Yanlei Zhu

Florent Delhommel

Florence Cordier

Susanne Lüchow

Ariel E Mechaly

Baptiste Colcombet-Cazenave

Virginie Girault

Elise Pepermans

Amel Bahloul

Candice Gautier

Sébastien Brule

Bertrand Raynal

Sylviane Hoos

Ahmed Haouz

Célia Caillet-Saguy

Ylva Ivarsson

Nicolas Wolff
email: nicolas.wolff@pasteur.frhighlights

Deciphering the Unexpected Binding Capacity of the Third PDZ Domain of Whirlin to Various Cochlear Hair Cell Partners

Keywords: Whirlin, Usher proteins, PDZ domain, stereocilium

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

Introduction

The sense of hearing is a mechanical and neurochemical process that requires highly specialized sensory cells (inner and outer hair cells) in the cochlea of the inner ear. At their apical surfaces, three rows of staircase-like actin-filled stereocilia are connected by different types of extracellular links. Two unconventional cadherins, protocadherin 15 (USH1F) and cadherin 23 (USH1D) form the interstereociliary tip link of auditory hair cell bundles, that conveys sound-evoked forces to the cation transducer channels [START_REF] Pepermans | The CD2 isoform of protocadherin-15 is an essential component of the tip-link complex in mature auditory hair cells[END_REF] [START_REF] Kazmierczak | Cadherin 23 and protocadherin 15 interact to form tip-link filaments in sensory hair cells[END_REF].These links are required to perform the mechanoelectrical transduction of the sound-induced vibrations [START_REF] Schwander | The cell biology of hearing[END_REF][4] [START_REF] Siletti | Daple coordinates organ-wide and cellintrinsic polarity to pattern inner-ear hair bundles[END_REF] [START_REF] Goodyear | Development and properties of stereociliary link types in hair cells of the mouse cochlea[END_REF]. During hair cell development, the hair bundle also harbours transient links called ankle links located at the stereocilia base. Both tip links and ankle links are anchored to large protein complex in the submembrane region. Most of the proteins that form these anchoring densities are called Usher proteins, as mutations altering their function cause the Usher syndrome (USH), that is the most common form of hereditary hearing-vision loss in humans. Usher proteins play fundamental roles in maintaining the particular sub-cellular structures and functions of hearing [START_REF] Mburu | Defects in whirlin, a PDZ domain molecule involved in stereocilia elongation, cause deafness in the whirler mouse and families with DFNB31[END_REF][8][9] [START_REF] Mathur | Usher syndrome: Hearing loss, retinal degeneration and associated abnormalities[END_REF], and same proteins are also found in retina photoreceptor cells. The submembrane scaffold proteins harmonin (USH1C) and SANS (Scaffolds protein containing Ankyrin repeats and SAM domain) (USH1G) anchor the upper part of this tip link to actin filaments directly or through interactions mediated by the actin-binding protein myosin 7a (USH1B). Usherin (USH2A) and ADGRV1 (USH2C)(adhesion G protein-coupled receptor V1). the two other large transmembrane Usher proteins make part of the ankle link complex [START_REF] Michalski | Molecular Characterization of the Ankle-Link Complex in Cochlear Hair Cells and Its Role in the Hair Bundle Functioning[END_REF] and are associated to the PDZscaffold proteins whirlin (USH2D) and PDZD7 (DFNB57) in the cytoplasm.

Whirlin is a multi-domain scaffolding protein, which is essential for the bundling and growing of stereocilia of the hair cells [START_REF] Mburu | Defects in whirlin, a PDZ domain molecule involved in stereocilia elongation, cause deafness in the whirler mouse and families with DFNB31[END_REF] [START_REF] Mustapha | DFNB31, a recessive form of sensorineural hearing loss, maps to chromosome 9q32-34[END_REF]. Human whirlin is composed of 907 amino acids and encompasses two harmonin Homology Domains (HHD) [START_REF] Delhommel | Structural Characterization of Whirlin Reveals an Unexpected and Dynamic Supramodule Conformation of Its PDZ Tandem[END_REF], three PDZ domains, a proline-rich (PR) region and a C-terminal PBM (PDZ binding motif) (Fig. 1) [START_REF] Mburu | Defects in whirlin, a PDZ domain molecule involved in stereocilia elongation, cause deafness in the whirler mouse and families with DFNB31[END_REF]. Whirlin is expressed in three main isoforms. The full-length (FL) isoform is found in both retina photoreceptor cells and auditory hair cells, which is an important component of the transient ankle links complex during development, and FL isoform has also been found at the tips of the tallest row of stereocilia [START_REF] Mathur | Distinct expression and function of whirlin isoforms in the inner ear and retina: An insight into pathogenesis of USH2D and DFNB31[END_REF]. The Cterminal isoform including the region from HHD2 to the C-terminal PBM is required for normal stereocilia length regulation, and is only expressed at the tips of tallest row of stereocilia [START_REF] Delprat | Myosin XVa and whirlin, two deafness gene products required for hair bundle growth, are located at the stereocilia tips and interact directly[END_REF][15] [START_REF] Mathur | Distinct expression and function of whirlin isoforms in the inner ear and retina: An insight into pathogenesis of USH2D and DFNB31[END_REF] [START_REF] Ebrahim | Alternative Splice Forms Influence Functions of Whirlin in Mechanosensory Hair Cell Stereocilia[END_REF]. Besides the Usher syndrome, the mutations in the Cterminal isoform region can also lead to non-syndromic deafness (DFNB31). Finally, the N-terminal isoform contains the N-terminal HHD1, a tandem of PDZ domains (PDZ1-2) and a second HHD domain (HHD2) but is only expressed in retina cells [START_REF] Mburu | Defects in whirlin, a PDZ domain molecule involved in stereocilia elongation, cause deafness in the whirler mouse and families with DFNB31[END_REF][8] [START_REF] Ebermann | A novel gene for Usher syndrome type 2: Mutations in the long isoform of whirlin are associated with retinitis pigmentosa and sensorineural hearing loss[END_REF].

Considering the various localizations of the protein according to the stages of development of hair bundle, whirlin is expected to participate in dynamical and transient complexes in rather different cellular environments. At the ankle links, whirlin interacts with usherin and ADGRV1, the two proteins critical for formation of these inter-stereocilia links [START_REF] Adato | Usherin , the defective protein in Usher syndrome type IIA , is likely to be a component of interstereocilia ankle links in the inner ear sensory cells[END_REF]. At the tip of tall stereocilia, whirlin controls the polymerization of F-actin, most likely together with others proteins such as EPS8 (epidermal growth factor receptor pathway substrate 8), myosin 15a and GPSM2 (Gprotein signalling modulator 2) [START_REF] Belyantseva | Myosin-XVa is required for tip localization of whirlin and differential elongation of hair-cell stereocilia[END_REF][14] [START_REF] Manor | Regulation of stereocilia length by myosin XVa and whirlin depends on the actinregulatory protein Eps8[END_REF] [START_REF] Mauriac | Defective Gpsm2/Gαi3signalling disrupts stereocilia development and growth cone actin dynamics in Chudley-McCullough syndrome[END_REF]. In the photoreceptor cells, SANS, usherin and whirlin have been reported to form a ternary complex [START_REF] Sorusch | Characterization of the ternary Usher syndrome SANS/ush2a/whirlin protein complex[END_REF]. Whirlin has been found enriched at inner segments and ciliary region but also distributed throughout the whole photoreceptor cells just like the harmonin a isoform [START_REF] Sorusch | Characterization of the ternary Usher syndrome SANS/ush2a/whirlin protein complex[END_REF] [START_REF] Reiners | Differential Distribution of Harmonin Isoforms and Their Possible Role in Usher-1 Protein Complexes in Mammalian Photoreceptor Cells[END_REF].

The PDZ domains of whirlin play a pivotal role in organizing these protein networks. PDZ domains are small modules of about 90 amino acids folded into five to six β-strands and one to two α-helices. The PDZ domain canonically recognizes short motifs composed of the last three C-terminal residues of the partners, known as PDZ Binding Motif (PBM). The C-terminus carboxylate of the motif directly contacts to the β1-β2 loop or 'GLGF' loop that is a main PDZ-PBM binding feature [START_REF] Harris | Mechanism and role of PDZ domains in signaling complex assembly[END_REF]. The two N-terminal PDZ domains of whirlin form a supramodule that enables fine-tuning of affinities for Usher proteins such as SANS and usherin [START_REF] Delhommel | 1H, 13C and 15N backbone resonance assignments and dynamic properties of the PDZ tandem of Whirlin[END_REF] [START_REF] Delhommel | Structural Characterization of Whirlin Reveals an Unexpected and Dynamic Supramodule Conformation of Its PDZ Tandem[END_REF]. The third PDZ domain of whirlin, present in both the FL and C-terminal isoforms, has been shown to interact with several partners but the molecular details of these interactions are not known. Whirlin PDZ3 binds to the class I canonical PBM of myosin 15a and participates to the F-actin polymerisation in a molecular complex at the stereocilia tip [START_REF] Delprat | Myosin XVa and whirlin, two deafness gene products required for hair bundle growth, are located at the stereocilia tips and interact directly[END_REF] [START_REF] Belyantseva | Myosin-XVa is required for tip localization of whirlin and differential elongation of hair-cell stereocilia[END_REF]. Early reports have also described whirlin is expressed in neuron cell soma and dendrites of the central nervous system; where its interaction with the Calcium/Calmodulin Dependent Serine Protein Kinase (CASK) has been confirmed [START_REF] Yap | CIP98, a novel PDZ domain protein, is expressed in the central nervous system and interacts with calmodulin-dependent serine kinase[END_REF]. In hair cells, the MAGUK proteins CASK and MPP1 (Membrane Palmitoylated Protein 1) have been shown to colocalize with whirlin [START_REF] Mburu | Whirlin complexes with p55 at the stereocilia tip during hair cell development[END_REF] [START_REF] Gosens | MPP1 links the Usher protein network and the Crumbs protein complex in the retina[END_REF]. Interestingly, the last 10 residues of CASK and MPP1 are identical and contain an internal class I PDZ binding motif (PBM) (-S-W-V-Y) albeit not at the C-terminus but shifted by one residue.

