To be able to perceive this world, we rely on our sensory systems, especially the vision and hearing. Both vision and hearing are based on highly differentiated sensory organ cells: the photoreceptor cells and auditory hair cells, whose functions depend on highly specialized cell structure and rapid signal transmission. Photoreceptor cell is one of the most metabolic active cells, which needs to constantly renew its photo-detector proteins on the subcellular membrane discs along the connecting cilium. The auditory hair cells function through the force-induced movement of hair bundles on their cell apical surface. It causes the opening of mechano-gated ion channel on the tip of the hair bundles and leads to the depolarization of the cells, then triggering the electrical signal transmission to the brain.

Usher syndrome is one of most common genetic neurosensory syndromes, which affect both vision and hearing. So far, nine genes mutations have been found related to the Usher syndrome. The encoded proteins are critical for the development and the molecular architectures in both photoreceptor cells and auditory hair cells. Usher proteins are found to form large protein densities in the tips and links anchoring points in the hair bundles. Among all the nine Usher proteins, seven of them contain PBM (PDZ Binding Motif) while two proteins have PDZ domains, which are Harmonin and Whirlin. PDZ domain is a well-known protein-protein interaction domain and PDZ-PBM interaction is the main protein-protein interaction way to form multi-protein complex in the Usher protein densities, but the protein network remains elusive. Deciphering the molecular mechanisms of Usher complexes would provide new clues to understand the physiopathogenesis of Usher mutations inducing hearing loss and might be valuable for developing new specific therapeutical approaches.

My PhD project focuses on the PDZ containing protein Whirlin, a crucial protein of the hearing/vision systems. Whirlin is a multi-domain scaffolding protein encompassing five structured globular domains linked with unstructured region, where three of them are PDZ domains. First, we characterised the function of the third PDZ domain-PDZ3 with its binding patterns, fully exposed at the C-terminus of the protein. PDZ3 recognizes at least seven proteins PBM: CASK, MPP1, Myosin 15a, Protocadherin 15 CD3, Cadherin 23, Harmonin a1 and Taperin, and we solved the high-resolution structures of four of them. We highlighted the unexpected binding capacity of the third PDZ domain of Whirlin to various cochlear hair cell partners coding for both canonical and non-canonical PBM sequences.

The first N-terminal PDZ1 is a domain of dual functions, which recognizes both protein PBM and lipids. We deeply characterised the PDZ1 and related PDZ1 containing multi-domain constructs in interaction with proteins and lipid membranes by various biophysical approaches. We solved the structures of PDZ1 in complex with partner PBM and with lipid, and highlighted the residues specifically involved in such interactions. In addition, we emphasized the role of the upstream N-terminal HHD1 domain adjacent to the PDZ1 domain and involved in the self-association of Whirlin. Altogether, these properties of Whirlin are related to its function as a scaffolding protein that ensures the anchoring of the membrane Usher proteins, a submembrane protein network essential for the development of hair bundle and for the photoreceptor functioning.

Remerciements/ Acknowledgements/ 致谢

First of all, I thank my supervisor Nicolas Wolff for his patient guidance and help for three years from lab work to find an accommodation for me to stay in Paris, too many things to list more than this thesis can hold.

I thank all my colleagues who kindly helped me for my life and study problems in our small group, especially Florent Delhommel, Célia Caillet-Saguy, Mariano Genera and Baptiste Colcombet-Cazenave, lots of sweet memories with you nice people in these three I especially thank our Chinese Society. With many brilliant ideas and questions, I truly enjoyed every week's Chinese seminar, where I learnt a lot about virology, immunology, epigenetics, microbiology and etc. This society broadened my horizon of scientific world and created a lot of future cross-discipline collaborators.

