MLN8054 (MLN8237) are ongoing.

The selectivity of MLN8054 can be considered as a function of two individual factors: bias towards Aurora-A, and selectivity towards the Aurora family. To investigate the first factor, we have studied the effects of MLN8054 on both Aurora-A and an Aurora-B active-site mimic (AurA x3 ). We find that MLN8054 exhibits a 6-fold stronger inhibition of Aurora-A than AurA x3 in vitro. The crystal structure of AurA x3 bound to MLN8054 exhibits a highly unusual conformation of the activation loop. The characteristic features of the conformation are that both the aspartic acid and phenylalanine residues of the DFG motif point into the interior of the protein structure, and the activation loop wraps around the inhibitor and makes contact using a valine sidechain five residues after the start of the DFG motif. The structural details of this conformation, and a comparison of the key residues involved with those found in other human kinases show that it provides a rationale for understanding the selectivity of MLN8054 towards Aurora kinases. The binding of MLN8054 is accompanied by the creation of a hydrophobic pocket equivalent to that filled by the DFG Phe sidechain in a DFG-out structure. Our analysis suggests that a subset of kinases could be targeted through the design of inhibitors to fill this pocket, and that this would be an alterative route to producing highly specific compounds. Chemical synthesis MLN8054 was synthesized as previously described [START_REF] Claiborne | Preparation of pyrimidoazepine derivatives and methods for inhibiting mitotic progression[END_REF].

MATERIALS AND METHODS

Protein production and crystallization

The Quikchange (Stratagene) method of site-specific mutagenesis was used to produce AurA x3 (Leu215Arg, Thr217Glu and Arg220Lys), AurA TE (Thr217Glu) and AurA WL (Trp277Leu). AurA, AurA x3 , AurA TE and AurA WL proteins were expressed and purified as previously [START_REF] Bayliss | Structural basis of Aurora-A activation by TPX2 at the mitotic spindle[END_REF]. Crystals of AurA x3 /MLN8054 were obtained using the hanging drop method of vapour diffusion. For the P3 1 form, the well buffer was 0.8 M NaH 2 PO 4 /1.2 M K 2 HPO 4 and 0.1 M acetate, pH 4.5. For the P6 1 22 form, the well buffer was 0.1 M Mes sodium salt pH 6.5, 2.0 M ammonium sulfate, 5% (w/v) PEG400. Drops comprised a 1:1 ratio of AurA x3 at 7 mg/ml and 1 mM MLN8054 in DMSO. Crystals were briefly soaked in well buffer supplemented with 20% glycerol (P3 1 ) or 30% glycerol (P6 1 22) before flash freezing in liquid nitrogen.

In vitro kinase assays

Kinase assays were carried out at room temperature using a Labchip EZ Reader II system (Caliper Life Sciences) and a fluorescein-labelled substrate peptide (5-FAM-LRRASLG-CONH2) in a buffer of 50 mM Tris pH 7.5, 200 mM NaCl, 5 mM MgCl 2 , 10% glycerol, 2% DMSO and 10 mM β-mercaptoethanol. 1 nM (AurA), 0.51 nM (AurA x3 ), 0.15 nM (AurA TE ) or 0.4 nm (AurA WL ) enzyme in duplicate were preincubated in 25 µl reaction volume with 1.5 µM substrate peptide and stated concentration of MLN8054 for 10 mins before starting the reaction with stated concentrations of ATP. Measurements of substrate phosphorylation were taken every 5 mins for 1 hr (AurA, AurA TE and AurA WL ) and 30 mins (AurA x3 ). In the experiments to measure the effect of TPX2 on the potency of MLN8054, 3.4 µM TPX2 (residues 1-43) was pre-incubated with AurA for 10 mins before the addition of MLN8054 and substrate. This concentration is 340 times the EC 50 of TPX2, which was determined to be 10 nM under conditions of saturating ATP. All measurements conformed to the linear initial rate assumption and best-fit rates were used to construct rate v [ATP] curves. Data were fitted to a global model for competitive inhibition using Prism5 (www.graphpad.com).

Crystallographic data processing and refinement

Diffraction data was reduced using Mosflm, scaled with Scala and the molecular replacement solutions were obtained with Phaser using the structure of ADP-bound Aurora-A as a model [START_REF] Ccp4 | The CCP4 (Collaborative Computational Project Number 4) suite: programmes for protein crystallography[END_REF]. Four AurA x3 molecules were located in the P3 1 asymmetric unit, exhibiting clear difference density corresponding to two different conformations of the activation loop. Coordinates and refinement parameter (.cif) files for the MLN8054 ligand were made using Phenix [START_REF] Adams | PHENIX: building new software for automated crystallographic structure determination[END_REF]. Iterative cycles of model building and refinement were carried out using Coot and Phenix [START_REF] Ccp4 | The CCP4 (Collaborative Computational Project Number 4) suite: programmes for protein crystallography[END_REF][START_REF] Adams | PHENIX: building new software for automated crystallographic structure determination[END_REF]. All structure figures were produced using PyMOL (http://www.pymol.org).

Sequence analysis of the human kinome

The human ePK alignment containing 491 kinase domains, available at http://www.kinome.com, was used as the basis for calculating the frequency of amino acids at specific positions within the sequence as shown in Table 3 [START_REF] Manning | The protein kinase complement of the human genome[END_REF]. The sequence alignment was interrogated using a custom Perl script, available upon request.

Accession Codes

RESULTS

Characterisation of an active-site model of Aurora-B

The catalytic domains of human Aurora-A and Aurora-B are 76% identical at the primary sequence level, and differ by just three amino acids in their ATP-binding pockets -residues Leu215, Thr217 and Arg220 in Aurora-A are replaced by Arg159, Glu161, and Lys164 respectively in Aurora-B. To date, comparative studies have been frustrated because human Aurora-B has poor physico-chemical properties relative to Aurora-A and has not been crystallized. We constructed a triple point mutant version of the catalytic domain of Aurora-A (residues 122-403, Leu215Arg, Thr217Glu, Arg220Lys), which we call AurA x3 , as an Aurora-B active site mimic for crystallographic studies and to assess the contribution of the three residues to selectivity of MLN8054. The resulting enzyme is catalytically active, with kinetic parameters very similar to those for wild-type Aurora-A catalytic domain, which we call AurA (Figure 1a, Table 1). MLN8054 acts as an ATP-competitive inhibitor for both enzymes (Lineweaver-Burke plots intersecting on y-axis, data not shown) and the resultant K i s show a 6-fold discrimination for AurA over AurA x3 , greater than that which can be explained by the relative K m s of the active site for ATP alone. This level of discrimination compares well with the 7-fold difference in dissociation constants reported in the absence of ATP using bead-based competition binding assays [START_REF] Karaman | A quantitative analysis of kinase inhibitor selectivity[END_REF] although we report a consistently tighter binding (0.3 nM compared with 6.5 nM and 1.8 nM compared with 43 nM for Aurora-A and Aurora-B respectively). We have used equation (1) and our measured K i to calculate the IC 50 we would expect to measure for AurA under the experimental conditions reported for radioactive FlashPlate assays [START_REF] Manfredi | Antitumor activity of MLN8054, an orally active small-molecule inhibitor of Aurora A kinase[END_REF]. The calculated value of 3 nM compares very well with the published IC 50 of 4 nM although we should point out that the extremely tight binding of MLN8054 means that the IC 50 contains a large contribution from the assay enzyme concentration. A similar calculation for Aurora-B using the K i for AurA x3 and a commercially reported K m for Aurora-B (5 µM, http://www.caliperls.com/products/aurb-h.htm) is within a factor of two (calculated 93 nM, reported 172 nM).

IC 50 = K i 1+ S K m ⎛ ⎝ ⎜ ⎞ ⎠ ⎟ + [enzyme] 2 ( 1 )
We can thus conclude that the three point mutations differentiating AurA x3 and AurA provide a good model for assessing the contribution of active site residues to the binding of MLN8054. Interestingly, the difference between our measured K m for ATP for AurA x3 (46 µM) and the reported value for AurB (5 µM) means that we would expect additional residues outside the active site pocket to influence the binding of ATP. 2). There are four AurA x3 molecules in the asymmetric unit that adopt two slightly different protein conformations of which chains A and B are representative (Figure 2a). The two conformations are the result of two different crystal packing contacts between the activation loop and a neighbouring molecule (stars in Figure 2a). The structure of Aurora-A catalytic domain mutated on the two sites of autophosphorylation (Thr287Ala, Thr288Ala), which we shall refer to as AurA AA , bound to the 8054-like compound (shown in Figure 1b), has recently been determined (PDB code 3H10, [24]). This complex crystallized in a different crystal form (P2 1 2 1 2 1 ) to the AurA x3 /MLN8054 (P3 1 ). In chains A and B of this structure the activation loop of AurA AA has a very similar conformation to that found in chain B of the AurA x3 /MLN8054 structure and is involved in different crystal packing contacts (Figure 2a). The activation loop is disordered between residues 284-291 in the third chain (D) of the AurA AA /8054-like structure, which presumably cannot form a stabilizing crystal packing contact.

Binding mode of MLN8054 in the active site of Aurora kinase

Additional electron density within the active site of AurA x3 was very clear, and the MLN8054 ligand was fitted unambiguously (Figure 2b). The two different conformations of AurA x3 are in fact very similar in the vicinity of MLN8054, with the exception of residue Glu217, one of the residues mutated to mimic Aurora-B. In chain A Glu217 is closest to the benzoic acid moiety of MLN8054 (3.8 Å shortest interatomic distance), whereas in chain B Glu217 is closest to the difluorophenyl group (4.7 Å shortest interatomic distance). The active site of AurA AA bound to the 8054-like compound is very similar in all three chains and is very similar to that of either of the two chains of AurA x3 bound to MLN8054 (Figure 2b). In the structure of AurA AA /8054-like, the methyl group of Thr217 is closest to the difluorophenyl group (3.6-3.8Å). The substitution of Glu for Thr at position 217 has no effect on the binding mode. However, the individual Thr217Glu point mutant of Aurora-A (AurA TE ) is almost 20-fold less potently inhibited by MLN8054 than the wild-type AurA (Figure 1, Table 1). The difference in potency of MLN8054 inhibition between AurA and AurA x3 is therefore primarily due to the Thr217Glu mutation. The difference is modest because there is no steric clash and hence no change in binding mode, and the flexibility of the Glu217 sidechain reduces the effectiveness of the repulsion between the ionised glutamate and the hydrophobic difluorophenyl or benzoic acid groups of MLN8054. With the exception of Glu217, the interactions between MLN8054 and AurA x3 chains A and B are identical, as are the contacts between AurA AA and the 8054-like compound. MLN8054 forms two H-bonds with the hinge region of AurA x3 at Ala213, and a water-mediated Hbond to Asp274 (Figure 2c). The aromatic benzoic acid group sits above the C α of Gly216 in a position similar to that occupied by aromatic rings in other Aurora inhibitor structures (e.g. PDB codes 2BFY [START_REF] Sessa | Mechanism of Aurora B activation by INCENP and inhibition by hesperadin[END_REF], 2W1I [START_REF] Howard | Fragment-based discovery of the pyrazol-4-yl urea (AT9283), a multitargeted kinase inhibitor with potent aurora kinase activity[END_REF]). The chlorine atom and the aromatic ring to which it is attached pack against the Gly-rich loop. The difluorophenyl group packs against Leu263, Ala273 and Val279, which also packs against the chlorine atom. With the exception of the acidic group, the entire molecule of MLN8054 is buried inside the active site pocket of AurA x3 . Remarkably, this is achieved in part by the remodeling of the activation loop conformation to wrap around part of the inhibitor.