In this study, we characterize at the molecular level the interaction of the whirlin PDZ3 domain with its different cellular partners. We identified harmonin a1 and taperin as interacting partners of this domain, using an in vitro high throughput Holdup assay [START_REF] Vincentelli | Quantifying domain-ligand affinities and specificities by high-throughput holdup assay[END_REF] and a proteomic peptide phage display (ProP-PD; PMID: 28002650), respectively. We also characterized the PDZ mediated interactions using a number of biophysical methods and solved the X-ray structures of whirlin PDZ3 in complex with four of its binding partners: myosin 15a, CASK, harmonin a1 and taperin. The comparison of these high-resolution structures highlights the plasticity of interaction of whirlin PDZ3, which is able to interact with several divergent PDZ binding motifs. These results extend our understanding of the binding plasticity of PDZ domains and further confirm the multiple roles of whirlin in the development of sensory cells and its relation with non-syndromic deafness (DFNB31).

Results

The C-terminal region of whirlin adopts an extended form with independent folded domains HHD2 and PDZ3

In the full-length protein and the short C-terminal isoform of whirlin (starting from HHD2 to PBM), the third PDZ domain is localized at the C-terminal end of the protein and linked to the HHD2 domain through a sequence of 309 residues including a proline rich (PR) region. As illustrated by the IUPRED plot (https://iupred2a.elte.hu; [START_REF] Dosztányi | Prediction of protein disorder based on IUPred[END_REF]) (Fig. 1), this large linker region is predicted to be intrinsically disordered and tethers the two well-defined folded structures of HHD2 domain and PDZ3 domain [START_REF] Delhommel | Structural plasticity of the HHD2 domain of whirlin[END_REF].

We investigated the conformation of several constructs of the C-terminal isoform using small angle X-ray scattering (SAXS) (Fig. 1). We produced five constructs of the Cterminal isoform; the proline rich region (PR), the second HHD domain (HHD2), the third PDZ domain (PDZ3), a segment from the PR region to the PDZ3 domain (PR-PDZ3) and a segment from the second HHD to the PR (HHD2-PR). The Guinier approximation of the radius of gyration at low q value and maximum distance values (Dmax) estimated from the pair-distance distribution function showed that the PR constructs corresponding to the large inter-domain linker yields a large radius of gyration (Rg, 57.1 Å) and a maximum distance of 250 Å. Similarly, for the constructs PR, PR-PDZ3 and HHD2-PR, large values of Rg and Dmax are found, which is consistent with the presence of a large flexible sequence that links the folded domains. By contrast, the two globular single domains HHD2 and PDZ3 showed much smaller Rg and Dmax values in agreement with their molecular weights. The dimensionless Kratky plot provides a sensitive means to evaluate the degree of compactness of proteins; interestingly, the PR region behaves in solution as an extended and flexible sequence with a plateau at large q-values while HHD2 and PDZ3 adopt folded and compact conformations as represented by their bell-shaped curves (Fig. 1).

Previously we reported the HHD2 domain does not interact with the N-terminal PDZ1 and PDZ2 domains and binding partners of the HHD2 are currently unknown [START_REF] Delhommel | Structural plasticity of the HHD2 domain of whirlin[END_REF]. Overall, our results demonstrated that the PDZ3 domain is independent from the other folded domain HHD2, in the larger molecular context of whirlin as it is linked to the rest of the molecule by flexible sequence. It is therefore useful to study this domain as an independent feature of the whirlin.

The PDZ3 domain of whirlin recognizes the PBM sequences of harmonin a1 and taperin as two newly identified binders 7

Using in vitro high-throughput approaches, we identified PDZ3 binding partners. Short peptides encompassing a biotinyl group, a (PEG)3 spacer and the C-terminal PBM sequence of Usher proteins (11 to 16 residues long) were synthetized. These peptides were used as bait in the automated high-throughput chromatography assay Holdup which allowed to quantify the interaction between the whirlin PDZ3 domain and the collection of stereociliary protein PBMs [START_REF] Vincentelli | Quantifying domain-ligand affinities and specificities by high-throughput holdup assay[END_REF]. The Holdup approach displayed a high sensitivity for low to medium-affinity pairs and presented the unique advantage of providing affinity-based ranking of the identified binders. Results are showed in binding intensities (BI) (Table 1), a measure of binding that correlates directly to the affinity of interaction that is comprised between 0 for no interaction and 1 for all observed species bound. The PBM of harmonin a1, myosin 15a and cadherin23 displayed significant BI values higher than 0.2, an empirical threshold defining relevant from unspecific interactions. (Table 1) [START_REF] Vincentelli | Quantifying domain-ligand affinities and specificities by high-throughput holdup assay[END_REF]. We concluded here that the PBM sequences of harmonin a1, myosin 15a and cadherin23 proteins have the capacity to bind to PDZ3 in vitro in the 1-100 M affinity range typically found for PDZ/PBM interactions. The third isoform of protocadherin 15 (CD3) shows a BI slightly above threshold, suggesting a possible weak interaction. We then quantified by surface plasmonic resonance (SPR) the interactions between PDZ3 and the PBMs of these Usher proteins. Holdup and SPR data were in perfect agreement. The ranking of affinity between the two methods are fully consistent. Among the PBM-containing Usher proteins, only myosin 15a, cadherin 23, harmonin a1 and protocadherin 15-CD3 are binders of PDZ3 with affinities ranging from 5 to 250 M. We identified partners of whirlin from the apical complexes in hair cells. To enlarge the panel of whirlin binders, we explore the capacity of its PDZ3 to recognize internal PDZ binding motifs. Some PDZ domains have been reported to also interact with non-canonical PDZ binding motifs that are found in intrinsically disordered regions of target proteins [START_REF] Zhang | Inhibition of Wnt signaling by Dishevelled PDZ peptides[END_REF][31] [START_REF] Hillier | Unexpected modes of PDZ domain scaffolding revealed by structure of nNOS-syntrophin complex[END_REF]. We used a previously described phage library that present a peptidome that covers the intrinsically disordered regions of the human proteome [START_REF] Davey | Discovery of short linear motif-mediated interactions through phage display of intrinsically disordered regions of the human proteome[END_REF]. The displayed peptides are 16 amino acids long, and the library design comprises about 500,000 unique peptides. MBP-tagged whirlin PDZ3 was used as bait protein for five cycles of phage display selection. Phage pool ELISA confirmed that there was an enrichment of binders over the days of selection. The peptide-coding region of the resulting binding enriched phage pool was analysed through next-generation sequencing (NGS). The analysis revealed that a single peptide from the inner ear stereocilia protein, taperin, was highly enriched. Taperin (DFNB79) is a non-syndromic deafness protein expressed at the base of stereocilia, which is critical for the length and orientation of stereocilia [START_REF] Rehman | Targeted Capture and Next-Generation Sequencing Identifies C9orf75, Encoding Taperin, as the Mutated Gene in Nonsyndromic Deafness DFNB79[END_REF][35] [START_REF] Zhao | Murine Fam65b forms ring-like structures at the base of stereocilia critical for mechanosensory hair cell function[END_REF]. The PDZ3 domain of whirlin is thus capable of binding internal ligands, and likely with high specificity. Analysis of the taperin peptide sequence (LPVTFIDEVDSEEAPQ) identified a putative internal class I binding motif followed by acidic residues.

Affinity determination of whirlin PDZ3 domain with various PBM cellular partners

Besides the high-throught methods, we determined the affinities of the four PBM sequences of CASK, myosin 15a, harmonin a1, and taperin for the PDZ3 domain using NMR and fluorescence titrations for better understanding of structural information. By NMR titration, Kd values of the interactions between PDZ3 and myosin 15a and harmonin a1 were determined following 1 H, 15 N chemical shift perturbations of about 15 resonances in the 1 H- 15 N HSQC spectra as a function of increasing concentrations of the PBM-containing peptide. The binding affinity with myosin 15a peptide is 2.9 ± 1.6 M, the strongest value among the four binding partners, while harmonin a1 peptide is the weakest binding partner with a Kd value 45-fold lower at 132 ± 13 M. This Kd value is in the lower range of affinity commonly found for PDZ domain interactions. For taperin, the Kd was determined by following the variation of the bound signal intensity because of the slow exchange binding process with this peptide. An affinity of 58 ± 31 M (Table 2) was estimated for taperin. For CASK, the slow/intermediate exchange regime observed in the NMR spectra led to severe line broadening during the course of titration and prevented an accurate Kd measurement. Therefore, we used fluorescence titration, following the emission fluorescence intensity of the unique tryptophan of the CASK peptide (-SWVYCOOH) affected by the binding to the PDZ3 domain. The Kd value is 7.1 ± 1.0 M for the CASK peptide, similar to the one obtained with the myosin 15a peptide.