It is a great honour to meet so many nice people in my PhD, which is very low chance as a Chinese student come from China and develop a network in Paris, in Chinese we call it '缘' (pronounced as ¨yuan¨) similar like fate or destiny, a predetermined principle that dictates a person's encounters, but this character is always positive in Chinese not like fate or destiny. I will cherish this period of time and people in the future sincerely. Human ear is a highly sensitive sound reception system, where less then 1 pm displacement vibration can be detected by our eardrum [1]. Beside the high sensitivity of sound wave amplification, the frequency detection is also highly accurate, which can distinguish the difference between pure tones of 1000 and 1003 Hz, from a total range of frequency between 20 to 20 000 Hz in childhood, and 15 000 Hz in young adults [2]. The acoustic wave is transmitted from the air to our ears, which sets the movement of eardrum and conveys the energy through the ossicles in the middle ear to the cochlea in the inner ear. The piston like motion of the last piece of the ossicles fluctuates the fluids in the cochlea, and thus causes the basilar membrane to flex up and down in the center of cochlea. Organ of Corti, where the sensory hair cells are located, is the sound-processing place. The movement of the hair cells actin-filled microvilli (stereocilia) causes the opening of the mechano-gated ion channels on the tip of the stereocilia, and triggers the depolarization of the hair cells, where the neuron signal is generated and transmitted to the brain [3]. The details of the anatomy of the inner ear will be detailed below.

Anatomy of inner ear

Human ears consist of three main anatomical parts: the outer, the middle and the inner ears. The outer ear is composed of the auricle, the auditory canal and the eardrum (tympanic membrane). The middle ear is an air-filled room called tympanic cavity, which has three ossicles connecting the eardrum to the cochlea of the inner ear, called malleus, incus and stapes. These three tiny bones transmit the vibration of eardrum to the cochlea of inner ear. The inner ear is covered with the bony labyrinth imbedded in the temporal bone, which contains the vestibular and cochlea (Fig. 1).

Figure 1: the anatomy of the ear. Image credit: Alila Medical Media Shutterstock.

Vestibular system

The vestibular system is a sensory system part of the inner ear, which is responsible for the spatial orientation and the sense of movement. To fulfil these two functions, the vestibular consists of two parts: the semicircular ducts and otolith organs, which detect the rotational movement and linear accelerations, respectively. The neural pathway of vestibular system is connected to the eyeball moving muscles. When tilt or move, the head can synchronously activate this circuit, resulting in an offset of opposite direction movement of the eyes. As a result, the eye can keep focusing during the head movement; this is called the Vestibuloocular reflex (VOR) (Fig. 2).

As described in last section, the actin filaments are linked with cross-linking proteins as a highly organized and high-dense structure. All actin filaments have the same polarity orientation with their barbed/plus ends facing the tip and pointed/minus ends facing the base. The actin filaments and cross-linking proteins act in a myosin driven actin molecular treadmill process, which means that actin, espin and other component proteins are involved in the moving and regeneration in the stereocilia, to maintain a stable equilibrium [START_REF] Rzadzinska | An actin molecular treadmill and myosins maintain stereocilia functional architecture and self-renewal[END_REF] [START_REF] Manor | Dynamic length regulation of sensory stereocilia[END_REF]. However, this hypothesis has been challenged, where [START_REF] Zhang | Multi-isotope imaging mass spectrometry reveals slow protein turnover in hair-cell stereocilia[END_REF] showed the absence of treadmill process in the stereocilia, except in the tip area. The stereocilia bundling can be divided into four stages in mice. In the stage I at E8 (embryonic day 8, E0 being the ovulation step), the hairy microvilli and one kinocilium (microtubule filled cilia) appear and start to differentiate. In the stage II at E8-E13, the kinocilium moves to the edge of hair cell surface, the internal actin filaments cross-linking increases, and stereocilia cover the full surface area. The stereocilia close to the kinocilium start to form linkers and grow taller than others; then the tip-links appear. At the end of stage II, the staircase hierarchy starts to set up. In stage III at E13-E16, stereocilia stop growing tall, but the internal actin filaments pack from around 100 filaments per stereocilium up to 900 filaments. At this stage, the cell apical surface forms the cuticular plate with the smaller microvilli disappearing. Stereocilia constitute the base taper, and actin filaments grow downwards into the cytoplasm. In the stage IV at E16-P3 (postnatal day 3), the stereocilia grow broader to the maturing size, while the kinocilium starts to disappear (Fig. 11). Since E17.5, the stereocilia have almost stop growing in length and size, with a dense coating but with less inter-stereocilia lateral links. At P2, the links at the base of the stereocilia called ankle links appear. At P9, the number of ankle links decreases and they disappear at P12. At this stage, only tip links and horizontal top connectors connect between different rows of stereocilia.