The conformation of the activation loop induced by binding of MLN8054

The conformations of the activation loop adopted by AurA x3 bound to MLN8054 and AurA AA bound to the 8054-like compound are highly unusual. The activation loop is flipped around compared to the DFG-in conformation observed in the ADP-bound AurA structure [START_REF] Bayliss | Structural basis of Aurora-A activation by TPX2 at the mitotic spindle[END_REF] and superficially resembles a DFG-out conformation, as observed in AurA/quinazoline-13 or cAbl bound to a Glivec variant (Figure 3a, PDB codes 2C6E and 1FPU respectively [START_REF] Schindler | Structural mechanism for STI-571 inhibition of abelson tyrosine kinase[END_REF][START_REF] Heron | SAR and inhibitor complex structure determination of a novel class of potent and specific Aurora kinase inhibitors[END_REF]). Upon closer inspection, the conformation of the activation loop of AurA x3 /MLN8054 is clearly different. In DFG-in and DFG-out conformations of kinases, the aspartic acid and phenylalanine sidechains sit on opposite sides of the backbone. In these conformations, when the phenylalanine sidechain points into the interior of the protein, the aspartic acid sidechain points out into the active site and vice versa (Figure 3b). By contrast, in the AurA x3 /MLN8045 and AurA AA /8054-like structures, both Asp274 and Phe275 project up into the interior of the kinase (Figure 3b,c). For this reason, and for the sake of brevity, we shall refer to this conformation as DFG-up.

The DFG-up conformation is characterized by a substantial reorganization of the activation loop between residues 274-293 and the HRD-motif at residues His254 and Arg255. Compared with the DFG-in conformation, the sidechain of Asp274 is rotated by approximately 180° so that it points into the interior of the protein, parallel to helix αC (Figure 3b). The sidechain of Phe275 points inwards and occupies a pocket above the Glu181 sidechain, whereas in the DFG-in conformation Phe275 occupies a pocket below Glu181. The Glu181 sidechain makes a H-bond with the indole ring of Trp277, which is twisted around from its outward position in the DFG-in conformation. Glu181 is a crucial residue for catalysis because it forms a salt bridge with Lys162 that positions the phosphates of ATP for transfer to substrate. In the DFG-up conformation, Lys162 forms a H-bond with Ser278 of the activation loop. Thus, although the salt bridge is broken in the DFG-up conformation, both residues find alternative partners with which to form H-bonds. The conformational change in the activation loop moves the C α of Val279 approximately 19 Å compared to its position in the DFG-in structure. Val279 can thus make two points of contact with MLN8054/8054-like. Beyond Val279, the conformations of chains A and B deviate until the end of the activation loop (Leu293), and the differences are likely due to the different crystal packing contacts (Figure 2a). The sidechains of the HRD motif are switched in the DFG-up structure. His254 points outwards, whereas in the DFG-in conformation it usually points inwards and stacks against Phe275. Arg255 points inwards and interacts with Asp274 (DFG motif) and Asn185 (helix αC), whereas it normally faces outwards, and recognises the phosphorylated Thr288 in an active AurA structure (PDB code 1OL5 [START_REF] Bayliss | Structural basis of Aurora-A activation by TPX2 at the mitotic spindle[END_REF]).

The DFG-up conformation is distinct from the DFG-out conformation that is observed in the structures of quinazoline-13 bound to Aurora-A or a Glivec variant bound to cAbl. Apart from the differences in the DFG motif, in the DFG-out conformation Trp277 interacts with the quinazoline-13 inhibitor and the arrangement of the HRDmotif is similar to that found in the DFG-in conformation. Additionally, structures of Aurora-A and cAbl in the DFG-out conformation are of dephosphorylated kinases, whereas the DFG-up is observed with either phosphorylated or unphosphorylated Aurora-A. We speculate that phosphorylated Aurora-A can adopt DFG-up (and probably DFG-out) conformations without much energetic penalty because the A c c e p t e d M a n u s c r i p t 8 phosphorylated activation loop of Aurora-A is not stably locked in the active conformation except in the presence of TPX2 [START_REF] Bayliss | Structural basis of Aurora-A activation by TPX2 at the mitotic spindle[END_REF]. One prediction from the AurA x3 /MLN8054 structure is that the binding of TPX2 should decrease the potency of MLN8054 either through competition or because more energy is required to move the activation loop from its stable, active conformation into DFG-up. We determined the K i for MLN8054 for AurA in the presence of a saturating concentration of TPX2 to be 3.3 ± 0.2 nM, a substantial increase on the K i in the absence of TPX2 comparable to that obtained with AurA x3 or AurA TE (Figure S1, Table 1, Table S1).

Insights into the selectivity of MLN8054 for Aurora kinases

Close inspection of the structure shows that many of the interactions between residues proximal to the active site are rearranged with respect to previous Aurora-A structures. This observation led us to consider whether other kinases might be able to adopt a similar conformation and if this might have any bearing on the selectivity of MLN8054. A search of the published alignment of human kinases and a search using the ProSite server reveals that only Aurora kinases possess a tryptophan at the equivalent position to 277, three residues after the start of the DFG motif (D+3, Table 3) [START_REF] Manning | The protein kinase complement of the human genome[END_REF]. Most of the more frequent residues at this position are hydrophobic and could not form a H-bond with Glu181. The exception to this is serine, found in 7.5% of kinases, although the position of the sidechain would have to be shifted by 2 Å to make the H-bond. In addition, Trp277 makes hydrophobic contacts with Phe144 and Val174 that could not be made by a serine sidechain. We mutated AurA at Trp277 to leucine, the most common residue at this position in human protein kinases. The K i for MLN8054 for AurA WL was determined to be 4.4 ± 0.4 nM, comparable to that determined for AurA x3 or AurA TE (Figure S1, Table S1). This supports our suggestion that the unusual tryptophan residue at position 277 contributes to MLN8054 selectivity towards Aurora kinases. Val279 at D+5 is also unusual at its position in the kinase domain and is found in less than 5% of kinases (Table 3). In most kinases that are regulated by phosphorylation of the activation loop the equivalent residue at this sequence position is most frequently a lysine or arginine, which interacts with the activating phosphate group (i.e. on a residue equivalent to Thr288) and helps order the activation loop. Full ordering of the activation loop of Aurora-A proceeds by a different mechanism than most kinases because binding of an activator such as TPX2 is required in addition to phosphorylation on Thr288 [START_REF] Bayliss | Structural basis of Aurora-A activation by TPX2 at the mitotic spindle[END_REF]. The interior surface of Aurora kinases that is contacted by the DFG motif in the DFG-up conformation is highly unusual. In most kinases, this region is hydrophobic, and in particular there is a conserved hydrophobic residue on the αC helix that forms part of the hydrophobic spine of kinases and interacts with the Phe sidechain of the DFG motif in the DFG-in conformation [START_REF] Kornev | Surface comparison of active and inactive protein kinases identifies a conserved activation mechanism[END_REF]. Across all human kinases, this residue is a leucine in 53% of cases, and a methionine in 25%. In the Aurora family, this hydrophobic residue is replaced by Gln185, which makes a H-bond with the backbone at Leu194. The presence of Gln185 is presumably important for the DFG-up conformation because it lies at the centre of a network of polar interactions that involves the sidechains of Asp274, Arg255 and the backbone at Leu194. Only COT, a mitogen-activated protein kinase kinase kinase, has both equivalent identical residues to Gln185 and Val279, and no other kinase has even a related combination (Ile or Leu for Val, His or Asn for Gln). The Aurora family thus has a unique set of features that create the capacity to adopt the MLN8054-bound activation loop conformation: the presence of the polar back pocket, the lack of a basic residue at D+5 and the tryptophan at D+3. This

A model to explain the structural and chemical basis for the conformational changes in DFG-out and DFG-up conformations of Aurora-A

We obtained a second crystal structure of the complex at 3.3Å resolution (AurA x3 /MLN8054*) in a P6 1 22 crystal form that is very commonly obtained with AurA (Table 2). In this structure, the activation loop adopts a DFG-in conformation (Figure 4a,b). This may simply represent a crystallization artefact, because the crystal packing is incompatible with the DFG-up conformation of the activation loop, which appears to be trapped in the DFG-in conformation by crystal packing contacts. When compared to the ADP-bound AurA structure, there is a steric clash between the Glyrich loop in the ADP-bound conformation and MLN8054 (star, Figure 4b). In the AurA x3 /MLN8054* structure the Gly-rich loop is displaced upwards due to contacts between MLN8054 and the Gly-rich loop at the sidechain at Val147 and the mainchain at residues 140-141 (Figure 4b). The AurA x3 /MLN8054* structure shows that the MLN8054 can be accommodated in the active site without movement of the activation loop, but requires displacement of the Gly-rich loop. Superposition of the higher resolution AurA x3 /MLN8054 or AurA AA /8054-like structures show the same steric clash between the Gly-rich loop of the ADP-bound structure with MLN8054 and a second steric clash between the Gly-rich loop of the ADP-bound structure (at residues 142-143) with the activation loop of the MLN8054-bound structure at Val279 (Figure 4c). The higher resolution structures show a displacement of the Glyrich loop similar to that observed at low resolution in the AurA x3 /MLN8054* structure. It appears that the upwards movement of the Gly-rich loop is a necessary adjustment to accommodate the activation loop in the DFG-up conformation, and could be an initial first step upon binding of MLN8054 to Aurora-A.

The fact that the DFG-up conformation has not been reported before suggested to us that there is something unusual about the binding of MLN8054 that induces this conformation. The superposition of AurA/ADP with AurA x3 /MLN8054 shows that the difluorophenyl group of MLN8054 occupies the equivalent space to that occupied by the α-PO 4 group of ADP, which we shall refer to as the α-PO 4 space (Figure 4d). The two groups have vastly different chemical properties, and the properties of this space are altered between DFG-in and DFG-up conformations. In the DFG-in conformation, Asp274 sits to one side of the α-PO 4 space and together with Asn261 coordinates two divalent cations (e.g. Mg 2+ ) that stabilise the ADP/ATP phosphates.