The PDZ3 domain is tethered to the C-terminal PBM of whirlin -VMLCOOH by a short linker of three residues. In order to evaluate if the PBM sequence of whirlin can affect the intermolecular binding properties of PDZ3 to its partner, we compared the affinity of PDZ3 and PDZ3 deleted from the PBM (PDZ3ΔPBM) for myosin 15a, which is the best binder. The Kd values of myosin 15a are similar for PDZ3ΔPBM and PDZ3 with 3.4 ± 1.8 M and 2.9 ± 1.6 M, respectively. We concluded that the whirlin PBM does not interfere with the binding of partner to the PDZ3 domain.

High resolution structures of PDZ3 complexes

Overall structure To investigate the structural determinants of binding, we determined the PDZ3 domain structure in complex with the PBMs of the four partners: myosin 15a, harmonin a1, CASK and taperin by X-ray crystallography. The crystal structures were solved by molecular replacement using the free form human PDZ3 from RIKEN Structural Genomics/Proteomics Initiative (RSGI) (PDB: 1UFX). Diffraction data collection and refinement statistics are summarized in Table S1. The overall RMSD of the backbone of PDZ3 between the free form (PDB: 1UFX) and the complexed forms are between 0.92 Å to 1.03 Å, which showed an equivalent overall backbone fold (Fig. 2). Figure 2: A: Backbone superimposition of 4 complexed PDZ3 and free PDZ3 (PDB: 1UFX), blue: myosin 15a, red: harmonin a1, green: CASK, yellow: taperin, white: free PDZ3. B : peptides superimposition with same colour set on the binding groove of apo PDZ3. C: overall RMSD values of PDZ3 backbone between free PDZ3 (PDB: 1UFX) and complexed PDZ3s. D: Crystal structures of PDZ3 in complex with PBMs from the four partners, myosin 15a, harmonin a1, CASK, and taperin with the PDB codes 6Y9N, 6Y9P, 6Y9O, 6Y9Q, respectively. E: Peptide sequence of each PBM, with position 0 corresponding to the residue forming H-bonds with 'TLGI' loop.

Structure and dynamics of the PDZ3/myosin 15a-PBM complex

Crystal structures of PDZ3ΔPBM, PDZ3/myosin 15a-PBM As for the affinity determination, we have compared the structures of PDZ3 with and without the intramolecular PBM in complex with the myosin 15a PBM peptide (NH2ERLTLPPSEITLLCOOH). The crystal structures of PDZ3/myosin 15a and PDZ3ΔPBM/myosin 15a were solved at 2.0 Å and 1.7 Å resolution, respectively, and are very similar. The asymmetric unit of the PDZ3/myosin 15a crystal contains one monomer, while PDZ3ΔPBM/myosin 15a is a dimer, where myosin 15a peptides form bridges tethering the two complexes (Fig. S1). As expected, PDZ3 of whirlin adopts a typical PDZ fold comprising five β strands and two α helices and the peptide is inserted into the PDZ-binding pocket through an interaction network specific to class I PDZ domains that recognize the consensus peptide sequence X-(S/T)-X-ΦCOOH, where X is any residue (position -1), and Φ is a hydrophobic residue (position 0). The last residue Leu(0) side chain is inserted in the hydrophobic core and the C-terminal carboxyl oxygen forms H-bonds with Gly(826) and Ile(827) amide nitrogens from the 'TLGI' loop, the 'GLGF' loop of whirlin. However, Leu(825) of 'TLGI' loop does not directly contact to the terminus carboxylate of myosin 15a, that contrasts with PDZ/PBM structures previously reported (Fig. 3). Notably, a water molecule forms two H-bonds between Leu(825) and one carboxylate oxygen atom. The side chain of Leu(-1) of myosin 15a peptide is facing out, toward the hydrophobic side chain of Ala(828) on the PDZ β2strand, that also contacts the side chain of Ile(-3). The three residues Leu(-1), Ile(-3) and Ala(828) compose with the Val(843) side chain from the 3-strand a hydrophobic patch exposed at the surface of the intermolecular -strand of complexed PDZ3. Thr(-2) forms an H-bond with the His(876) side chain on α2-helix as previously reported for a typical Class I of PBM binding [START_REF] Harris | Mechanism and role of PDZ domains in signaling complex assembly[END_REF]. In addition, the residue Glu(-4) side chain carboxyl group forms H-bonds with the Lys(877) side chain on α2-helix, most likely contributing to the high affinity of binding [START_REF] Maisonneuve | Molecular basis of the interaction of the human protein tyrosine phosphatase non-receptor type 4 (PTPN4) with the mitogen-activated protein kinase p38γ[END_REF] (Fig. 3A3). Lys(877), found in mouse gene, is replaced by an Arg in other species from zebra fish, xenopus laevis to mammals like horse, bovin, rat and human. This substitution could establish a stronger double-bond with the Glu(-4) side-chain of myosin 15a (Fig. S2). In both PDZ3/myosin 15a and PDZ3ΔPBM/myosin 15a structures, all the 13 residues of the myosin 15a peptide are visible in electron density maps, and the last 5 residues inserted in the binding groove attached to the β2-strand fully overlay (Fig. 3A4). However, the N-terminal of the myosin 15a peptide forms H-bonds with β2β3 loop, while in the crystal of PDZ3ΔPBM/myosin 15a, such contacts were not observed. Instead, the myosin 15a peptide binds to another PDZ3 domain organizing a dimer in the crystal (Fig. S1). We concluded that the structural data on binding modes are consistent with the affinity determination, and the PBM of whirlin does not affect the binding of myosin 15a peptide to the PDZ3 domain.

PDZ3ΔPBM construct of whirlin is monomeric in solution

The PDZ3ΔPBM/myosin 15a complex formed a crystallographic dimer. In order to know the oligomeric state of the complex in solution, we performed biophysical tests on the purified PDZ3ΔPBM domain. To estimate the oligomeric state of the PDZ3ΔPBM domain, Analytical Ultracentrifugation (AUC) and SAXS experiments were achieved with the PDZ3ΔPBM domain in free form and in complex with the PBM peptide of the myosin 15a protein. Only one species of PDZ3ΔPBM was detected for both free state or complexed, with a similar sedimentation coefficient of 1.21 S for the free form and 1.25 S for the complexed form. Both constructs show a frictional ratio of 1.4. showing similar shape with an onset of elongation. Taken together, these data are relatively consistent with a monomeric protein of 11kDa and a one to one complex of 12.6kDa, for the free and complexed PDZ3ΔPBM, respectively. In agreement with the AUC experiments, we found by SAXS that the free and complexed PDZ3ΔPBM behaves as a mono-disperse distribution of monomers in solution (Table 3). The estimated molecular mass of free and complexed PDZ3 derived from the extrapolated intensity I(0) at the origin is consistent with the theoretical value of 11.3 kDa. In conclusion, both techniques showed that the PDZ3ΔPBM in buffer (Tris 50mM, NaCl 150mM and TCEP 0.5mM) is on monomeric state in free and bound forms. This predominant monomeric state of PDZ3 in solution was also observed by measuring its rotational correlation time (τc) by NMR experiments. The 15 N relaxation parameters T1, T2 were determined for PDZ3ΔPBM in apo form and in complex with myosin 15a. The τc values, estimated from the average T1/T2 ratio of non-flexible residues, are 6.1 and 7.3 ns for free PDZ3ΔPBM and the PDZ3ΔPBM /myosin 15a complex, respectively. In the free form, this value is in good agreement with the theoretical value of 5.4 ns expected for a 11.0 kDa globular domain (18.2 Å Stokes radius) (Table 3). We conclude that the PDZ3/myosin 15a and PDZ3ΔPBM/myosin 15a complexes are monomeric in solution and have a 1:1 stoichiometry in our NMR, SAXS and AUC experiments. Dynamics in solution of PDZ3ΔPBM in free form and in complex with myosin 15a PBM We have assigned the backbone NMR signals of PDZ3ΔPBM. Over the 99 expected nonproline residues, 115 amide signals were visible in the HSQC spectrum and 95 backbone amide peaks could be assigned (96.0%). Backbone dynamics of PDZ3ΔPBM in apo form and in complex with myosin 15a has been evaluated by measuring 15 N relaxation parameters (T1, T2 and { 1 H}- 15 N heteronuclear NOE). The 15 N relaxation parameters were analysed in terms of internal dynamic parameters (order parameter S 2 , internal correlation time i and exchange parameter Rex) using the Lipari-Szabo model-free approach (Fig. S5) [START_REF] Lipari | Model-free approach to the interpretation of nuclear magnetic resonance relaxation in macromolecules[END_REF]. Overall, the data showed a quite rigid PDZ domain in both free and complexed states. Within each form, increased flexibility on the fast timescale (ps to ns) is observed in the N-and C-terminus ends (residues 802-813, 902-903, respectively) and in the loop connecting α2-β5 (891-894). In the complexed form, loops of PDZ3 are globally more rigid (slightly higher S 2 values for residues 834-839, 858-860, 867-868, 890-892) on the fast timescale. Interestingly, in the presence of myosin 15a partner, conformational exchange on the microsecond to millisecond time-scale has been detected by shorter T2 values in the region around the binding site encompassing the β2-β3 loop (residues 830-832; 837) and the helix α2 (residues 877-887), that are in direct contact with the ligand, while the end of helix α1 (852-856) is more rigid.