Chapter 2: Deafness related proteins 2.1. Background of hearing loss Human auditory system is a sensitive device, which can distinguish very subtle difference in frequency and amplitude. The sound loudness unit dB (decibel, i.e. 1/10 Bell) is used to characterize the level of amplitude. It is a logarithmic scale unit, meaning that the difference between 20dB to 0dB is a 100-fold of power or 10-fold of amplitude increase. At 0dB, the average sound pressure is 2x10 -5 Pa, which is also the hearing threshold of young adults. For noise between 0-70dB, there is no hearing damage regardless of the exposure time, but it is potentially hazardous for exposure at 85dB or louder for more than 8 hours, while 85dB is similar to the noise made from the busy traffic road [START_REF] John | Noise exposure and self-reported hearing impairment among gasfired electric plant workers in Tanzania[END_REF] [START_REF] Goines Lisa | Noise Pollution: A modern plague[END_REF]. The threshold of pain is 140dB (Fig. 13).

Figure 13: Table of noise levels and sources.

A normal hearing person has a threshold of less than 22 dB in both ears. The disabling hearing is defined as in the good hearing ear with listening threshold above 40 dB in adults and 30 dB in children. The degrees of hearing loss have been divided into five levels: mild (25-40 dB), moderate (41-55 dB), moderately severe (56-70 dB), severe (71-90 dB) and profound (above 90 dB) [START_REF] Koffler | Genetics of Hearing Loss -Syndromic[END_REF]. Deafness is the most prevalence sensory disorder among human beings, which affects 2 to 3 out of 1000 newborns in United States, 25% of the population between 65 and 74 years, and over 50% of elderly over 75 years (Quick Statistics About Hearing. of calcium level in hair cells, which subsequently lead to a stimulation of reactive oxygen species (ROS) production by mitochondria [START_REF] Böttger | The mitochondrion: A perpetrator of acquired hearing loss[END_REF] [START_REF] Peng | Oxidative stress caused by mitochondrial calcium overload[END_REF]. The ROS is one of the most common causes of hair cell death; antioxidant molecules have been shown to attenuate the noiseinduced hair cell damage, preventing potential hearing loss [START_REF] Oishi | Emerging treatments for noise-induced hearing loss[END_REF]. By contrast, the hair cells of frog or chicken that can be regenerated, hair cells in mammals are permanent and irreparable once been damaged [START_REF] Gale | Survival of bundleless hair cells and subsequent bundle replacement in the bullfrog's saccule[END_REF].

Other hearing losses

Ototoxic and neurotoxic drugs induced hearing loss is responsible for the large proportion of hearing loss in China around 1960-1970, when aminoglycoside antibiotics, such as neomycin and kanamycin, were routinely administered at high doses. An estimation of ototoxicity caused hearing loss ranges from 2% to 25% of hearing deficits and 1-10% of vestibular deficits [START_REF] O'sullivan | Towards the prevention of aminoglycoside-related hearing loss[END_REF]. Aminoglycosides form a large group of cationic charged small molecules with three-or four-ringed compound displaying specific potent activity against gram-negative bacteria. It has been reported that the mitochondrial 12S ribosomal RNA (MT-RNA1) has a high susceptibility to aminoglycoside, especially for four variant: A1555G, C1494T, T1095C, 961delT+C(n). These variants are more similar to the bacterial 12SrRNA, and may be targeted by aminoglycosides [START_REF] Ealy | The prevalence of mitochondrial mutations associated with aminoglycoside-induced sensorineural hearing loss in an NICU population[END_REF]. Nowadays, the hearing loss caused by aminoglycoside declined especially in developed centuries [START_REF] Rybak | Ototoxicity[END_REF]. Another example concerns the maternal women infected by "TORCH" organisms, which are Toxoplasmosis, Rubella, Cytomegalovirus, and Herpes. These are well-known causes of newborn's hearing loss [START_REF] Smith | Sensorineural hearing loss in children[END_REF]. Some other physical and physiological diseases can cause hearing loss, such as head trauma, tumours, etc, but I will not address them all here.