In the DFG-up conformation, Asp274 is flipped and the Mg 2+ binding sites are eliminated. The movement of Val279 completes the conversion of the α-PO 4 space to a hydrophobic pocket, which is occupied by the difluorophenyl group of MLN8054 (Figure 3b,4d). The DFG-up conformation of Aurora-A could therefore be driven by the occupation of the α-PO 4 space by the difluorophenyl group of MLN8054. Interestingly, a similar change to the chemical properties of the α-PO 4 space occurs in the DFG-out conformation. In fact, the DFG-up and DFG-out conformations are clearly related and they possess two equivalent hydrophobic pockets that can be filled by a phenyl group (yellow, green dashed circles Figure 4e). The groups which occupy these two pockets in the DFG-up or DFG-out conformations display a degree of symmetry. In the DFG-out conformation, the α-PO 4 space is occupied by the Phe275 sidechain and the second pocket is filled by the quinazoline-13 compound (Figure 3b, ). In the DFG-up conformation, the α-PO 4 space is occupied by the difluorophenyl group of MLN8054 and the second pocket is filled by the sidechain of Phe275. Further exploration of the chemical, structural and biophysical relationship between these two conformations may be beneficial in the production of more selective Aurora compounds.

DISCUSSION

The observation of the same, unusual activation loop conformation induced by two similar compounds bound to variants of the protein which are unphosphorylated (AurA AA ) or phosphorylated with mutations in the active site (AurA x3 ), and which were crystallized in two different crystals forms, is strong evidence that this conformation is neither an artefact of any of the protein modifications nor an artefact of crystal packing, but is driven by binding of the MLN8054/8054-like compounds. This raises the concern that selectivity testing for MLN8054 may be difficult to interpret because the inhibitor might be expected to bind more tightly to off-target kinases in the inactive state rather than the active proteins which are used in assay screens. Our analysis of the sequence requirements for the DFG-up conformation raises the question of how MLN8054 is able to inhibit several other kinases, albeit with lower potency than the Aurora family, such as Abl (76% inhibition at 1 µM) and Yes (80% inhibition at 1 µM) [START_REF] Manfredi | Antitumor activity of MLN8054, an orally active small-molecule inhibitor of Aurora A kinase[END_REF]. It is possible that these kinases are able to adopt the DFG-up conformation, and that the lower potency is partly because this conformation is a less favoured, higher energy state. Alternatively, MLN8054 could bind to these kinases in a different conformation, such as the DFG-in conformation observed in the low-resolution AurA x3 /MLN8054* structure. The determination of the selectivity profile of this class of inhibitor is therefore perhaps most accurately achieved by using a direct binding screen against inactive and active forms of the kinases [START_REF] Karaman | A quantitative analysis of kinase inhibitor selectivity[END_REF]. Rational design and prediction of selectivity profiles remains an extremely difficult challenge given the existence of multiple inactive conformations accessible to protein kinases and the possibility that some compounds might be able to target more than one conformation.

It has been suggested that Thr217 might be the basis for the higher potency of MLN8054 for Aurora-A over Aurora-B[24]. We have now confirmed that Thr217 is the main determinant of its bias towards Aurora-A in vitro and shown the relative disposition of a glutamic acid sidechain at this position in the crystal stucture. MLN8054 does not fully utilize the potential of the Thr217Glu substitution to generate selectivity, because the compound does not sit very close to residue 217 and can still be accommodated when the residue is substituted for glutamic acid. The selectivity of MLN8054 for Aurora-A over Aurora-B in vitro is not high, only approximately 6-fold using strictly comparable K i or K d measurements. In contrast, MLN8054 is over 100-fold selective for Aurora-A over Aurora-B in cell-based assays and the compound can be used to obtain a cellular phenotype consistent with clear discrimination [START_REF] Manfredi | Antitumor activity of MLN8054, an orally active small-molecule inhibitor of Aurora A kinase[END_REF][START_REF] Leroy | Localization of human TACC3 to mitotic spindles is mediated by phosphorylation on Ser558 by Aurora A: a novel pharmacodynamic method for measuring Aurora A activity[END_REF]. The Aurora-A activator TPX2 reduces the potency of MLN8054 versus Aurora-A, although only a specific pool of Aurora-A localized to mitotic spindle microtubules is thought to be bound to TPX2. The cellular potency of Aurora-A might be due to targeting the non-TPX2-bound pool of Aurora-A. The mechanism by which Aurora-B is only poorly inhibited by MLN8054 in cells is presently unclear. It is possible that Aurora-B is prevented from adopting the DFG-up conformation through binding to a protein partner, but it is not obvious how this could The structure of AurA x3 bound to MLN8054 suggests that the specificity of this compound for the Aurora family is at least partly a function of the protein conformation it induces. This does not imply that we believe it to be impossible to produce inhibitors that target other kinases in a DFG-up conformation. On the contrary, it might be possible to exploit the principle behind the DFG-up conformation to generate compounds that induce the DFG-up conformation of other kinases, just as it has been possible to generate potent inhibitors that bind the DFGout conformation of a number of kinases. This is because the conformation induced by these compounds would be DFG-up, but would differ in the precise details of the conformation adopted by the activation loop, especially the residues just after the DFG motif. One feature that is potentially vital for adopting a DFG-up conformation is the polar residue on the C-helix (Gln185), and an equivalent polar residue is present in just under 10% of human kinases. We predict that these would be the most likely targets for DFG-up inhibitors. The other key features of the DFG-up conformation are the interactions within the kinase domain and between the activation loop at D+5 (Val279) and the inhibitor. To design a DFG-up targeting inhibitor, we predict that one should modify a Type I inhibitor with the addition of a hydrophobic group to fit the α-PO 4 pocket that is complementary to the residue at D+5. One might expect that this would only work with kinases that possess residues at D+3 and D+4 capable of making compensating interactions with the disrupted Lys-Glu salt bridge residues. Future studies will elaborate which other kinases can be induced to form the DFG-up conformation. Furthermore, the discovery of the DFG-up conformation, and our recent discovery of multiple, stable, ligand-induced conformations of Nek2 kinase [START_REF] Westwood | Insights into the conformational variability and regulation of human Nek2 kinase[END_REF] suggest to us that the conformational space of the activation loop should be further explored, and exploited to produce selective inhibitors.

AUTHOR CONTRIBUTION

Charlotte Dodson carried out the enzyme kinetics studies, contributed to the interpretation of kinetic data and contributed to the writing of the paper. Magda Kosmopoulou prepared the proteins, generated crystals, determined the X-ray crystal structures and contributed to the interpretation of structural data. Mark Richards carried out DNA manipulations and mutagenesis. Butrus Atrash synthesized the MLN8054 compound. Vassilios Bavetsias advised on the design of the synthetic route to MLN8054 and contributed to the interpretation of data in relation to MLN8054 Aurora isoform selectivity. Julian Blagg provided leadership in the generation of MLN8054 and contributed to the interpretation of data in relation to MLN8054 Aurora isoform selectivity. Richard Bayliss was the principal investigator on the project, contributed to the X-ray crystal structure determination, interpreted the data and wrote the paper. A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Biochemical

  Journal Immediate Publication. Published on 12 Jan 2010 as manuscript BJ20091530 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20091530 A c c e p t e d M a n u s c r i p t 4

Biochemical

  Journal Immediate Publication. Published on 12 Jan 2010 as manuscript BJ20091530 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20091530 A c c e p t e d M a n u s c r i p t Coordinates and structure factors of AurA x3 /MLN8054 structures have been deposited in the Protein Data Bank (PDB) with accession code 2WTV (P3 1 ) and 2WTW (P6 1 22).

BiochemicalA c c e p t e d M a n u s c r i p t 6 The

 6 Journal Immediate Publication. Published on 12 Jan 2010 as manuscript BJ20091530 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20091530 chemical structure of MLN8054 is shown in Figure 1b. AurA x3 was cocrystallised with MLN8054 and the structure determined to a resolution of 2.4 Å (Table

  Biochemical Journal Immediate Publication. Published on 12 Jan 2010 as manuscript BJ20091530 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20091530 A c c e p t e d M a n u s c r i p t

Biochemical

  Journal Immediate Publication. Published on 12 Jan 2010 as manuscript BJ20091530 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20091530

Biochemical

  Journal Immediate Publication. Published on 12 Jan 2010 as manuscript BJ20091530 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20091530 A c c e p t e d M a n u s c r i p t suggests that the conformation of AurA x3 bound to MLN8054 might not be easily accessible to other kinases, and might thus contribute to the selectivity of MLN8054.

Biochemical

  Journal Immediate Publication. Published on 12 Jan 2010 as manuscript BJ20091530 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20091530 A c c e p t e d M a n u s c r i p t 10 4e

Biochemical

  Journal Immediate Publication. Published on 12 Jan 2010 as manuscript BJ20091530 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20091530 A c c e p t e d M a n u s c r i p t be mediated from current structures of the Aurora-B/INCENP complex. The elucidation of this mechanism may be important for understanding the cellular regulation of these kinases.

A c c e p t e d M a n u s c r i p t 12 Boxall,

 12 Ian Collins, Amir Faisal, Spiros Linardopoulos and Rob van Montfort at the Centre for Cancer Therapeutics. The authors are employees of The Institute of Cancer Research which has a commercial interest in a number of drug discovery programs for which further information is available from the ICR Enterprise Unit. Julian Blagg is a consultant and stock holder of both Pfizer and Solace Pharmaceuticals.

Figure 1 .

 1 Figure 1. Comparison of AurA, AurA x3 and AurA TE kinetic parameters and MLN8054 inhibition a) Global fits of apparent ATP K m in presence of varying concentrations of MLN8054: 0 nM (black), 0.02 nM (dark grey, AurA only), 0.06 (light grey, AurA x3 and AurA TE only, filled circles), 0.2 nM (black), 0.6 nM (dark grey), 2 nM (light grey, open circles), 5 nM (black), 16 nM (dark grey), 50 nM (light grey, AurA x3 and AurA TE only, diamonds). Error bars and quoted errors are fitting errors of duplicate measurements. The average of three repeat measurements in duplicate for AurA x3 did not alter the K i or K m , and standard errors of these values were the same magnitude as fitting errors quoted. b) Chemical structures of MLN8054, the 8054-like compound [24] and quinazoline-13 [27].

Figure 2 .Figure 3 .