Structure of the PDZ3/harmonin a1-PBM complex

The crystal structure of PDZ3/harmonin a1 (NH2PKEYDDELTFFCOOH) was solved at 3.2 Å resolution with 6 copies in the asymmetric unit. Depending on the monomer, between 5 and 11 amino acids are observed in the electronic density. The C-terminal -TFFCOOH sequence is a Class I PBM as the one of myosin 15a. The peptide inserted in a same way as myosin 15a peptide in the PDZ binding groove. Notably, the Phe and Leu side chains at positions -1 and -3, respectively, are facing outwards of the binding groove, and form similar hydrophobic interactions with Ala(828) and Val(843) of the -strand. We noted that the proximity of the benzyl group from Phe(-3) triggers a strong chemical shift perturbation (CSP) of the NH group from Ala(828) as monitored by NMR in solution (Fig. S3). The peptide C-terminal carboxylate can only form weak H-bonds with the 'TLGI' loop (Fig. 3B2). In addition, 3 out of 6 copies in the asymmetric unit have deformed α1 helix, and in these 3 complexes (Fig. 3B4), 1 of them formed H-bonds with harmonin a1 carboxyl oxygen via its hydroxy oxygen of Thr of 'TLGI' loop. Meanwhile, the positions -3 and -4 are conserved with hydrophobic and anionic residues, respectively, -EI-for myosin 15a and -EL-for harmonin a1 sequences. However, we observed no electron density for Glu(-4) in harmonin a1 and Lys(877) on α2-helix that reflects the flexibility of these side-chains. Thus, the absence of stable salt bridges between these residues could explain the lower binding affinity of PDZ3 for harmonin a1 peptide than the one for myosin 15a.

Structure of the PDZ3/CASK-PBM complex

The crystal of PDZ3 complexed to the internal PBM of CASK (NH2TAPQWVPVSWVYCOOH) diffracted at 1.6 Å resolution. The asymmetric unit only contains one copy of the complex, and the last 7 residues -VPVSWVYCOOH are well defined in the electron density.

In this structure, PDZ3 still recognized a Class I -SWV-as a PBM shifted by one position. The amide oxygen of Val(0) is located at the same position as the myosin 15a and harmonin a1's carboxyl oxygen; the side chains of CASK-Val(0), harmonin a1-Phe(0) and myosin 15a-Leu(0) perfectly superimpose, pointing toward the PDZ pocket. The Val(0) carbonyl oxygen and the Tyr(+1) carboxyl oxygen form three H-bonds, mimicking the Cterminus carboxyl group on 'TLGI' loop (Fig. 3C2). The Trp(-1) side chain is facing out of the binding groove towards the hydrophobic surface of the β2 and β3-strands. Interestingly, the indole side chain is packed to the Tyr(+1) side chain to form a π-π offset packing, where the Tyr side chain hydroxyl group is 3.3 Å away from the indole group of Trp (Fig. 3C3). This aromatic packing seems to be very important for the backbone and side-chain orientations of CASK peptide in the binding groove to satisfy the network of hydrogen bonds with PDZ3. Ser(-2) is positioned in a reminiscent way of Thr(-2) of myosin 15a and harmonin a1, and forms the classical H-bond with His side chain on the α2-helix. The Pro(-5) side chain of CASK peptide is located near the side chain of His on α2-helix, creating potential CH-π interaction [START_REF] Zondlo | Aromatic-proline interactions: Electronically tunable CH/π interactions[END_REF], and the Pro(-5) Cγ is 3.8 Å away from the His(876). The Val(-6) side chain points toward the aliphatic side chain of Arg(836) from the 2-3 loop (Fig. 3C4).

Structure of the PDZ3/taperin-PBM complex

The crystal structure of the complex formed by PDZ3 and the internal (NH2GLPVTFIDEVDSEEAPQCONH2) peptide of taperin was solved at 1.3 Å resolution. The N-terminal 11 residues, -GLPVTFIDEVD-, are well ordered in the crystal (Fig. 3D). PDZ3 recognises the internal PBM of taperin peptide (-TFI-) as a class I PBM. Thr(-2) forms a H-bond with His imidazole nitrogen on PDZ3 α2-helix as a classical class I PDZ interaction motif. The Phe(-1) side chain is closed to the hydrophobic surface of β3strand and facing outside the binding grove in a similar position to the one adopted by Phe(-1) of the harmonin a1 peptide with only a slight shift of the phenyl group by 1.6 Å. Ile(0) has its side chain pointing towards the binding grove with its hydrophobic side chain as observed for typical last residue of C-terminal PBM. Ile(0) however participated to the formation of a short α-helix of three residues encompassing the two downstream residues Asp(+1) and Glu(+2). Interestingly, side chain carboxyl group of Asp(+1) turns back and forms H-bonds with the backbone amide of Leu and Gly with 'TLGI' loop, and consequently mimics the C-terminal PBM hydroxyl binding similar to the harmonin a1 peptides but stronger H-bonds (Fig. 3D2). In the short helix of taperin peptide, Ile(0) with Glu(+2) and Asp(+1) with Val(+3) formed two intramolecular H-bonds to maintain the helical conformation. In this structure, the Pro(-4) side chain positioned as a 90° vertical to the side chain of His on α2-helix, at the same location and same position as the Pro in the PDZ/CASK-PBM complex. Moreover, as also observed in the PDZ/CASK-PBM complex, the Leu(-5) side chain is stabilized by hydrophobic contacts involving the aliphatic side chains of Arg(836) in the β2-β3 loop and of Glu(830) from the β2-strand (Fig. 3D4).

Interestingly, the two internal motifs of CASK and taperin have sequences close to the consensus peptide; Φ-P-Φ-S/T -Ω-Φ (position 0)-, where Φ is a hydrophobic residue, and Ω is an aromatic residue, which is similar as predicted whirlin internal PBM binding pattern -T-[FLV]-I-previously reported by yeast two hybrid screening [START_REF] Mu | Characterization of diverse internal binding specificities of PDZ domains by yeast two-hybrid screening of a special peptide library[END_REF]. As a consequence, we noted that the N-terminals part of taperin-PBM and CASK-PBM, encompassing the hydrophobic position -5, Leu and Val, respectively, are in close contact with the β2-β3 loop and well superimposed while a shift between 2.4 and 3.2 Å of this loop toward the binding groove is observed for PDZ3 in complex with the two canonical PBMs from myosin 15a and harmonin a1.

Discussion

Proteins encoded by Usher genes contain numerous scaffolding domains and proteinprotein interaction motifs necessary to the intricacy of the molecular networks in hair cells. Remarkably, out of the nine Usher proteins involved in the development of stereocilia and the mechano-transduction machinery, eight possess short C-terminal PBM motifs, but only 2 Usher proteins have PDZ domains -whirlin and harmonin. Through their PDZ domains, as well as other scaffolding domains, these two paralogous proteins are qualified to assemble transient multi-protein complexes that coordinate the development of stereocilia as well as the mechano-transduction signalling. PDZ domains are dynamic tools for PPI, suited for multi-target binding environment. As illustrated by our results with the PDZ3 of whirlin, PDZ domains interact with their PBM-containing partners mainly with short residence time resulting in weak to medium binding affinities (1-100 M). In the hair cells, whirlin controls the stereocilia elongation and the planar polarity during development and also participates in the mechano-transduction signalling, stereocilia bundle cohesion and neuronal connection at the ribbon synapse [START_REF] Mburu | Defects in whirlin, a PDZ domain molecule involved in stereocilia elongation, cause deafness in the whirler mouse and families with DFNB31[END_REF][41][10][42] [START_REF] Ebrahim | Alternative Splice Forms Influence Functions of Whirlin in Mechanosensory Hair Cell Stereocilia[END_REF]. In photoreceptors, whirlin is involved in the renewal of the lightsensitive receptor protein rhodopsin, while it is also involved in the mechanosensory signalling of proprioceptors [START_REF] De Nooij | The PDZ-Domain Protein Whirlin Facilitates Mechanosensory Signaling in Mammalian Proprioceptors[END_REF]. This pleiotropic function of whirlin relies on its capacity to participate in various protein complexes. Whirlin is found at different locations in the hair cells, such as at the tip, ankle links and synapse, with many different isoforms whose expressions are regulated in time and space. As illustrated by in vitro investigations, the PDZ1 domains of whirlin and harmonin are multi-PBM binders, by contrast with their second PDZ domain [START_REF] Delhommel | Structural Characterization of Whirlin Reveals an Unexpected and Dynamic Supramodule Conformation of Its PDZ Tandem[END_REF].