Genetic Hearing Loss 2.2.1. Syndromic hearing loss

Syndromic SNHL accounts for around 20% of genetic hearing loss; it not only affects the auditory system, but also other tissues, like eye, kidney, musculoskeletal or nervous systems. There are more than 700 different syndromes that include hearing loss, but syndromic hearing loss is still not as common as non-syndromic hearing loss. Among the 700 syndromes, the three most common syndromes are Pendred, Usher and Waardenburg syndromes. Pendred syndrome is a type of autosomal recessive syndromic hearing loss (ARSHL), which is the most prevalent syndrome affecting 7.5 per 100,000 people and accounts for up to 8% of the congenital hearing loss. Usher syndrome is also a type of ARSHL, with 1/6,000 to 1/10,000 cases in the overall population; more details about the Usher syndrome will be discussed below. The Waardenburg syndrome is an autosomal dominant syndromic hearing loss (ADSHL), which affects 1 per 42,000 individuals.

Non-syndromic hearing loss

Non-syndromic hearing loss accounts for 80% of genetic cases of hearing loss, where autosomal recessive counts for 80%, autosomal dominant counts for 19% and the rest 1% is X-linked, miRNA or mitochondrial linked.

Autosomal dominant (DFNA)

According to the homepage of Hereditary Hearing Loss (hereditaryhearingloss.org), up to date (17/Jan/2020), 47 genes are associated with autosomal dominant non-syndromic hearing loss (ADNSHL). Several forms are characterized by a loss of frequency detection. For instance, the Wolfram syndrome 1 gene (WFS1) (DFNA6) causes a characteristic low frequency (< 2kHz) hearing loss [START_REF] Cryns | Mutations in the WFS1 gene that cause low-frequency sensorineural hearing loss are small non-inactivating mutations[END_REF], while the KCNQ4 gene mutation (DFNA2A) causes a high frequency hearing loss [START_REF] Kubisch | KCNQ4, a novel potassium channel expressed in sensory outer hair cells, is mutated in dominant deafness[END_REF], [START_REF] Kamada | A novel KCNQ4 one-base deletion in a large pedigree with hearing loss: Implication for the genotype-phenotype correlation[END_REF].

Autosomal recessive (DFNB)

Autosomal recessive non-syndromic hearing loss (ARNSHL) is the most common form of genetic hearing loss (80% of cases). Up to now, 76 genes have been reported to cause ARNSHL (hereditaryhearingloss.org). In contrast to autosomal dominant with postlingual onset, the ARNSHL is most often reported with prelingual onset. The most common form of ARNSHL, which is caused by mutation in GJB2 gene, leads up to 50% of total ARNSHL cases. GJB2 is the first ARNSHL to be reported in 1994 [START_REF] Guilford | neurosensory , recessive deafness region of chromosome 13q[END_REF]. GJB2 gene encodes for the protein connexin 26, which forms the channel at the gap junctions; it is vital for the ion recycling in the inner ear maintaining the cochlear homeostasis [START_REF] Mammano | Inner ear connexin channels: Roles in development and maintenance of cochlear function[END_REF]. The protein Whirlin, the key protein in my PhD project, causes one of the ARNSHL form called DFNB31, involving mutations located in the C-terminal region of the protein [START_REF] Mustapha | DFNB31, a recessive form of sensorineural hearing loss, maps to chromosome 9q32-34[END_REF] [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]. Some other mutations of essential proteins expressed in the stereocilia are also related to the ARNSHL, like myosin 7a (DFNB2), myosin 15a (DFNB3), TMC1 (Transmembrane Channel-like Protein 1)(DFNB7/11), Cadherin 23 (DFNB12), Harmonin (DFNB18), Protocadherin 15 (DFNB23), Taperin (DFNB79), etc.