 23 Figure 2. Binding mode of MLN8054a) Two different conformations of AurA x3 are shown as yellow cartoons, with MLN8054 shown as spheres using the colour scheme carbon grey, nitrogen blue, oxygen red, chlorine green and fluorine cyan. The structure of AurA AA is shown as an orange cartoon, with the 8054-like compound shown in the same colour scheme as MLN8054. Crystal contacts that direct the different paths of activation loops are marked with stars, and the neighbouring protein molecule is depicted as grey cartoon. b) Magnified view of the active site. The mainchain of AurA x3 /AurA AA is shown as a C α trace. MLN8054/8054-like and residues lining the active site of AurA x3 /AurA AA are shown in stick representation. The final 2mF o -dF c electron density map around MLN8054 is shown as a wiremesh contoured at 1.0σ. c) Magnified view of the active site shown in a view rotated through approximately 90º about the horizontal axis relative to (b). Putative H-bonds are shown as dashed lines, and an ordered water molecule that mediates contact between the ligand and the protein is shown as a red sphere. The sidechain of Tyr212 is omitted so that the Hbonds between Ala213 and the ligand are not obscured.

Figure 4 .

 4 Figure 4. Activation loop conformational differences between Aurora-A structures are perhaps driven by the chemical group that occupies the α-PO4 space a) Superposition of AurA/ADP (protein dark teal cartoon, ADP dark teal sticks, PDB code 1OL7[START_REF] Bayliss | Structural basis of Aurora-A activation by TPX2 at the mitotic spindle[END_REF]) with the 3.3 Å resolution AurA x3 /MLN8054* structure (protein pink cartoon, MLN8054 pink sticks). b) Magnified view of the superposition, with mainchain shown as ribbon and key residues shown as sticks and the ADP molecule omitted for clarity. The final 2mF o -dF c electron density map around MLN8054 is shown as a wiremesh contoured at 1.0σ. The steric clash between the Gly-rich loop (shown as a thicker ribbon) in the ADP-bound structure and MLN8054 is highlighted with a black star. c) Superposition of chain A of AurA x3 /MLN8054 structure (chain A, protein yellow ribbon, MLN8054 grey sticks) with structure of phosphorylated, ADP-bound AurA (protein dark teal ribbon, ADP dark teal sticks, PDB code 1OL7[START_REF] Bayliss | Structural basis of Aurora-A activation by TPX2 at the mitotic spindle[END_REF]). Key regions of conformational difference are marked. d) Magnified view of superposition to show detail of active site. The upper panel is depicted in the same orientation as (c), the view in the lower panel is rotated through 90º about the vertical axis. Steric clashes between MLN8054 or the activation loop of MLN8054-bound AurA x3 and the Gly-rich loop of ADP-bound AurA are highlighted with single and double stars respectively. Magnesium ions in the ADP-bound structure are depicted as spheres. In the lower panel, the deviation between the two structures starting from Asp274 in the activation loop is clearly evident. e) Comparison of the phenyl-binding pockets of the three different DFG motif conformations. The left-hand images show simplified structural models of the three conformations viewed from the back of the active site looking out to solvent (i.e. 180º rotation relative to the top panel of Figure4d). The mainchain of the DFG motif is shown as a magenta cartoon, the Asp274 sidechain is shown as magenta space-filling, the sidechain of Phe275 as dark grey space-filling and the ligand as space-filling with the following colour scheme: carbon light grey, nitrogen blue, oxygen red, phosphorous orange, fluorine cyan. The right-hand images show a schematic diagram of the comparison. The α-PO 4 space is shown as a dashed yellow circle and the second phenyl-binding space is shown as a dashed green circle. The pocket occupied

1 )

 1 Only residues that occur in more than 10 kinase domains are shown, with the exception of Trp at position 277. Aurora kinase residues are marked in bold and underlined. Biochemical Journal Immediate Publication. Published on 12 Jan 2010 as manuscript BJ20091530 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20091530 A c c e p t e d M a n u s c r i p t Publication. Published on 12 Jan 2010 as manuscript BJ20091530 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20091530 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2010 The Authors Journal compilation © 2010 Portland Press Limited Publication. Published on 12 Jan 2010 as manuscript BJ20091530 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20091530

Table 1 . Summary of parameters determined for kinase activity and inhibition by MLN8054

 1 Errors on k cat are propagated from the fitting error on V max , assuming a 10% error in the determination of the final concentrations of enzyme and peptide. The K m for ATP for AurA is in excellent agreement with the previously published value30 .

	AurA 20 ± 2 4.8 ± 0.7 0.27 ± 0.03 a) Biochemical Journal Immediate Publication. Published on 12 Jan 2010 as manuscript BJ20091530 AurA x3 AurA TE K m, ATP ( M) 46 ± 3 72 ± 5 k cat (min -1 ) a 8.1 ± 1.2 52.4 ± 7.5 K i (nM) 1.8 ± 0.2 5.2 ± 0.3

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Table 2 . Summary of crystallographic analysis

 2 Biochemical Journal Immediate Publication. Published on 12 Jan 2010 as manuscript BJ20091530 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20091530 A c c e p t e d M a n u s c r i p t

	Crystals	AurA x3 /MLN8054 AurA x3 /MLN8054*
	Spacegroup	P3 1	P6 1 22
	Lattice constants a (Å)	118.71	84.04
	b (Å)	118.71	84.04
	c (Å)	135.89	171.06
	Data collection
	Resolution range (Å)	59.3 -2.4	72.9 -3.3
	(Highest resolution shell)	(2.53 -2.40)	(3.30-3.48)
	Unique reflections	83351 (11952)	5790 (815)
	Completeness(%)	99.5 (97.6)	99.6 (99.5)
	Multiplicity	3.4 (2.7)	14.8 (14.8)
	Rmerge (%)	8.3 (34.6)	11.6 (46.7)
	I/ (I)	12.3 (3.2)	20.6 (7.7)
	Refinement
	Resolution range	19.8 -2.4	67 -3.3
	Number of reflections	79178	5738
	Rfactor (%)	16.4	23.4
	Rfree a (%)	21.1	28.8
	R.m.s. deviations
	Bond lengths (Å)	0.005	0.004
	Bond Angles ( )	1.05	0.820
	Ramachandran outliers (%)	0.0	0.4
	Ramachandran favoured (%)	98.4	92.9

a Free Rfactor was computed using 5% of the data assigned randomly.

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Table 3 . Frequency of amino acids in the N-terminus of human kinase domain activation loops

 3

	Aurora-A kinase residue numbering
	D	D+1	D+2	D+3	D+4	D+5
	274	275	276	277	278	279
	Frequency of occurrence in human ePK kinase domain sequences 1
	Asp (458)	Phe (403)	Gly (446)	Leu (245)	Asp (247)	Arg (157)
		Leu (50)		Phe (58)	Ser (144)	Lys (109)
		Tyr (18)		Ser (37)	Cys (42)	Thr (33)
				Met (34)	Thr (18)	Ala (30)
				Val (31)	Gly (15)	Val (23)
				Ile (23)		Ser (22)
				.		Cys (16)
				.		Asn (16)
				Trp

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© 2010 The Authors Journal compilation © 2010 Portland Press Limited

ACKNOWLEDGEMENTS

We are indebted to the staff of ESRF beamlines ID14.1 and ID14.4 for their support during data collection. We wish to thank our colleagues at The Institute of Cancer Research for their helpful comments and support, especially Wynne Aherne, Kathy

FUNDING

This study was supported by a Royal Society University Research Fellowship, the Career Development Faculty Programme of The Institute of Cancer Research, a Cancer Research UK project grant (C24461/A8032) and a Breakthrough Breast Cancer project grant (AURA 05/06) to R.B., and the core Cancer Research UK support to the Centre for Cancer Therapeutics (C309/A8274) to J.B. We acknowledge infrastructural support for structural biology at The Institute of Cancer Research by Cancer Research UK and NHS funding to the National Institute for Health Research (NIHR) Biomedical Research Centre.


Clément Faye

Antonio Inforzato

Marine Bignon

Daniel J Hartmann

Laurent Muller

Lionel Ballut

Bjorn R Olsen

Anthony J Day

Sylvie Ricard-Blum
email: s.ricard-blum@ibcp.fr

TRANSGLUTAMINASE-2: A NEW ENDOSTATIN PARTNER IN THE EXTRACELLULAR MATRIX OF ENDOTHELIAL CELLS

Keywords: Endostatin, Protein-protein interaction, SPR binding assays, Transglutaminase-2

Endostatin, the C-terminal domain of collagen XVIII, binds to transglutaminase-2 (TG-2) in a cation-dependent manner. Recombinant human endostatin binds to TG-2 with an affinity in the nanomolar range (K D = 6.8 nM). Enzymatic assays indicated that, in contrast to other extracellular matrix proteins, endostatin is not a glutaminyl substrate of TG-2 and is not cross-linked to itself by the enzyme. Two arginine residues of endostatin, R27 and R139, are crucial for its binding to TG-2. They are also involved in the binding to heparin (Sasaki et al., , and to α5β1 and αvβ3 integrins (Faye et al., J Biol Chem 284:22029-22040), suggesting that endostatin is not able to interact simultaneously with TG-2 and heparan sulfate, or with TG-2 and integrins. Inhibition experiments support the GTP binding site of TG-2 as a potential binding site for endostatin. Endostatin and TG-2 are colocalized in the extracellular matrix secreted by endothelial cells under hypoxia, that stimulates angiogenesis. This interaction occurring in a cellular context might participate in the concerted regulation of angiogenesis, and tumorigenesis by the two proteins.

INTRODUCTION

Endostatin (ES) is a C-terminal fragment of the α1 chain of collagen XVIII, which inhibits angiogenesis, and tumor growth [START_REF] Folkman | Antiangiogenesis in cancer therapy--endostatin and its mechanisms of action[END_REF][START_REF] Cao | Molecular mechanisms and therapeutic development of angiogenesis inhibitors[END_REF]. It binds to α5β1 and αvβ3 integrins [START_REF] Rehn | Interaction of endostatin with integrins implicated in angiogenesis[END_REF][START_REF] Faye | Molecular interplay between endostatin, integrins, and heparan sulfate[END_REF] glypicans 1 and 4 [START_REF] Karumanchi | Cell surface glypicans are low-affinity endostatin receptors[END_REF], and to vascular endothelial growth factor receptor-2 (VEGFR-2, Flk1/KDR) [START_REF] Kim | Endostatin blocks vascular endothelial growth factor-mediated signaling via direct interaction with KDR/Flk-1[END_REF] on the surface of endothelial cells. The binding of human endostatin to α5β1 integrin leads to the inhibition of focal adhesion kinase/c-Raf/MEK1/2/p38/ERK1 mitogenactivated protein kinase pathway [START_REF] Sudhakar | Human tumstatin and human endostatin exhibit distinct antiangiogenic activities mediated by alpha v beta 3 and alpha 5 beta 1 integrins[END_REF]. Endostatin also inhibited the binding of VEGF165 to both endothelial cells and to the purified extracellular domain of KDR/Flk-1. Furthermore, endostatin blocks VEGF-induced tyrosine phosphorylation of KDR/Flk-1 and activation of ERK, p38 MAPK, and p125(FAK) in human umbilical vein endothelial cells [START_REF] Kim | Endostatin blocks vascular endothelial growth factor-mediated signaling via direct interaction with KDR/Flk-1[END_REF].