Here, we report that the PDZ3 domain of whirlin, present in both the full-length and short C-terminal isoforms expressed in hair and photoreceptors cells, is also a multipartner binder, thanks to its unique capacity to accommodate non-canonical binding motifs. By contrast with the two N-terminal PDZ domains of whirlin involved in heterotypic and homotypic supramodule [START_REF] Delhommel | Structural Characterization of Whirlin Reveals an Unexpected and Dynamic Supramodule Conformation of Its PDZ Tandem[END_REF], we showed that its PDZ3 is fully independent from other domains at the C-terminal end of the protein, facilitating its accessibility to potential ligands. We quantified for the first time the in vitro interactions between the last PDZ domain of whirlin and all PBM-containing peptides from Usher proteins using the high-throughput Holdup chromatographic assay and SPR. Four Usher binders have been identified, myosin 15a and cadherin 23 with high affinity of few M, harmonin a1 with a moderate affinity 20 to 40-fold lower, and protocadherin 15 CD3 with a low affinity (250 M). In addition, non-Usher binders have been pinpointed; CASK, a well-known neuronal kinase, and taperin, a known deafness protein, which respectively interact with moderate to high affinity. Interestingly, these two partners possess internal PDZ binding motifs that efficiently recognize whirlin. By deeply characterising the dynamical and structural properties of the PDZ3 domain of whirlin in this work, we identified some features that could contribute to the promiscuity of the domain. We solved the four high-resolution structures of the binders myosin 15a, harmonin a1, taperin and CASK complexed to whirlin PDZ3. These proteins have been reported to interact or co-localize with whirlin in hair cells or photoreceptor cells [START_REF] Delprat | Myosin XVa and whirlin, two deafness gene products required for hair bundle growth, are located at the stereocilia tips and interact directly[END_REF] [18] [START_REF] Yap | CIP98, a novel PDZ domain protein, is expressed in the central nervous system and interacts with calmodulin-dependent serine kinase[END_REF][25] [START_REF] Gosens | MPP1 links the Usher protein network and the Crumbs protein complex in the retina[END_REF] [21] [START_REF] Sorusch | Characterization of the ternary Usher syndrome SANS/ush2a/whirlin protein complex[END_REF].

The four PDZ/ligand complexes, comprising two C-terminal (myosin 15a and harmonin a1) and two internal motifs (CASK and taperin), adopt a class I network of interactions; with their hydrophobic residue at position 0 inserted in a dedicated pocket of the PDZ and their Thr/Ser (-2) interacting with the conserved His on the PDZ α2 helix [START_REF] Harris | Mechanism and role of PDZ domains in signaling complex assembly[END_REF]. Interestingly, the PDZ3 domain is very permissive concerning the network of hydrogen bonds between the residue at position 0 and the 'TLGI' loop, as well as the recognition of the PBM at position -1, which could result from two distinct structural features. (A simplified LigPlot version is in supplementary Fig. S6) First, whirlin PDZ3 possesses a singular long α2-helix (13 residues) in comparison with the PDZ1 and PDZ2 domains (7 and 8 amino acids long, respectively), resulting in between one to two supplementary C-terminal helical turns. Among the six PDZ domains encoded by Usher proteins, this longer α2-helix is also observed in the PDZ3 of harmonin and PDZD7 (Fig. S4). Interestingly, Phe(887) in the last C-terminal turn of α2-helix is facing the binding groove. The orientation of the Phe side chain is constrained by hydrophobic contacts with Lys(820), Leu(825) and Ile(895) on the 'TLGI' loop and the α2-β5 loop. One consequence of this side-chain orientation is the shift of the 'TLGI' loop away from the binding groove due to steric hindrance. The aliphatic core of Lys(820) on the upstream 'TLGI' loop and the aromatic group of Phe form a strong antiparallel lock to stabilize the conformation of both loops and the α2-helix. In addition, Lys(820) further stabilizes the extra-long α2-helix by forming three H-bonds with the backbone carbonyl oxygens of the α2-helix and the α2-β5 loop. Interestingly, Thr(889) has its backbone carbonyl oxygen facing towards amine group of Lys(820) with a C-N-O angle nearly 180° and only 2.8 Å away from each other. This network of hydrogen bonds involving the lysine NH3 + group and backbone carbonyl has been documented before for its role in capping the C-terminus of α-helices [START_REF] Rogacheva | A new structural arrangement in proteins involving lysine NH3+ group and carbonyl[END_REF]. This original conformation of the top of the binding groove is observed in our four complexes structures but also in the absence of ligand, in the NMR structure of the free PDZ3 (PDB: 1UFX). This 'Lys-Phe lock' is strictly conserved in all orthologous whirlin sequences, the Phe being substituted by a Trp in harmonin and a Tyr in PDZD7 (Fig. S4). This lock features found in all four complexes most likely increase the rigidity of the upper part of the binding groove in agreement with our NMR dynamics measurements (Fig. 4,S5). We propose that this stable structure of the extra-long α2-helix facilitates the protein-protein interaction with a greater diversity of binders. The upper part of the binding groove is more constrained and is entropically favourable to accommodate non-canonical peptide such as the internal PBM of taperin. The slightly far-away Leu on the 'TLGI' loop extended the upper part of the PDZ3 binding pocket, thus suitable for internal PBM recognition (Fig. 3). In the case of the canonical PBM of myosin 15a, only one H-bond is formed between this 'TLGI' loop and the COOH of PBM, which is unique for PDZ-PBM binding. However, in the non-canonical binding CASK and taperin PBM, the 'TLGI' loop forms two H-bonds respectively. In addition, the carboxyl side-chain at position +1 of taperin internal PBM curves back and mimics the C-terminus carboxyl group (Fig. 3D2). The PBM of CASK, shifted by a single residue, which has never been reported before, in which the final residue Tyr is exposed to the solvent together with the Trp at position -2, forms a π-π offset, a well-documented type of interaction [START_REF] Burley | Aromatic-aromatic interaction: A mechanism of protein structure stabilization[END_REF][46] [START_REF] Daeffler | Functionally important aromaticaromatic and sulfur-π interactions in the D2 dopamine receptor[END_REF], that contributes to the stability and function of biomolecules. Samanta and coworkers mentioned that the π-π stacking of Tyr and Trp appears as an edge stacking like the OH-π interactions, as exhibited in the CASK structure [START_REF] Samanta | Packing of aromatic rings against tryptophan residues in proteins[END_REF].

Second, the exposed and flat hydrophobic surface of the β2-strand also facilitates the ligand accommodation, which could explain part of the versatility of PDZ3 to bind various PBMs. The β2 strand is formed by 4 residues: Ile-Ala-Ile-Glu. The small hydrophobic side chain of Ala(828) is exposed onto the surface with potential interactions with the positions (0) to (-2) of the PBM peptide. Thus, this hydrophobic surface makes it possible for the large hydrophobic (Leu) or aromatic residues (Phe or Trp) to adapt at these positions, as observed for the PBM sequences of all the four partners we studied (Fig. 3). Val(843) from the 3-strand of PDZ3 fill-up the hydrophobic patch exposed at the surface of the -strand and also participates to the PBM binding, mainly with the conserved position -3 of all four partners, which is systematically a hydrophobic residue, Leu or Val.

In this study, we showed that the PDZ3 of whirlin interacts with myosin 15a, harmonin a1, CASK and taperin PBM via either canonical or non-canonical PDZ-PBM binding, but also with cadherin 23 and protocadherin15-CD3. This variety of ligands likely reflects the different functions of whirlin in its various environments in hair cells and photoreceptors. Whirlin fulfils a function of scaffolding, participating in different complexes along the stereocilia. During the stereocilia development, it is well documented that whirlin localizes at the transient ankle links at the root region and at the tips of the tallest stereocilia row [START_REF] Ebrahim | Alternative Splice Forms Influence Functions of Whirlin in Mechanosensory Hair Cell Stereocilia[END_REF]. At the tip, whirlin has been previously reported to co-localize and interact with actin motor protein myosin 15a, two related MAGUK family proteins CASK and MPP1, GPSM2, and the actin regulation protein EPS8. The myosin 15a is a well-known binding partner of whirlin. It helps transporting whirlin to the tip of stereocilia, and remains in the protein complexes at the tip [START_REF] Delprat | Myosin XVa and whirlin, two deafness gene products required for hair bundle growth, are located at the stereocilia tips and interact directly[END_REF]. Altogether, these proteins play a crucial role in the regulation of actin dynamics according to their localization along the stereocilia. At ankle region of stereocilia, the scaffolding whirlin is proposed to adopt a polar organization in the cytoplasm, where its N-terminus is facing the cell membrane to form the USH2 complex with PDZD7, Usherin and ADGRV1, mainly through PDZ/PBM interactions [START_REF] Chen | Whirlin and PDZ domain-containing 7 (PDZD7) proteins are both required to form the quaternary protein complex associated with Usher syndrome type 2[END_REF], while the C-terminus is in contact with the actin filaments, potentially by the interaction with actin-binding protein such as myosin 7a even though the implication of PDZ3 is unclear in this process. The non-syndromic deafness DFNB79 related protein taperin is also expressed at the base of the stereocilia and its depletion affects the length and orientation of stereocilia and eventually causes stereocilia loss [START_REF] Rehman | Targeted Capture and Next-Generation Sequencing Identifies C9orf75, Encoding Taperin, as the Mutated Gene in Nonsyndromic Deafness DFNB79[END_REF][35] [START_REF] Zhao | Murine Fam65b forms ring-like structures at the base of stereocilia critical for mechanosensory hair cell function[END_REF]. Taperin is also reported interacting with GRXCR2 (Glutaredoxin domain-containing cysteine-rich protein 2) in the taper region. In the GRXCR2 knockout mice, the taperin has two main distinct localizations in the early development stage: at the base and tip of stereocilia, exactly where whirlin localizes [50].