X-linked (DFNX)

Up to now, 5 genes have been found related to X-linked non-syndromic hearing loss; PRPS1 (DFNX1), POU3F4 (DFNX2), SMPX (DFNX4), AIFM1 (DFNX5) and COL4A6 (DFNX6). SMPX (DFNX4) is a postlingual onset gene, and all others are prelingual genes.

Mitochondrial linked

Mitochondrion is the power plant in the cell, which is also important in cell growth and cell death [START_REF] Mcbride | Mitochondria: More Than Just a Powerhouse[END_REF]. Mitochondria have their own mitochondrial DNA (mtDNA), with potential mutations that lead to different types of diseases. Some of them can affect the auditory system, causing hearing loss. For example, the single point mutation 3243 A-to-G in the MTTL1 gene has been found related to diabetes mellitus. Among 61% of patients sharing this mutation will develop hearing loss only after onset of diabetes mellitus [START_REF] Shearer | Hereditary Hearing Loss and Deafness Overview[END_REF].

Usher syndrome

The scottish ophthalmologist Charles Usher, firstly described Usher syndrome, which is one of the most common genetic neurosensory disorder and affects hearing, vision and balancing systems (Fig. 15). Usher (USH) syndrome has been further divided into three types depending on the clinical symptoms. Ten Usher genes have been identified so far, where 5 genes are involved in USH type 1 (USH1), 3 genes in USH type 2 (USH2) and 1 gene in USH type 3 (USH3) (source; hereditaryhearingloss.org). However, some USH genes are debatable. CIB2, for example, has been considered as a USH1 gene (USH1J) along with the other 5 USH genes [START_REF] Riazuddin | Alterations of the CIB2 calcium-and integrin-binding protein cause Usher syndrome type 1J and nonsyndromic deafness DFNB48[END_REF]. However, recent researchs showed that CIB2 is an autosomal recessive non-syndromic hearing loss gene (DFNB48) [START_REF] Booth | Variants in CIB2 cause DFNB48 and not USH1J[END_REF]. PDZD7 (PDZ domain-containing 7) has been suggested to be an USH2 protein [START_REF] Ebermann | PDZD7 is a modifier of retinal disease and a contributor to digenic Usher syndrome[END_REF], but some mutations in PDZD7 cause the DFNB57 instead of the Usher syndrome [START_REF] Booth | PDZD7 and hearing loss: More than just a modifier[END_REF]. The USH3 protein HARS (Histidyl-tRNA synthetase) has been shown causing Usher syndrome due to its over activity. HARS is also not been counted as an USH3 gene by some teams [START_REF] Mathur | Usher syndrome: Hearing loss, retinal degeneration and associated abnormalities[END_REF] [START_REF] Mathur | Usher syndrome and non-syndromic deafness : Functions of different whirlin isoforms in the cochlea , vestibular organs , and retina[END_REF]. The three different types of Usher syndrome are closely related to the respective proteins locations in the auditory hair cells. During early stage (P2-P12) of hair cell development, there are both ankle links at the root region of stereocilia and tip links on the top of second and third rows regions of stereocilia. However, in the mature hair cells (P12+), the ankle links disappear and only tip-links remain [START_REF] Goodyear | Development and properties of stereociliary link types in hair cells of the mouse cochlea[END_REF]. The five USH1 proteins are mostly found associated with the developement of the tip links anchoring cytoplasmic densities. The three USH2 proteins are located at the ankle links region, except Whirlin, which has been also found expressed at the tip of the tallest row of stereocilia. Finally, the USH3 protein Clarin1 (USH3A) is expressed at the synapse region [START_REF] Joensuu | Mutations in a novel gene with transmembrane domains underlie usher syndrome type 3[END_REF] [START_REF] Adato | USH3A transcripts encode clarin-1, a four-transmembrane-domain protein with a possible role in sensory synapses[END_REF].