Endothelial cells are a rich source of transglutaminase-2 (TG-2) [START_REF] Korner | Bovine aortic endothelial cell transglutaminase. Enzyme characterization and regulation of activity[END_REF], and this enzyme plays an important role at the surface of those cells. For example, evidence has been reported for its externalization and its co-localization with the β1 integrin [START_REF] Gaudry | Tissue transglutaminase is an important player at the surface of human endothelial cells: evidence for its externalization and its colocalization with the beta(1) integrin[END_REF]. Cell surface TG-2 interacts with integrins of the β1 and β3 subfamilies in focal adhesion sites [START_REF] Akimov | Tissue transglutaminase is an integrin-binding adhesion coreceptor for fibronectin[END_REF], and the formation of a complex between TG-2 and VEGFR-2 has been proposed as a mechanism for modulation of endothelial cell response to VEGF [START_REF] Dardik | Complex formation between tissue transglutaminase II (tTG) and vascular endothelial growth factor receptor 2 (VEGFR-2): proposed mechanism for modulation of endothelial cell response to VEGF[END_REF]. Conflicting in vivo studies have proposed that the enzyme both stimulates and inhibits angiogenesis [START_REF] Haroon | Tissue transglutaminase is expressed, active, and directly involved in rat dermal wound healing and angiogenesis[END_REF][START_REF] Haroon | Tissue transglutaminase is expressed as a host response to tumor invasion and inhibits tumor growth[END_REF]. However application of exogenous TG-2 blocks angiogenesis in a dose-dependent manner in an in vitro angiogenesis assay without causing cell death, by a mechanism that involves increased accumulation of extracellular matrix proteins [START_REF] Jones | Matrix changes induced by transglutaminase 2 lead to inhibition of angiogenesis and tumor growth[END_REF]. This Ca 2+ -dependent enzyme catalyzes post-translational modification of proteins by the formation of γ-glutamyl-ε-lysine bonds between glutamine and lysine residues [START_REF] Griffin | Transglutaminases: nature's biological glues[END_REF][START_REF] Lorand | Transglutaminases: crosslinking enzymes with pleiotropic functions[END_REF][START_REF] Collighan | Transglutaminase 2 cross-linking of matrix proteins: biological significance and medical applications[END_REF]. TG-2 is a multifunctional enzyme which undergoes a GTP-binding/GTPase cycle, with guanine nucleotide and calcium binding reciprocally regulating its transamidation activity [START_REF] Ruan | The Differential Effects of R580A Mutation on Transamidation and GTP Binding Activity of Rat and Human Type 2 Transglutaminase[END_REF].

Endostatin and TG-2 share several extracellular partners such as nidogen, SPARC, collagen VI, and the β-amyloid peptide [START_REF] Griffin | Transglutaminases: nature's biological glues[END_REF][START_REF] Sasaki | Structural basis and potential role of heparin/heparan sulfate binding to the angiogenesis inhibitor endostatin[END_REF][START_REF] Dieterich | Cross linking to tissue transglutaminase and collagen favours gliadin toxicity in coeliac disease[END_REF][START_REF] Hartley | Transglutaminase induces protofibril-like amyloid beta-protein assemblies that are protease-resistant and inhibit long-term potentiation[END_REF][START_REF] Faye | The first draft of the endostatin interaction network[END_REF]. Both proteins are also able to bind to heparin [START_REF] Sasaki | Structural basis and potential role of heparin/heparan sulfate binding to the angiogenesis inhibitor endostatin[END_REF][START_REF] Feraud | Characterization of endostatin binding to heparin and heparan sulfate by surface plasmon resonance and molecular modeling: role of divalent cations[END_REF][START_REF] Gambetti | Interaction with heparin protects tissue transglutaminase against inactivation by heating and by proteolysis[END_REF], α5β1 and αvβ3 integrins [START_REF] Rehn | Interaction of endostatin with integrins implicated in angiogenesis[END_REF][START_REF] Faye | Molecular interplay between endostatin, integrins, and heparan sulfate[END_REF][START_REF] Akimov | Tissue transglutaminase is an integrin-binding adhesion coreceptor for fibronectin[END_REF], and VEGF receptor-2 [START_REF] Kim | Endostatin blocks vascular endothelial growth factor-mediated signaling via direct interaction with KDR/Flk-1[END_REF][START_REF] Dardik | Complex formation between tissue transglutaminase II (tTG) and vascular endothelial growth factor receptor 2 (VEGFR-2): proposed mechanism for modulation of endothelial cell response to VEGF[END_REF]. They are involved in angiogenesis, and are increased in brain after trauma [START_REF] Deininger | Endostatin/collagen XVIII accumulates in patients with traumatic brain injury[END_REF][START_REF] Wang | Tissue Transglutaminase, Protein Cross-linking and Alzheimer's Disease: Review and Views[END_REF]. Furthermore they may play a role in Alzheimer's disease. Endostatin is released by neurons and accumulates in amyloid plaques [START_REF] Deininger | Aberrant neuronal and paracellular deposition of endostatin in brains of patients with Alzheimer's disease[END_REF] and TG-2 co-localizes with the pathological lesions in Alzheimer's disease brain [START_REF] Zhang | Immunohistochemical demonstration of tissue transglutaminase in amyloid plaques[END_REF][START_REF] Wilhelmus | Transglutaminases and transglutaminasecatalyzed cross-links colocalize with the pathological lesions in Alzheimer's disease brain[END_REF]. All these common properties prompted us to investigate the possible existence of an interaction between these two proteins, and we have recently shown that endostatin interacts with TG-2 [START_REF] Faye | The first draft of the endostatin interaction network[END_REF]. We report here that endostatin binds to TG-2 with an affinity in the nanomolar range in a calcium-dependent manner. Endostatin is not a glutaminyl substrate of TG-2 in vitro, and it is not cross-linked to itself by the enzyme. Since endostatin and transglutaminase-2 are both involved in the regulation of angiogenesis, and have been implicated in Alzheimer's disease, it is likely that their interaction is of major importance for the modulation of endothelial cell migration and/or proliferation, and for the formation and/or the stabilization of amyloid plaques in neurodegenerative diseases.

EXPERIMENTAL

Proteins and antibodies

Recombinant human TG-2 was purchased from two different suppliers (Immundiagnostik, Germany, and Covalab, France). Guinea pig TG-2 extracted from liver was from Sigma-Aldrich (St Quentin Fallavier, France). Recombinant endostatin, the Cterminal domain of collagen XVIII (NC1), and NC1 mutants (D104N and R27A/R139A) were produced in human embryonic kidney cells expressing Epstein-Barr virus nuclear antigen (HEK 293-EBNA cells, [START_REF] Faye | Molecular interplay between endostatin, integrins, and heparan sulfate[END_REF][START_REF] Faye | The first draft of the endostatin interaction network[END_REF][START_REF] Feraud | Characterization of endostatin binding to heparin and heparan sulfate by surface plasmon resonance and molecular modeling: role of divalent cations[END_REF]. Transfected HEK 293 cells expressing wild type or mutant endostatin and the NC1(XVIII) domain were a generous gift of Naomi Fukai and Reidunn Jetne (Harvard Medical School, Boston, USA). To avoid the confusion due to two different numberings reported for endostatin in the literature, amino acids residues are numbered starting from the first amino acid residue of endostatin (i.e. His 1 , corresponds to His 132 when numbering starts from the first amino acid of the entire C-terminal domain NC1 of collagen XVIII). Both proteins were tagged by the peptide Flag at the N-or the C-terminus for endostatin and at the C-terminus for the NC1 domain. The conditioned culture media were filtered through 0.22-µm filters and applied to a 5-ml heparin HiTrap (GE Healthcare, Uppsala, Sweden) for wild-type and mutant endostatin, and to an anti-Flag M2 affinity column (Sigma-Aldrich) for wild-type and mutant NC1. Endostatin was further purified by gel filtration on a Superdex S75 column (60 cm x 2.6 cm, GE Healthcare), and the NC1 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

domain was further purified on a Sephacryl S200 column (60 cm x 2.6 cm, GE Healthcare). The purity of proteins was assessed by SDS-PAGE and immunoblotting. The purified proteins were concentrated by ultrafiltration and stored at -80°C. Recombinant human endostatin expressed in Pichia pastoris was from Calbiochem (San Diego, CA). Anti-TG-2 monoclonal antibody (CUB7402) was from NeoMarker (Fremont, CA, USA). Bovine collagen XI was a generous gift from Dr. Marie-Claire Ronzière (UMR 5086, CNRS, University Lyon 1, France).

Preparation of a polyclonal antibody against human endostatin

Recombinant human endostatin with a Flag peptide at the C-terminus was used for the immunization of New Zealand white rabbits. Endostatin diluted in phosphate buffered saline (PBS, 660 µg/ml) emulsified with an equal volume of Freund's complete adjuvant was injected intra-dermally. Two booster injections with Freund's incomplete adjuvant were performed 14 and 28 days after the first injection. The IgG fraction was purified from the immunserum by affinity chromatography on Protein A Ceramic Hyper D®F (Pall Life Sciences, Saint-Germain en Laye, France). IgGs were eluted by 0.1 M citric acid pH 3, and neutralized with 1 M potassium phosphate buffer pH 9. The polyclonal antibody was assayed by immunoblotting, and solid-phase assays. The antibody reacted with wild type and mutant endostatin, and with the wild type and mutant NC1(XVIII) domains (Supplementary material, Fig S1).