Here, we identified the interaction between taperin and whirlin in vitro, and we propose a potential interplay between the taperin/GRXCR2 and USH2 complexes at the basal region of stereocilia.

The C-terminal region of whirlin coding for its third PDZ domain undoubtedly participates to the interactions with others proteins in the ankle link complex, with USH1 proteins forming USH1/USH2 complexes, as already observed with SANS and myosin 15a in photoreceptors, and potentially with cadherin 23 and protocadherin 15-CD3 along the stereocilia, but also with other deafness proteins such as taperin or catalytic protein such as CASK. Further studies are needed to unravel these different interactions in the development and functioning of sensorial cells.

METHODS

Constructs cloning, expression and purification

The constructs used: HHD2 (420-499), Proline-rich (PR) region (500-808), PDZ3ΔPBM (809-903), PDZ3 (809-906), HHD2-PR (420-808), PR-PDZ3 (500-906). The constructs of whirlin were synthesized from cDNA of Mus musculus whrn isoform 4 (UniprotKB number Q80VW5-4). Constructs HHD2, PDZ3ΔPBM and PDZ3 were subcloned into a pGST//2 expression vector (derived from pGEX-4T-1; Amersham) with an ampicillin resistant cassette. Constructs PR, HHD2-PR and PR-PDZ3 were subcloned pHTP1 vector by commercial kit: NZYEasy Cloning kit at NZYtech company. A TEV protease cleavage site was introduced between the N-terminal GST or His tag and the protein sequence. The constructs plasmids were transformed into E. coli BL21 (DE3) star (Invitrogen) strain. Bacteria were grown in LB or isotope ( 15 NH4Cl and/or 13 C-glucose) labelled M9 minimal media at 37°C. The expression was induced by 1mM IPTG (Isopropyl β-D-1thiogalactopyranoside) at OD600nm 0.8 to 1.0, at 30°C for 3 hours. Bacteria were then harvested and resuspended in lysis buffer (Tris pH 7.5 50mM, NaCl 250mM, βmercaptoethanol 2mM and protease inhibitor 1 table per 50ml (Complete, EDTA-free, Roche Diagnostics)). Then, the cells were broken by sonication, and the debris were pelleted by centrifugation (30,000g, 1 h, 4°C). The GST tag fused proteins were purified by affinity chromatographies column: GSTrap HP (GE Healthcare) or nickel chelated HiTrap Chelating HP (GE Healthcare), followed by a TEV protease cleavage at 4°C overnight. Then, the next day, a final step of a size-exclusion chromatography (SEC) was achieved by using Sephacryl S-100 HR 16/600 (GE Healthcare) with buffer Tris pH 7.5 50mM, NaCl 150mM, TCEP 0.5mM.

Holdup assay with human PDZ domains

The peptides were synthesized in solid phase using Fmoc strategy (Proteogenix) encompassing a biotinyl group, a (PEG)3 spacer and the C-terminal PBM sequence of Usher proteins (11 to 16 residues long). All PDZ domains (244 to 259 over 266 PDZ domains identified in the human genome) were expressed following the highthroughput protocol previously described [START_REF] Vincentelli | Quantifying domain-ligand affinities and specificities by high-throughput holdup assay[END_REF][51]. The holdup assay was carried out as previously described [START_REF] Vincentelli | Quantifying domain-ligand affinities and specificities by high-throughput holdup assay[END_REF]. The minimal Binding Intensity (BI) threshold value to define a significant interaction is 0.2.

SPR experiments

Interactions between whirlin and Usher peptides were characterized by SPR using a ProteOn XPR 36 system (Biorad) equilibrated with a buffer containing NaCl 250mM, Tris pH 7.5 50mM, TCEP 0.5mM. Biotinylated peptides were captured as ligands on a Neutravidin-functionalized NLC sensor chip (Biorad), over which the different whirlin fragments were flowed as analytes at 20 L/min for 2 minutes. The steady-state SPR responses (Req, experimental or extrapolated) were plotted against the concentration (C) of analyte and fitted using the following equation, Req = (Rmax * C)/(Kd + C), where Kd is the equilibrium dissociation constant, and Rmax the maximal binding capacity of the surface, using BIAevaluation 4.1 software (Biacore).

Phage display

Protein purification for phage display His-MBP-tagged whirlin PDZ3 fusion protein was expressed in E. coli BL21-gold (DE3) in 2YT media (1.6 % (w/v) tryptone, 0.5 % NaCl, 1 % yeast extract) supplemented with carbenicillin (100 µg/ml). Bacteria were pelleted by centrifugation and incubated at -20°C 1h before lysis with Bacterial Cell Lysis Buffer (NZYtech). The protein was purified by affinity chromatography using Ni-sepharose beads and eluted with 300 mM imidazole.

ProP-PD selections ProP-PD selections were carried out following a published protocol [START_REF] Davey | Discovery of short linear motif-mediated interactions through phage display of intrinsically disordered regions of the human proteome[END_REF] with few alterations. The MBP-tagged PDZ domain (30 µg in 100 µl PBS) was immobilized in a MaxiSorp flat-bottom 96-well plate (Nunc). For the use in pre-selection, 200 µl blocking solution (PBS with 0.5 % BSA) was immobilized in a different well. The proteins were immobilized overnight at 4°C, before the PDZ coated well was blocked with 200 µl blocking solution. The wells were washed four times with 200 µl PBS. An M13 phage library displaying 16 amino acid peptides covering the intrinsically disordered regions of the human proteome [START_REF] Davey | Discovery of short linear motif-mediated interactions through phage display of intrinsically disordered regions of the human proteome[END_REF] was added to the pre-selection well for 1 h, before being transferred to the MBP-PDZ3 coated well. After 2 h incubation at 4°C, unbound phage particles were removed by four times washing with 200 µl PT buffer (PBS, 0.05% (v/v) Tween-20), while bound phage particles were eluted with 100 µl log-phase OMNImax TM E. coli (Thermo Fischer Scientific). After a 30 min incubation at 37°C, 10 µl M13KO7 helper phage (1x1011 p.f.u./ml, New England Biolabs) were added and the culture was incubated at 37°C for another 45 min. Finally, the culture was added to a deep-well 96well plate with 1.1 ml 2YT media supplemented with 100 µg/ml carbenicillin, 30 µg/ml kanamycin and 1 mM IPTG, which was incubated at 37°C overnight. The next day, the bacteria were spun down at 2,000 g and 800 µl phage supernatant was transferred to a new well and incubated 10 min at 65°C to kill remaining bacteria. The phage solution was chilled, and pH adjusted by the addition of 1/10 volume of 10 x PBS, before being used in a new round of selection. Five rounds of selection were completed. The phage supernatant from each selection day was analysed for enrichment of binding phage by phage ELISA. MBP-PDZ3 (10 µg in 50 µl PBS) was immobilized in a MaxiSorp plate and incubated overnight at 4°C. The MBP-PDZ3 coated well and a control well were then blocked using the blocking solution. After 1 h incubation at 4°C, the wells were washed four times with PT buffer, and then incubated 2 h with 50 µl phage pool per well. Subsequently, the wells were washed four times with 200 µl PT buffer, before 100 µl horseradish peroxidase-conjugate anti-M13 antibody (BioSite) (1:5,000 dilution in PT buffer with 0.5 % BSA) was added for a 1h incubation. The wells were washed five times with 200 µl PT buffer. 50 µl of 1:1 mix of TMB (3,3',5,5'-Tetramethylbenzidine) and peroxide solution (VWR) was added as HRP substrate. The reaction was stopped using 50 µl 0.6 M H2SO4. The signal from both control and MBP-PDZ3 well was measured spectrophotometrically at 450 nm. The ratio between the signal from the MBP-PDZ3 and control well was calculated to evaluate the enrichment of binders over the days of selection, and the peptide coding region from the binding enriched phage pools were amplified and barcoded by PCR as described elsewhere [START_REF] Davey | Discovery of short linear motif-mediated interactions through phage display of intrinsically disordered regions of the human proteome[END_REF] before being analysed by next-generation sequencing on the Ion torrent platform.

Small Angle X-ray Scattering (SAXS)

X-ray scattering data were collected at the SWING beamline at Soleil synchrotron (Saclay, France). 50μl of sample (protein concentration 9.75 g/l, the mixture has protein and peptide 1:2 ratio) was injected on a Superdex-75 5/150GL (GE) size exclusion column in-line with the SAXS measuring cell. Frames with a duration of 1s were recorded during the whole elution time. Approximately 100 frames recorded in the dead volume of the column were averaged to generate the buffer subtraction curve and 10 to 30 frames corresponding to the top of the elution peak were averaged to generate the result curve. Primary data reduction was performed using the program FOXTROT (Xenocs SAS). The one-dimensional scattering intensities are expressed as a function of the modulus of the scattering vector Q = 4πsinθ/λ with 2θ being the scattering angle and λ the X-ray wavelength. Buffer intensities were subsequently subtracted from the respective sample intensities using the software package PRIMUS [52]. The radii of gyration were evaluated using Guinier approximation [53]. Dmax was determined from distance distribution function P(r) obtained with the program GNOM [54].

