Using in vitro high-throughput approaches, we identified PDZ3 binding partners. Our high-throughput chromatography assayHoldup allowed BIOLOGY AND HEALTH SCIENCES SYNCHROTRON SOLEIL HIGHLIGHTS 2020 us to quantify the interaction between the whirlin PDZ3 domain and the collection of stereociliary protein PBMs [5]. In parallel, we have used a previously described phage library that covers the intrinsically disordered regions of the human proteome to detect potential internal SWING, PROXIMA-1 & PBM. Altogether, we identified seven stereociliary proteins that encode PBMs and interact with whirlin PDZ3, where four of them are first reported, PROXIMA-2A BEAMLINES in the 3-130 µM affinity range measured by NMR, Holdup and SPR. Associated publication To investigate the structural determinants of binding, we determined the Deciphering the unexpected binding PDZ3 domain structure in complex with the PBMs of the four partners: capacity of the third PDZ domain of myosin 15a, harmonin a1, CASK and taperin by X-ray crystallography whirlin to various cochlear hair cell (Fig. 2). The complexed forms showed an equivalent backbone fold of partners. the PDZ3 domain. Whirlin PDZ3 possesses a singular long α2-helix Y. Zhu, F. Delhommel, F. Cordier, in comparison with others PDZ domains. In agreement with our NMR S. Lüchow, A. Mechaly, dynamics measurements, the structure of this extra-long α2-helix is B. Colcombet-Cazenave, V. Girault, stabilized by a ‘Lys-Phe’ lock in its upper part (Fig. 3). The binding groove E. Pepermans, A. Bahloul, C. Gautier, is thus more constrained and is entropically favourable to accommodate S. Brûlé, B. Raynal, S. Hoos, A. Haouz, a greater diversity of binders such as the non-canonical PBM of taperin. C. Caillet-Saguy, Y. Ivarsson & N. Wolff. In addition, the exposed and flat hydrophobic surface of the ß2-strand J Mol Biol., 432, 5920 (2020). also facilitates the ligand accommodation for large hydrophobic or aromatic residues. References FIGURE 2 FIGURE 3 [1] M. Schwander et al., J. Cell Biol., 190, 9 (2010). [2] P. Mathur & J. Yang, Biochim. Biophys. Acta - Mol. Basis Dis. 1852, 406 (2015). [3] N. Michalski et al., J. Neurosci, 27, 6478 (2007). [4] P. Mburu et al., Nat. Genet., 34, 421 (2003). [5] R. Vincentelli et al., Nat. Methods. 12, 787 (2015). Corresponding author Nicolas Wolff Institut Pasteur, CNRS CONCLUSION Unité Récepteurs-Canaux 75015 Paris, France The C-terminal region of whirlin encompassing the third PDZ domain nicolas.wolff@pasteur.fr undoubtedly participates to the interactions in the ankle link complex, with others USH proteins, as already observed with SANS and myosin 15a Captions in photoreceptors, and potentially with cadherin 23 and protocadherin FIGURE 1: DThe C-terminal PDZ3 domain of whirlin is 15-CD3 along the stereocilia, but also with other deafness proteins such independent in the multi-domain organization of the Usher as taperin or catalytic protein such as CASK. Several structural features protein. PDZ3 domain is separated by a long proline rich region (PR) from N-terminal domains. Upper pannel: the five of PDZ3 we highlighted in this study are responsible for the versatility of generated constructs are underlined. Middle pannel: size this domain to bind various partners. parameters Rg and Dmax extracted from SAXS data, and predicted and theoretical molecular weight (MW) for whirlin constructs. Lower pannel: dimensionless Kratky plots of PR involved constructs. SAXS data were collected at the SWINGbeamline at Soleil synchrotron (Saclay, France). FIGURE 2: Comparison of high resolution structure of complexes between whirlin PDZ3 and four motifs from stereociliary partners. X-ray diffraction data were collected at the PROXIMA beamlines at Soleil synchrotron (Saclay, France). A: Backbonesuperimposition of four complexed PDZ3 and free PDZ3 (PDB: 1UFX). B : peptides superimposition on the bindinggroove of apo PDZ3. C: overall RMSD values of PDZ3 backbone between free PDZ3 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. FIGURE 3: A unique ‘Lys-Phe lock’ increases the rigidity of the PDZ3 binding groove. Cover page of Journal of Biology, 55 Volume 433, Issue 4, 19 february 2021.