Solid-phase binding assays

Transglutaminase was diluted in 25 mM Tris•HCl, 150 mM NaCl, pH 7.4 (TBS) for coating. Endostatin and the NC1(XVIII) domain were diluted in 10 mM PBS pH 7.4 containing 138 mM NaCl, 27 mM KCl, or in 25 mM Tris•HCl, 150 mM NaCl, pH 7.4 (TBS). Aliquots (100 µl) were added to the wells of a 96-well microplate (MaxiSorp, Nunc, Thermo Fisher Scientific, Roskilde, Denmark). Plates were incubated overnight at 4°C, and wells were blocked for 2 h with 5% (w/v) BSA in TBS. The plates were incubated for 2 h at room temperature with TG-2 diluted in TBS containing 1 mM EDTA with or without cations (8 mM CaCl 2 , MgCl 2 , or MnCl 2 ). The wells were washed three times with TBS containing 0.1% (v/v) Tween 20. Bound transglutaminase was detected by a monoclonal anti-TG-2 antibody diluted 1:1000 in TBS + 0.1% (v/v) Tween 20 for 1 h at room temperature. Bound endostatin was detected with the polyclonal anti-endostatin antibody diluted 1:1000. The wells were washed 3 times with TBS, 0.1% (v/v) Tween 20, and were then incubated with either alkaline phosphatase-conjugated or peroxidase-conjugated secondary antibodies. The immunological reaction was detected by adding p-nitrophenyl phosphate (absorbance measured at 405 nm), or 3,3',5,5'-tetramethylbenzidine (absorbance was measured at 450 nm). Nonspecific binding was measured in BSA-coated wells, and was subtracted from the values measured in endostatin-, or transglutaminase-coated wells. For inhibition experiments by GTP, endostatin was diluted in 25 mM Tris•HCl, 150 M NaCl pH 7.4 (0.1 µg/well), and incubated overnight at 4°C in a 96-well microplate (MaxiSorp, Nunc). Wells were blocked for 2 h with 5% (w/v) BSA in TBS. TG-2 was preincubated in TBS containing 8 mM CaCl 2 with several concentrations of GTP (0-100 µM) for 1 h at room temperature, before incubation for 2 h at room temperature. The binding of TG-2 to endostatin was performed as described above. All assays were performed in triplicate.

Surface plasmon resonance arrays

SPR arrays were performed using a Biacore Flexchip system (GE Healthcare, Sweden) as described previously [START_REF] Faye | The first draft of the endostatin interaction network[END_REF]. This available high-density array platform is able to follow the binding of a single analyte to 400 target spots at a time. Proteins (50 and 200 µg/ml) were spotted in triplicate at two concentrations onto the surface of a Gold Affinity chip (GE Healthcare) using a non-contact PiezoArray spotter (Perkin Elmer, Courtaboeuf, France). Six drops of 330 pl each were delivered to the surface of the chip (spot diameter: 250-300 µm, spotted amount: 100-400 pg/spot). The chips were then dried at room temperature and stored under vacuum at 4°C until their insertion into the Biacore Flexchip instrument. The chip was blocked with Superblock buffer (Pierce, Thermo Scientific, Brebières, France) for 5 x 5 min. The blocked chip was then equilibrated with PBS, 0.05% (v/v) Tween 20 at 500 µl/min for 90 min. TG-2 (500 nM) was flowed at 25°C over the chip surface for 25 min at the same flow rate. The dissociation of the endostatin-TG-2 complex was monitored in buffer flow -PBS, 0.05% (v/v) Tween 20 -for 40 min. Data collected from reference spots (gold surface), and from buffer spots were subtracted from those collected on spotted proteins to obtain specific binding curves.

Surface plasmon resonance binding assays

The SPR binding assays were performed in a Biacore T100 instruments (GE Healthcare). Recombinant human endostatin (50 µg/ml in 10 mM maleate buffer, pH 6.2) was covalently immobilized to the dextran matrix of a CM5 sensor chip via its primary amine groups according to the manufacturer's instruction (amine coupling kit, GE Healthcare) at a flow rate of 5 µl/min with 10 mM Hepes buffer pH 7.4 containing 0.15 M NaCl and 0.05% (v/v) P20 (HBS-P + buffer, GE Healthcare) as running buffer. An immobilization level ranging between 1800 and 2000 resonance units (RU) was obtained; a control flow cell was prepared with 10 mM maleate buffer pH 6.2. ZnCl 2 (1 mM) was added to the running buffer for binding experiments. Sensorgrams collected on the control flow cell were automatically subtracted from the sensorgrams obtained on immobilized endostatin to yield specific binding responses. Binding assays were performed at 25 °C, but the sample compartment of the Biacore T100 was kept at 4°C to maintain transglutaminase-2 in its native state. Kinetic and affinity constants were calculated by injecting several concentrations of transglutaminase-2 (8-130 nM) in the presence of 2 mM CaCl 2 , over immobilized endostatin at 60 µl/min. The complexes were dissociated by a pulse (60 s) of 1.5 M NaCl + 2 M guanidinium chloride. The kinetic rates, k a and k d (association and dissociation rate constants, respectively), were calculated using the Biacore T100 evaluation software 2.0.1. Apparent equilibrium dissociation constants (K D ) were calculated as the ratio of k d /k a for a 1:1 (Langmuir) interaction model. Inhibition experiments were carried out by preincubating TG-2 (130 nM) with 1 mM GTP for 15 min at room temperature before injecting the mixture over endostatin immobilized on the sensor chip.

Cross-linking experiments

Guinea pig TG-2 (5 mU/ml, Sigma-Aldrich) was incubated with recombinant human endostatin expressed in mammalian cells (10 µg/ml), the biotinylated peptide TVQQEL used as an acyl donor (30) (Assay mix A5731, Sigma-Aldrich) and 1 mM dithiothreitol. The reaction was carried out for 2, 5, 10, 20, 30 and 60 min at room temperature and was stopped by addition of 20 mM EDTA. Control experiments were performed with TG-2 and endostatin alone. The reaction products were analyzed by SDS-PAGE and by immunoblotting with either streptavidin-peroxidase, or with the polyclonal anti-endostatin antibody, followed by chemiluminescence detection (ECL, GE Healthcare). In another series of experiments, TG-2 (10 mU/ml) was incubated with endostatin (10 µg/ml), biotin-X-cadaverin (2 mM, Sigma-Aldrich), 8 mM CaCl 2 and 1 mM dithiothreitol. The reaction was carried out for 120 min at room temperature, and was stopped by addition of 20 mM EDTA. Control experiments were performed with either TG-2 or endostatin alone. The reaction products were analyzed as described above. Cross-linking experiments: Reaction between endostatin (476 nM) and biotinylated peptide TVQQEL-OH mediated by guinea pig TG-2 (5 mU/ml). Reactions were carried out for 60 min at room temperature with or without GTP (µM). Results were analyzed by immunoblotting using A) extravidin-peroxidase, and B) polyclonal antibody against endostatin.

Immunocytochemistry

Primary cultures of human umbilical vein endothelial cells (HUVEC) were grown in endothelial cell growth medium 2 (PromoCell, Heidelberg, Germany). The cells were used for immunofluorescence experiments between passages 2 and 5. HUVEC were seeded on glass coverslips and cultivated in complete medium in normoxia or hypoxia (1% O 2 ). The cells were then fixed with cold methanol for 5 minutes before blocking with 10% (v/v) normal goat serum in PBS. They were then incubated either with the anti-endostatin antibody, or with the antibody directed against TG-2 diluted in PBS containing 1% (v/v) normal goat serum. Secondary goat antibodies directed against rabbit or mouse IgG coupled to AlexaFluor-488 or -555 were from Invitrogen (Cergy Pontoise, France). Nuclei were stained with TO-PRO-3 (Invitrogen). Coverslips were mounted with Mowiol and observed with a TCS SP2 confocal microscope (Leica Microsystems, Nanterre, France).

RESULTS

Transglutaminase binds to monomeric endostatin and to the C-terminal domain of collagen XVIII

The binding of human TG-2 to endostatin and to the trimeric NC1 domain of collagen XVIII was demonstrated by SPR arrays (Figure 1A). Under the same experimental conditions, TG-2 was found previously to bind to known ligands, heparin and heparan sulfate [START_REF] Scarpellini | Heparan sulfate proteoglycans are receptors for the cellsurface trafficking and biological activity of transglutaminase-2[END_REF], collagen XI [START_REF] Kleman | Transglutaminase-catalyzed cross-linking of fibrils of collagen V/XI in A204 rhabdomyosarcoma cells[END_REF] (Supplementary material, Fig. S2), α5β1 and αvβ3 integrins [START_REF] Akimov | Tissue transglutaminase is an integrin-binding adhesion coreceptor for fibronectin[END_REF], and amyloid Aβ peptide [START_REF] Wang | Tissue Transglutaminase, Protein Cross-linking and Alzheimer's Disease: Review and Views[END_REF]. The interaction was confirmed by solid-phase assays (Figure 1B), and was also observed with guinea pig TG-2 (Figure 2B). Several sources of TG-2 and endostatin were used to support the existence of the interaction between these two molecules.

The binding properties of the D104N mutant were investigated because individuals with the homozygous D104N polymorphism in the COL18A1 gene have a high risk of occurrence of cancer [START_REF] Lourenco | A high risk of occurrence of sporadic breast cancer in individuals with the 104NN polymorphism of the COL18A1 gene[END_REF][START_REF] Nascimento | D104N polymorphism in endostatin, an angiogenesis inhibitor, in acute and chronic myeloid leukaemia[END_REF]. The D104N mutation did not alter the folding of endostatin and of the NC1(XVIII) domain as assessed by intrinsic fluorescence (Supplementary material Fig. S3). This mutation did not affect significantly the ability of endostatin, or of the NC1 domain of collagen XVIII, to bind to transglutaminase-2 (Figures 1B, and3B). In contrast, the double R27A/R139A NC1 mutant that has lost its capacity to bind heparin [START_REF] Sasaki | Structural basis and potential role of heparin/heparan sulfate binding to the angiogenesis inhibitor endostatin[END_REF] but is properly folded as assessed by fluorescence spectroscopy (Supplementary material Fig. S3), bound to TG-2 to a lesser extent (31% compared to the wild type NC1 domain) (Figure 1B).

The binding of recombinant endostatin (i.e. expressed in yeast or in mammalian cells) to immobilized TG-2 was concentration-dependent (Figure 2A). Similar results were obtained in the reverse orientation when TG-2 was incubated with endostatin-coated wells (Figure 2B).

The binding of endostatin to transglutaminase-2 is dependent upon cations

Binding assays were performed with human recombinant TG-2 in the presence of 1 mM EDTA to remove residual cations from the sample and to analyze the effects of different cations added at 8 mM. In absence of any cation, TG-2 did not bind significantly to endostatin (Figure 3A). The addition of cations increased the binding by ~ 2-fold for MgCl 2 , ~ 10-fold for MnCl 2 , and ~ 38-fold for CaCl 2 (Figure 3A). Similar increase in binding was obtained with guinea pig TG-2 in the presence of cations (Supplementary material Fig. S4). Calcium was used for further experiments because it was the most efficient in promoting endostatin binding. In presence of 8 mM CaCl 2 , the binding of TG-2 to wild-type endostatin and to the D104 N endostatin mutant was saturable (Figure 3B).

Endostatin binds to transglutaminase with high affinity

Kinetic analysis was performed by injecting guinea pig TG-2 at several concentrations over immobilized endostatin produced either in mammalian cells (Supplementary material Fig. S5A) or in yeast (Supplementary material Fig. S5B). The sensorgrams were fitted to a 1:1 Langmuir model using the Biacore T100 evaluation software (2.0.1), and kinetics and affinity constants were calculated (Table 1). The affinity constants were 6.8 nM, and 5.6 nM for recombinant endostatin produced from mammalian cells and yeast, respectively (Table 1).