Fluorescence measurements

Measurements of fluorescence emission were performed at 25°C in a LS50 spectrofluorometer (PerkinElmer) using a 109F cuvette (Hellma Analytics). Excitation is set at 295 nm to excite only Tryptophan and emission spectrum is recorded from 300 nm to 450 nm, with a 5 nm bandwidth for both. Peptide (CASK-PBM: TAPQWVPVSWVY) was diluted in the buffer (Tris 50 mM pH 7.5, NaCl 150 mM, TCEP 0.5 mM) at concentration 2.4 μM. Buffer fluorescence is subtracted from peptide emission spectra and the spectra were corrected according to the measured concentration. The titration curve fitting was done with software Origin 7.5.

Sedimentation velocity -Analytical Ultracentrifugation (AUC)

Sedimentation velocity experiments were performed on an XL-I ultracentrifuge (Beckman-Coulter, Brea, CA, USA). Oligomerization state of PDZ3ΔPBM in presence and absence of 0.6 mM myosin 15a peptide were determined. Samples at 4 mg/ml were loaded in cells equipped with 1.2 cm double-sector epoxy centerpieces and sapphire windows. Samples were equilibrated for 2h at 20°C in an 4-hole AN60-Ti rotor and then spun at 42,000 rpm for 15h. Rayleigh interference profiles were recorded. Sedimentation profiles were analyzed using the continuous size distribution model c(s) of the software Sedfit [55]. All the c(s) distributions were calculated with a fitted frictional ratio f/f0 and a maximum entropy regularization procedure with a confidence level of 0.68. The protein partial specific volume of 0.740 ml/g was theoretically determined with Sednterp. Samples were prepared in 50 mM TRIS, 50 mM NaCl pH 8 and buffer density and viscosity at 20°C of 1.005 g/ml and 1.016 cP, respectively, were also calculated with Sednterp.

NMR Spectroscopy

All NMR experiments were performed at 25°C on a 600 MHz Bruker Avance III spectrometer equipped with a TCI cryoprobe and analyzed with CcpNmr Analysis 2.4 software [56].

Protein assignment. The backbone 1 H N , 15 N, 13 C , 13 C and 13 CO resonance assignment of PDZ3 PBM was performed on a 0.8 mM 15 N/ 13 C-labeled sample (sodium phosphate, pH 6.5, 2mM TCEP and 150mM NaCl) using 2D 1 H- 15 N HSQC and the following BEST-or BEST-TROSY-based 3D experiments: HNCO, HNCO+, HN(CA)CO, HN(CO)CACB, HNCACB. Protein dynamics. The 15 N relaxation times (T1 and T2) and { 1 H}- 15 N heteronuclear NOE were measured on a 0.2 mM 15 N-labeled sample of PDZ3 PBM (Tris 50m, pH7.0, 150mM NaCl and 2mM TCEP) in the absence and presence of a 2-fold excess of myosin 15a peptide. Experiments were recorded by standard methods [57], in an interleaved manner with a recycling time of 3 s and with 7 relaxation delays for T1 [START_REF] Sorusch | Characterization of the ternary Usher syndrome SANS/ush2a/whirlin protein complex[END_REF]100,200, 400, 600, 800, 1200 ms) and 8 for T2 (17, 34, 51, 68, 85, 119, 153, 238 ms). The heteronuclear NOE were recorded in the presence and absence of a 3 s 1 H saturation sequence (120° 1 H pulse train), with a recycling delay of 5 s. The relaxation parameters were analyzed using the model-free formalism of Lipari and Szabo [START_REF] Lipari | Model-free approach to the interpretation of nuclear magnetic resonance relaxation in macromolecules[END_REF] with the program TENSOR2 [58] to extract internal dynamical parameters (amplitude of the picosecond to nanosecond time scale motion S 2 , internal correlation time i and exchange parameter Rex). An isotropic model, with a correlation time c, was sufficient to describe the global reorientation of the protein and an anisotropic model did not improve the fit.

Peptide titration. PDZ3 (and PDZ3 PBM) samples initially contained about 200 mM 15N-labeled PDZ3 in 300 L of buffer containing Tris 50mM, pH7.0, 150mM NaCl and 2mM TCEP. Unlabeled peptide was added stepwise to a final PDZ/peptide ratio of 1:4, 1:10 and 1:11 for myosin 15a, harmonin a1 and taperin peptides, respectively. 1 H- 15 N HSQC spectra were recorded at 13, 15 and 9 different titration points, respectively. For myosin 15a and harmonin a1, average ( 1 H, 15 N) chemical shift changes were calculated as ∆δavg = ((∆δH) 2 +(∆δN×0.159) 2 ) 1/2 . For taperin, the variations of the bound signal intensity were followed because of the slow exchange binding process with this peptide. The dissociation constants (Kd) of the respective complexes were obtained by fitting the titration data (∆δavg or intensities) with a model assuming a 1:1 complex formation and with nonlinear regression.

Crystallization and diffraction data collection

Crystallization screening trials were performed at the Crystallography Platform of the Institut Pasteur [59]. For the initial screenings, sitting drops of 400 nL (1:1 protein to precipitant ratio) were set up in 96-well Greiner plates with a Mosquito automated nanoliter dispensing system (TTP Labtech, Melbourn, UK). The plates were then stored in a RockImager (Formulatrix, Bedford, USA) automated imaging system to monitor crystal growth. Crystallization conditions corresponding to the different complexes between whirlin PDZ3 and peptides are detailed in Table S1. Diffraction data were collected at beamlines PROXIMA 1, PROXIMA 2a (SOLEIL synchrotron, St. Auban, France), and ID-29 (Synchrotron ESRF, Grenoble, France) and processed with XDS [60] and Aimless [61].

Structure determination and model refinement

The crystal structures of whirlin PDZ3 in complex with the different peptides were solved by the molecular replacement technique using the PDZ3 domain of the human KIAA1526 protein (PDB 1UFX) as search model with Phaser [62]. Final models of the complexes were obtained through interactive cycles of manual model building with Coot [63] and reciprocal space refinement with Buster [64] and phenix.refine [65]. X-ray diffraction data collection and model refinement statistics are summarized in Table S1. Figures showing the crystallographic models were generated with PyMol (Schrodinger, LLC, version 2.4.0a0).

Figure 1 :

 1 Figure 1: PDZ3 domain is separated by a long proline rich region (PR) from N-terminal domains. A: The IUPRED predicted structured and unstructured regions of FL-whirlin. The five generated constructs are underlined (sequence based on Uniprot ID Q80VW5-4). B: size parameters Rg and Dmax extracted from SAXS data, and predicted and theoretical molecular weight (MW) for whirlin constructs. C: dimensionless Kratky plots of PR involved constructs. Five constructs are in the same colours in Fig 1A and 1B.

Figure 3 :

 3 Figure 3: Binding details of four PDZ3-PBM peptides structures. Only hydrogen bonds distance lower than 3.7 Å are represented. A. PDZ3-myosin 15a. A2. Detailed the accommodation of PBM at 'TLGI' loop. A3. An extra side-chain interaction between Glu(-4) of myosin 15a and Lys(877). A4. An overlay view of PDZ3/PDZ3ΔPBM-myosin 15a, where last three residues -VML are in stick-shape. PDZ3ΔPBM-myosin 15a complex are in colour cyan and yellow. B. PDZ3-harmonin a1. B2. Detailed the 'TLGI' loop with PBM. B3. Phe(-1) and Leu(-3) of harmonin a1 at the hydrophobic surface of β2-and β3strands. B4. Overlay of all six copies in the asymmetric unit, where three of them have the deformation of α-helix 1 coloured in blue. C. PDZ3-CASK. C2. Detailed the 'TLGI' loop with peptide C-terminus. C3. π-π offset packing between Tyr (1) and Trp(-1). Hydrophobic stacking of Trp(-1) and Val(-3) at the surface of β2-and β3-strands. C4. Val(-5) at the pocket formed with Glu(830), Arg(836) and Gln(837) aliphatic sidechains. D. PDZ3-taperin. D2. Side-chain of Asp1 mimics C-terminus PBM carboxylate group, forming 2 H-bonds with 'TLGI' loop. D3. Phe(-1) and Val(-3) of taperin at the surface of β2 and β3-strands. D4. Hydrophobic contacts between Leu(-5) and a pocket formed by Glu(830), Arg(836) and Gln(837) aliphatic side-chains.

Figure 4 :

 4 Figure 4: the 'Lys-Phe lock ' at the top of 'TLGI' loop and α2-helix in the four PDZ-PBM complexes. A. PDZ3/myosin 15a. B. PDZ3/harmonin a1. C. PDZ3/CASK. D. PDZ3/taperin. Yellow: the peptide. Orange: the α2-helix. Magenta: Lys820. Blue: Phe887. Distances are shown by dash lines with threshold 3.8 Å.