TG-2 is regulated by calcium and GTP. The transamidating activity of the enzyme is inhibited by GTP in an allosteric fashion, whereas CaCl 2 partially reverses GTP inhibition [START_REF] Achyuthan | Identification of a guanosine triphosphate-binding site on guinea pig liver transglutaminase. Role of GTP and calcium ions in modulating activity[END_REF]. We performed inhibition experiments with GTP, a reversible inhibitor of the crosslinking activity of TG-2, to determine if it was able to interfere with the binding of TG-2 to endostatin. Recombinant TG-2 in TBS containing 8 mM CaCl 2 was preincubated with increased concentrations of GTP (0-100 µM) for 1 h at room temperature. It was then added to a 96-well microplate coated with recombinant human endostatin expressed in mammalian cells and incubated for 2 h at room temperature. We observed a decrease in the binding level of TG-2 to endostatin upon the addition of increasing concentrations of GTP (Figure 4A). This inhibition was confirmed by SPR experiments where the binding of guinea pig TG-2 to recombinant human endostatin expressed in mammalian cells was nearly abolished (96.7% inhibition) by 1 mM GTP (Figure 4B). A similar effect was observed with human endostatin produced in yeast (100% inhibition, data not shown). The above results suggest that either endostatin binds to TG-2 at the same site as GTP, or that the interaction with endostatin requires TG-2 to be in an open conformation, fully activated by calcium.

Endostatin is not a glutaminyl substrate of transglutaminase-2

We performed enzymatic assays to determine if endostatin was a substrate of TG-2. Endostatin was incubated with the enzyme in the presence of an acyl donor, i.e. the biotinylated peptide TVQQEL. Analysis of the reaction products by SDS-PAGE and immunoblotting showed that the biotinylated peptide was cross-linked to endostatin, and to a lesser extent to the enzyme (Figure 5A). The amount of incorporated peptide increased as a function of time. In absence of TG-2, or in presence of a Ca 2+ ion chelator (20 mM EDTA), the biotinylated peptide was not cross-linked with either endostatin or TG-2. Immunodetection with a polyclonal anti-endostatin antibody showed that endostatin was not cross-linked to itself or to TG-2 under those experimental conditions (Figure 5B). When endostatin was reacted with the biotinylated peptide TVQQEL in the presence of TG-2 and GTP, the amount of peptide incorporated into endostatin decreased when GTP concentration increased (Figure 6A), whereas the amount of endostatin did not change in these conditions (Figure 6B). The incorporation of the peptide was totally inhibited by 500 µM GTP as shown in Figure 6 A. Since GTP is an inhibitor of the transamidating activity of TG-2, these results confirm that the cross-linking of endostatin to TVQQEL is mediated by the transamidating activity of TG-2 and that endostatin behaves as an acyl acceptor in the transamidation reaction. A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

When endostatin was incubated with TG-2 and cadaverin, an acyl acceptor, no crosslinking reaction was detected, even when the reaction was performed with an increased amount of enzyme (10 U/ml instead of 5 mU/ml) and for a longer period time (120 min versus 60 min, Figure 7A andB). These results show that endostatin is an acyl acceptor, but not an acyl donor in the transamidation reaction catalyzed by TG-2. Preliminary results showed that endostatin did not inhibit the cross-linking activity of transglutaminase-2 in vitro (Supplementary material Fig. S6), suggesting that endostatin does not bind to the transamidating active site of TG-2.

Endostatin and transglutaminase-2 are co-localized in the extracellular matrix secreted by endothelial cells and in vessel walls

To determine if the binding of endostatin with TG-2 occurs within the extracellular matrix secreted by endothelial cells and/or at the cell surface, immunostaining experiments were performed with anti-endostatin and anti-transglutaminase antibodies. Since hypoxia induces COL18A1 gene in a HIF-1α dependent manner in endothelial cells [START_REF] Paddenberg | Hypoxiainduced increase of endostatin in murine aorta and lung[END_REF], human umbilical cord endothelial cells (HUVEC) were incubated in a hypoxic environment for six days to enhance detection of endostatin (i.e. by immunofluorescence). Indeed an increased level of endostatin was detected in the extracellular matrix synthesized by cultured endothelial cells under hypoxia, that stimulates angiogenesis, compared to normoxia (Figure 8A andB). As can be seen from Figure 8, transglutaminase-2 co-localized with endostatin/collagen XVIII in the extracellular matrix secreted by HUVEC cultured under hypoxic conditions (Figure 8C-E). We have confirmed the co-localization of transglutaminase-2 and endostatin in tissue by performing double immunostaining of foreskin sections with antibodies directed against TG-2 and endostatin. TG-2 and endostatin were co-localized in vessel walls (Supplementary material Fig. S7).

DISCUSSION

The interaction between endostatin and TG-2 was demonstrated by SPR and by solidphase assays with two different sources of transglutaminase (human recombinant TG-2 and guinea pig TG-2 extracted from liver), and two different sources of recombinant human endostatin (produced by HEK 293 EBNA cells or in yeast). These experiments revealed that endostatin binds to transglutaminase with affinity in the nanomolar range and in a cationdependent manner, calcium ions being the most potent among those assayed.

TG-2 catalyzes post-translational modification of proteins by the formation of γglutamyl-ε-lysine bonds between glutamine and lysine residues. Endostatin contains eight glutamine residues that could potentially act as glutaminyl substrates (acyl donors) in the transamidation reaction. However, endostatin is not a glutaminyl substrate of TG-2 in vitro, but rather an acyl acceptor providing lysine residues in the transamidation reaction. This is in agreement with the fact that none of the glutamine residues of endostatin is found within a QXP sequence, a preferred sequence for glutamine-donor substrates where X represents a variable amino acid [START_REF] Fleckenstein | Gliadin T cell epitope selection by tissue transglutaminase in celiac disease. Role of enzyme specificity and pH influence on the transamidation versus deamidation process[END_REF] and with the fact that the N-terminus of endostatin contains a QP (Q 7 P 8 ) sequence reported to abolish transamidation [START_REF] Fleckenstein | Gliadin T cell epitope selection by tissue transglutaminase in celiac disease. Role of enzyme specificity and pH influence on the transamidation versus deamidation process[END_REF]. The LGQS sequence found in the Npropeptide of the α1 chain of procollagen III, a physiological substrate of TG-2 [START_REF] Bowness | Identification of a substrate site for liver transglutaminase on the aminopropeptide of type III collagen[END_REF], is present in endostatin (residues 158-161) but it is located on the side of endostatin that is not involved in the binding to TG-2. Therefore the glutamine residue 160 does not appear to be a glutaminyl substrate of TG-2.

GTP inhibits the binding of endostatin to tranglutaminase-2, suggesting that endostatin could bind to the GTP binding site on TG-2. However, the binding of endostatin to TG-2 is calcium-dependent and calcium is known to activate TG-2 by switching on its transamidating (i.e. cross-linking) activity [START_REF] Griffin | Transglutaminases: nature's biological glues[END_REF]. Since the activation of TG-2 by Ca 2+ can be counteracted by the allosteric inhibitor GTP [START_REF] Achyuthan | Identification of a guanosine triphosphate-binding site on guinea pig liver transglutaminase. Role of GTP and calcium ions in modulating activity[END_REF], the inhibition of endostatin binding by GTP might alternatively suggest that endostatin binds to the transamidating active form of the enzyme. Thus, we used TG-2 in an extended, active, conformation [START_REF] Pinkas | Transglutaminase 2 undergoes a large conformational change upon activation[END_REF] to further investigate the location of the endostatin binding site on TG-2. We have shown here that R27 and/or R139 residues participate in the binding of endostatin to TG-2. These residues both form part of a large basic patch on endostatin, comprising the heparin-binding site [START_REF] Sasaki | Structural basis and potential role of heparin/heparan sulfate binding to the angiogenesis inhibitor endostatin[END_REF], that is likely to interact with negatively charged amino acids on TG-2 surface. In addition to our experimental data, we have used the Connolly molecular surface of TG-2 to have clues on possible binding site(s) of endostatin on TG-2. There are three negatively charged areas on the enzyme: one in the N-terminal β-sandwich domain, one close to transamidating catalytic core, and a third one close to the GTP-binding site. We have shown that endostatin does not interfere with TG-2 cross-linking activity in vitro and that GTP blocks the binding of endostatin to TG-2. These results suggest that endostatin may bind to, or close to, the GTP binding site but not to the transamidating active site of TG-2.

The requirement of calcium ions for endostatin binding to TG-2 suggests as discussed above that endostatin binds to the open form of the enzyme. Extracellular TG-2 is predominantly maintained in the catalytically inactive closed conformation under ordinary physiological conditions, and is able to promote cell adhesion, spreading, migration, or differentiation independently of its catalytic activity [START_REF] Griffin | Transglutaminases: nature's biological glues[END_REF]. Extracellular TG-2 is transiently activated in response to innate immune signals such as exposure to polyinosinic-polycytidylic acid, a potent ligand of the toll-like receptor TLR-3 [START_REF] Siegel | Extracellular transglutaminase 2 is catalytically inactive, but is transiently activated upon tissue injury[END_REF]. The stimulation of innate immune pattern recognition receptors such as TLR3 triggers the release of extracellular TG-2 from cell surface integrins and the concomitant enzyme activation [START_REF] Siegel | Extracellular transglutaminase 2 is catalytically inactive, but is transiently activated upon tissue injury[END_REF]. It is thus possible that in the course of angiogenesis up-regulated innate immune receptors trigger the activation of extracellular TG-2 leading in turn to endostatin binding. This hypothesis is supported by the fact that multiple human endothelial cell types express surface TLR3, which play a key role in disrupting the haemostasis balance on endothelial cells [START_REF] Shibamiya | A key role for Toll-like receptor-3 in disrupting the hemostasis balance on endothelial cells[END_REF], and by the suppression of sequence-and target-independent angiogenesis by siRNA via TLR3 [START_REF] Kleinman | Sequence-and target-independent angiogenesis suppression by siRNA via TLR3[END_REF].

Endostatin might cooperate with TG-2 in several physio-pathological processes either through direct interactions or through more complex mechanisms involving multimolecular complexes. Direct interactions between endostatin and TG-2 might play a role in organizing the extracellular matrix in skin basement membrane, endostatin being present in this location [START_REF] Elamaa | Endostatin overexpression specifically in the lens and skin leads to cataract and ultrastructural alterations in basement membranes[END_REF], and TG-2 stabilizing basement membranes [START_REF] Aeschlimann | Cross-linking of laminin-nidogen complexes by tissue transglutaminase. A novel mechanism for basement membrane stabilization[END_REF]. At the endothelial cell surface, endostatin inhibits both the extracellular activation of proMMP-2 by inhibition of MT1-MMP as well as the catalytic activity of MMP-2, thus blocking the invasiveness of tumor cells [START_REF] Kim | Endostatin inhibits endothelial and tumor cellular invasion by blocking the activation and catalytic activity of matrix metalloproteinase[END_REF]. MMP-2, functioning in concert with MT1-MMP, hydrolyzes cell-surface-associated TG-2, and the cleavage eliminates both the adhesion and the enzymatic activity of TG-2 [START_REF] Belkin | Cell-surface-associated tissue transglutaminase is a target of MMP-2 proteolysis[END_REF]. It should be noted that endostatin and TG-2 bind to the catalytic domain of MMP-2, and both proteins inhibit MMP-2 maturation [START_REF] Belkin | Cell-surface-associated tissue transglutaminase is a target of MMP-2 proteolysis[END_REF][START_REF] Lee | Endostatin binds to the catalytic domain of matrix metalloproteinase-2[END_REF].

Besides MMP-2, endostatin and TG-2 share heparan sulfate, VEGF-R2 and integrins as binding partners at the cell surface, and both regulate endothelial cell adhesion [START_REF] Rehn | Interaction of endostatin with integrins implicated in angiogenesis[END_REF][START_REF] Kim | Endostatin blocks vascular endothelial growth factor-mediated signaling via direct interaction with KDR/Flk-1[END_REF][START_REF] Akimov | Tissue transglutaminase is an integrin-binding adhesion coreceptor for fibronectin[END_REF][START_REF] Dardik | Complex formation between tissue transglutaminase II (tTG) and vascular endothelial growth factor receptor 2 (VEGFR-2): proposed mechanism for modulation of endothelial cell response to VEGF[END_REF][START_REF] Dixelius | Endostatin regulates endothelial cell adhesion and cytoskeletal organization[END_REF]. As reported here, two arginine residues implicated in the binding of endostatin (R27 and/or R139) to TG-2 are also involved in the binding to α5β1 and αvβ3 integrins [START_REF] Faye | Molecular interplay between endostatin, integrins, and heparan sulfate[END_REF], and to heparin [START_REF] Sasaki | Structural basis and potential role of heparin/heparan sulfate binding to the angiogenesis inhibitor endostatin[END_REF]. This suggests that endostatin is not able to bind simultaneously to TG-2 and α5β1/ αvβ3 integrins or to TG-2 and heparan sulfate. The co-localization of endostatin/collagen XVIII and TG-2 in the extracellular matrix secreted by cultured endothelial cells indicates that this interaction may occur in a cellular context and supports its physiological relevance. These proteins are co-localized in the extracellular matrix synthesized under hypoxia that stimulates angiogenesis. This raises the possibility that endostatin and TG-2 regulate endothelial cell behaviour and control angiogenesis in a concerted fashion. However, their molecular interplay in the extracellular matrix secreted by endothelial cells and/or at the surface of these cells remains to be deciphered.

Table 1: Kinetic and equilibrium dissociation constants of the binding of transglutaminase-2 to endostatin.

Rate and affinity constants of the binding of soluble transglutaminase-2 to immobilized endostatin calculated from SPR binding assays using the Biacore T100 evaluation software 2.0. Reaction between endostatin (476 nM) and the biotinylated peptide TVQQEL-OH mediated by guinea pig TG-2 (5 mU/ml). Reactions were carried out for 60 min at room temperature in absence of GTP or in presence of increased GTP concentrations (10 -500 µM). Results were analyzed by immunoblotting using A) extravidin-peroxidase, and B) polyclonal antibody against endostatin. A c c e p t e d M a n u s c r i p t

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Biochemical

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Biochemical

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Biochemical

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Biochemical

  Journal Immediate Publication. Published on 15 Feb 2010 as manuscript BJ20091594 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20091594

Biochemical

  Journal Immediate Publication. Published on 15 Feb 2010 as manuscript BJ20091594 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20091594A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Figure 1 :

 1 Figure 1: TG-2 binds to endostatin and to the trimeric C-terminal NC1 domain of collagen XVIII A) Overlay of sensorgrams resulting from the injection of human TG-2 (500 nM) over immobilized wild-type NC1 (-, 154 pg) and wild-type endostatin (---, 300 pg) (flow rate 500 µl/min). B) Solid-phase binding assay. Binding of human TG-2 (350 nM) to immobilized wild type and mutant endostatin and NC1(XVIII) domain (0.5 µg/well).

Figure 2 :

 2 Figure 2: Soluble and immobilized endostatin bind to TG-2 Solid-phase binding assays. A) Binding of recombinant human endostatin (500 nM) produced in mammalian ( ) or yeast ( ) expression systems to immobilized TG-2 (mean of 3 experiments ± SD). B) Binding of human ( , 350 nM) and guinea pig TG-2 ( , 350 nM) to immobilized mammalian human endostatin. The values are expressed as mean ± SD (n=3).

Figure 3 :

 3 Figure 3: The binding of endostatin to TG-2 is cation-dependent A) Solid-phase assays: effect of cations. Binding of human TG-2 (64 nM) to coated endostatin produced in HEK 293 cells (0.1 µg/well) in the presence of cations (8 mM). (mean of 3 experiments ± SD) B) Binding of human TG-2 (0, 1, 2, 4, 8, 16, 32, 64, 128, 256 nM to immobilized human endostatin (---wild-type endostatin, -D104N mutant endostatin; 0.1 µg/well) in the presence of 8 mM CaCl 2 . The values are expressed as mean ± SD (n=3).

Figure 4 :

 4 Figure 4: GTP inhibits the binding of TG-2 to endostatinA) Solid-phase binding assays: Effect of GTP (0-100 µM) on the binding of human TG-2 (64 nM) to endostatin (expressed in HEK cells; 0.1 µg/well) in presence of 8 mM CaCl 2 . The values are expressed as mean ± SD (n=3); B) SPR binding assays: injection of guinea pig TG-2 (130 nM) with (-) or without (---) 1 mM GTP over immobilized human endostatin.

Figure 5 :

 5 Figure 5: Endostatin is as an acyl acceptor in the transamidation reaction catalyzed by TG-2Cross-linking experiments: Reaction between endostatin (476 nM) and the biotinylated peptide TVQQEL-OH mediated by guinea pig TG-2 (5 mU/ml). The reaction was carried out for 0, 2, 5, 10, 20, 30 and 60 min at room temperature. Results were analyzed by immunoblotting using A) extravidin-peroxidase, and B) polyclonal antibody against endostatin.

Figure 6 :

 6 Figure 6: GTP inhibits the cross-linking between endostatin and an acyl donor mediated by guinea pig TG-2.Reaction between endostatin (476 nM) and the biotinylated peptide TVQQEL-OH mediated by guinea pig TG-2 (5 mU/ml). Reactions were carried out for 60 min at room temperature in absence of GTP or in presence of increased GTP concentrations (10 -500 µM). Results were analyzed by immunoblotting using A) extravidin-peroxidase, and B) polyclonal antibody against endostatin.

Figure 7 :

 7 Figure 7: Endostatin is not a glutaminyl substrate of TG-2Cross-linking experiments: Reaction between endostatin (ES, 476 nM) and biotin-Xcadaverin (Cad) in presence of human or guinea pig TG-2 (10 mU/ml). Analysis of the reaction products by immunoblotting using A) extravidin-peroxidase, and B) anti endostatin antibody.

Figure 8 :

 8 Figure 8: Endostatin and TG-2 are co-localized in the extracellular matrix synthesized by HUVECs under hypoxia Confocal microscopy: immunostaining of endostatin/ collagen XVIII (A-C), and TG-2 (D), merge (E) in the extracellular matrix secreted by cultured HUVECs. Cells were grown to confluence in normoxia (A) or hypoxia (B-E). Nuclei (blue) were stained using TO-PRO-3. Bar = 30 µm.

Figure 1

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Figure 8 Biochemical

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  Biochemical Journal Immediate Publication. Published on 15 Feb 2010 as manuscript BJ20091594 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20091594

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		Biochemical Journal Immediate Publication. Published on 15 Feb 2010 as manuscript BJ20091594
	THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20091594	Endostatin (Immobilized on the chip) Guinea pig TG-2 Injected in soluble form ka (M -1 s -1 ) kd (s -1 ) K D (M) Chi 2 U-value A c c e p t e d M a n u s c r i p t HEK 293 EBNA cells P. pastoris + 2 mM CaCl 2 + 2 mM CaCl 2 1.12 x 10 5 M -1 s -1 6.10 x10 4 M -1 s -1 7.65 x 10 -4 s -1 3.41 x 10 -4 s-1 6.8 nM 5.59 nM 4.1 8.81 1 1

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' 2010 The Authors Journal compilation ' 2010 Portland Press Limited

ACKNOWLEDGEMENTS

We thank Dr. C. Marquette and Prof. L. Blum (UMR 5246 CNRS-University Lyon 1) for their help in spotting on Gold affinity chips, Sylviane Guerret (Novotec, Lyon, France) for her skillful assistance in the production of the polyclonal anti-endostatin antibody, and Naomi Fukai and Reidunn Jetne for their generous gift of transfected HEK 293 cells expressing wild type or mutant endostatin and the NC1(XVIII) domain. The support of GE Healthcare (Dr. C. Quétard, P. Linden, T. Salomon, X. Rousselot and A. Sylvan) is gratefully acknowledged. SPR experiments were performed in the facility of IFR 128 Gerland Lyon Sud (France).

FUNDING

This work was supported by the Association pour la Recherche contre le Cancer [grant 3652 to SRB], GIS Maladies Rares [INSERM A04115SP to SRB], CPER Rhône-Alpes to CF and SRB), the Emergence Research Program (Région Rhône-Alpes to SRB), a BQR grant from University Lyon-1 [to SRB], and NIH [36820 to BO].

Abbreviations

BSA: Bovine serum albumin, ES: endostatin, HBS: Hepes-Buffered-Saline, GTP: Guanosine 5'-triphosphate, HEK: Human embryonic kidney; HIF: Hypoxia-inducible factor; HUVEC: Human umbilical vein endothelial cells; MMP-2: Matrix metalloproteinase-2; MT-MMP1: Membrane type matrix metalloproteinase-1; PBS: Phosphate-buffered-saline, PNPP: pnitrophenyl phosphate; RU: Resonance unit, SPR: Surface plasmon resonance; TBS: Trisbuffered-saline; TG-2: Transglutaminase-2; TLR3: Toll-like receptor-3; TMB: 3,3',5,5'-Tetramethylbenzidine; VEGF: Vascular endothelial growth factor Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.