Table 1 :

 1

	Measured affinities between the whirlin PDZ3 domain and the collection of
	stereociliary protein PBMs. A: Holdup assay results are reported as Binding Intensities
	(BI) which correlate with binding affinities scale [27]. B: Surface Plasmon Resonance
	(SPR)

data are reported as Kd (µM). PBM peptides Holdup (BI) Kd by SPR (μM)

	myosin 15a	0.67	4.4±0.8
	cadherin 23	0.57	4.3±0.3
	harmonin a1	0.45	67±25
	protocadherin15-CD3 0.26	230±30
	SANS	0.19	No Binding
	whirlin	0.03	No Binding
	protocadherin15-CD2 0.01	No Binding
	protocadherin15-CD1 -0.03	No Binding
	ADGRV1	-0.03	No Binding
	usherin	-0.05	No Binding

Table 2 :

 2 Binding affinity of PDZ3 to various PBMs with sequences and binding pattern. The three residues that form the PBM are in bold. *: The CASK Kd value was determined using fluorescence titration, and others were measured by NMR titration.

		Peptide	Sequence	Kd (μM)	binding pattern
	PDZ3ΔPBM myosin 15a	ERLTLPPSEITLL	3.4 ± 1.8	Class I
	PDZ3	myosin 15a	ERLTLPPSEITLL	2.9 ± 1.6	Class I
	PDZ3	harmonin a1 PKEYDDELTFF	132 ± 13 Class I
	PDZ3	CASK*	TAPQWVPVSWVY	7.1 ± 1.0	Class I (internal PBM)
	PDZ3	taperin	GLPVTFIDEVDSEEAPQ 58 ± 31	Class I (internal PBM)

Table 3 :

 3 Oligomeric state of PDZ3 monitored by SAXS, AUC and NMR rotational correlation time measurements.

		Rg (Å) Dmax (Å) Sedimentation coefficient (S) τc(ns)
	PDZ3ΔPBM	15.18	66.94	1.21	6.1
	PDZ3ΔPBM+myosin15a 15.87	65.25	1.25	7.3

ACKNOWLEDGMENTS

The project has received funding from the European Union's Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement PDZnet No 675341. The authors also acknowledge the Conseil Reǵional d'Ile de France for financial support through the SESAME 2014 NMRCHR program No. 14014526 (800 MHz spectrometer). The authors acknowledge Marie-Christine Vaney for technical assistance concerning the analysis of X-ray diffraction data and Patrick England for technical assistance in SPR experimental design and analysis. The authors are grateful to Sylvie Nouaille for providing the initial plasmid encoding for PDZ3 PBM of whirlin. We acknowledge SOLEIL and ESRF for provision of synchrotron radiation facilities, and we would like to thank the staff of the SWING and PROXIMA1&2 beamlines in synchrotron SOLEIL and ESRF-ID29 beamline for assistance during the SAXS and X-ray diffractions measurements.

Data and PDB code availability

Atomic coordinates for the whirlin PDZ3/PDZ3ΔPBM in complexes with ligand PBMs have been deposited in the protein data bank with accession code 6Y38, 6Y9N, 6Y9O, 6Y9P and 6Y9Q. 6Y38: PDZ3ΔPBM+myosin 15a peptide, 6Y9N: PDZ3+myosin 15a peptide, 6Y9O: PDZ3+CASK peptide, 6Y9P: PDZ3+harmonin a1 peptide, 6Y9Q: PDZ3+taperin peptide

AUTHOR CONTRIBUTIONS

Concept


Asako Isogawa

Robert P Fuchs

Shingo Fujii

Chapter 16. Chromatin pull-down methodology based on DNA triple helix formation

Keywords: DNA-based chromatin capture, Reverse-ChIP, Replication, Repair, Transcription, Proteomics, Chromatin, Epigenetics, Drug screening

Identification of the protein complexes associated with defined DNA sequence elements is essential to understand the numerous transactions in which DNA is involved, such as replication, repair, transcription, and chromatin dynamics. Here we describe two protocols, IDAP (Isolation of DNA Associated Proteins) and CoIFI (Chromatin-of-Interest Fragment Isolation), that allow to isolate DNA/protein complexes (i.e., nucleoprotein elements) by means of a DNA capture tool based on DNA triple helix (triplex) formation. Typically, IDAP is used to capture proteins that bind to a given DNA element-of-interest (e.g., a specific DNA sequence, an unusual DNA structure, a DNA lesion) that can be introduced at will into plasmids. The plasmids are immobilized by means of a triplex forming probe on magnetic beads and incubated in nuclear extracts; by using in parallel a control plasmid (that lacks the DNA element-of-interest), proteins that preferentially bind to the DNA element-of-interest are captured and identified by Mass Spectrometry. Similarly, CoIFI also uses a triplex forming probe to capture a specific chromatin fragment from a cultured cell line that has been engineered to contain multiple copies of the DNA element-of-interest.

Introduction

Characterization of proteins involved in various DNA transactions is needed to understand functional control of protein networks during all DNA transactions. For example, when taking into account numerous studies, the control of transcription in vivo is best described as a complex protein network [1].

A straightforward way to delineate a protein network on DNA is to isolate and characterize nucleoprotein fragments of interest from living cells. In these years, such approaches have been successfully applied in various studies [2][3][4][5][6].

The formation of DNA triple helices has been well characterized and implemented in numerous applications including therapeutic purposes [7,8]. The principle of triplex formation is that a third strand (i.e., Triplex Forming Oligonucleotide (TFO)) consisting of either a pyrimidine-rich or a purine-rich sequence, forms Hoogsteen (or reverse Hoogsteen) base pairs with a complementary purine-rich strand in the major groove of double-stranded DNA (dsDNA) without disrupting its canonical Watson-Crick base pairs. In order to practically utilize triplex formation in applications, numerous efforts were implemented to increase stability of triplexes. As a consequence, the stability of triplexes was largely improved by using modified TFO probes composed of chemically altered nucleotides (e.g., peptide nucleic acid (PNA), locked nucleic acid (LNA)) [9,10] and by the introduction of a DNA intercalator (e.g., acridine, psoralen) to one of the extremities of the probe [START_REF] Sun | Sequence-specific intercalating agents: intercalation at specific sequences on duplex DNA via major groove recognition by oligonucleotide-intercalator conjugates[END_REF][12][13].

By utilizing the unique feature of triplex formation, we developed a robust experimental workflow in order to capture, identify and characterize proteins assembled, in vivo (CoIFI) or in vitro (IDAP), on a given DNA sequence-of-interest [14]. Here, we introduce some applications of IDAP and CoIFI as model cases.

Materials

All buffers are freshly prepared unless otherwise stated. When using H2O, it is ultrapure water. 13. During this heat step, it is expected that the CAG triplet repeat forms a stem-loop as drawn in Fig. 6a. Owing to the stem-loop formation, 6 nt ssDNA regions flanking the CAG triplet repeat are closely placed. Subsequently, the 12-mer oligo is annealed to the proximate 6 nt ssDNA regions. 14. Before using the beads, they are washed two times with CB3.

15. Outcomes of sonication (i.e., patterns of genomic DNA fragmentation) are largely influenced by buffer components, types of cell lines, cell density, cross-link conditions, and so on. The sonication condition needs to be optimized. [START_REF] Georgakilas | p21: A Two-Faced Genome Guardian[END_REF]. Before using the resin, it is washed two times with CB1.3 with 50 mM NaCl. 17. Before mixing the sample, the beads are washed two times with CB3.1. Then, the beads are resuspended in 10 µl of CB3.1 with 1.25 mg/ml BSA. Fig. 8 The constructed plasmids capture via TFO-mediated triplex formation. 40% of the elution products are analyzed on 2% agarose gel containing 0.5 µg/ml EtBr. As a control, 13 ng of each input (pAS200.2, pAS203, pAS203(rcc), pAS203(gap), and pAS203(cSL)) are loaded on the same gel.

Figure legends

Recovery yields of all plasmids through the TFO-mediated plasmid capture are nearly same (≈65% of input). Thus, the constructed plasmids are suitable to use as DNA substrates in the IDAP approach.

Fig. 1 A

 1 Fig. 1 A schematic view of triplex formation. A TFT region on DNA is shown in blue. A red line in the

Fig. 2

 2 Fig. 2 Construction of pAS114.1γ. ori: high copy number pUC origin in E. coli. Ap: ampicillin resistance

Fig. 3 A

 3 Fig. 3 A schematic view of IDAP approach.

Fig. 4

 4 Fig. 4 Nucleoprotein isolation in IDAP. (a) 40% of the elution products are analyzed via silver staining

Fig. 5

 5 Fig. 5 Construction of pAS200.2 and pAS203. pUC ori: high copy number pUC origin in E. coli. Tet:

Fig. 6

 6 Fig. 6 Construction of pAS203 with a closed stem-loop. (a) pAS203 is introduced double nicks by using

Fig. 7

 7 Fig. 7 Confirmation of characteristic properties of pAS203 derivatives. (a) Lane sc is a super coiled

Fig. 9

 9 Fig. 9 Schematic views of construction of a human stable cell line and isolation of nucleoproteins in

Fig. 10

 10 Fig. 10 Nucleoprotein isolation in CoIFI. (a) Genomic DNA is purified from the input sample for CoIFI

Fig. 1 Fig. 2 20Fig. 3 Fig. 4 22Fig. 5

 12345 Fig.1

Fig. 6 24Fig. 7

 67 Fig. 6

Fig. 8 26Fig. 9

 89 Fig.8

Fig. 10

 10 Fig. 10