-Myosin 7a (USH1B) Myosin 7a is an unconventional actin-based motor of 2215 amino acids long (254 kDa) encompassing a motor domain at its N-terminus followed by five IQ (Ile, Gln Calmodulinbinding) motifs, one SAH (Single α helix), and two MyTH4-FERM tandems with a SH3 in between. These unconventional myosins form a type of myosins with extra diverse functions besides the actin-based motor, like intracellular trafficking and mechanical support [START_REF] Li | Structural Basis of Cargo Recognition by Unconventional Myosins in Cellular Trafficking[END_REF]. Myosin 7a has been identified to cause USH1B in 1995 as the first confirmed Usher gene [START_REF] Gibson | A type VII myosin encoded by the mouse deafness gene shaker-1[END_REF][102] [START_REF] Hasson | Expression in cochlea and retina of myosin VIIa, the gene product defective in usher syndrome type 1B[END_REF]. It is also the most common form of USH1 accounting for more than 40% of all cases. The N-terminal motor binds to the actin-filaments in the stereocilia, while the following five IQ motifs and SAH are Calcium modulated modules, which stiffness changes upon Ca 2+ binding [START_REF] Li | Ca2+-Induced Rigidity Change of the Myosin VIIa IQ Motif-Single α Helix Lever Arm Extension[END_REF]. The MyTH4 and FERM domains form supramodules; the crystal structures of complexes between MyTH4-FERM1 and MyTH4-FERM2 tandems interacting with binding partners such as SANS and Harmonin, respectively, have been solved [START_REF] Wu | Structure of myTH4-FERM domains in myosin VIIa tail bound to cargo[END_REF] [START_REF] Yu | Myosin 7 and its adaptors link cadherins to actin[END_REF]. It is also reported that the absence of Myosin 7a in mouse leads to elongated, and splay-organized morphology of stereocilia, dramatically affecting the distribution of USH2 protein Whirlin and the DFNB protein PDZD7 at the ankle link region [START_REF] Zou | The roles of USH1 proteins and PDZ domain-containing USH proteins in USH2 complex integrity in cochlear hair cells[END_REF]. Mutations of Myosin 7a not only cause USH1B, but also may result in autosomal non-syndromic hearing loss DFNA11 [START_REF] Sun | Novel missense mutations in MYO7A underlying postlingual high-or lowfrequency non-syndromic hearing impairment in two large families from China[END_REF] and DFNB2 [START_REF] Liu | Mutations in the myosin VIIA gene cause non-syndromic recessive deafness[END_REF] [START_REF] Riazuddin | Mutation spectrum of MYO7A and evaluation of a novel nonsyndromic deafness DFNB2 allele with residual function[END_REF].

-Harmonin (USH1C) Harmonin is the second identified Usher syndrome type 1 gene after Myosin 7a, in 2000 [START_REF] Verpy | A defect in harmonin, a PDZ domain-containing protein expressed in the inner ear sensory hair cells, underlies Usher syndrome type 1C[END_REF]. Eight isoforms of Harmonin can be classified in three classes: a, b and c. In retina photoreceptors, only isoforms from classes a and c are detected. The isoform b is the longest sequence, which consists of a Harmonin Homology Domain (HHD) at the N-terminus followed by two PDZ domains, two coiled-coil (CC) regions, a Proline/Serine/Threonine rich region (PST) and the PDZ3 at the C-terminus. The isoform a1 is the shorter isoform, which lacks one CC and PST regions but has a PBM at the C-terminus. The isoform c is the shortest class, which only has a HHD, the PDZ1-PDZ2 tandem and a CC [START_REF] Verpy | A defect in harmonin, a PDZ domain-containing protein expressed in the inner ear sensory hair cells, underlies Usher syndrome type 1C[END_REF]. The N-terminal HHD domain is a small helical domain conserved among different species, i.e. a five helix bundle, that was first described in Harmonin, and later found also in USH2D protein Whirlin, and another PDZ-containing protein PDZD7 (DFNB57) in hair cells [START_REF] Pan | Assembling stable hair cell tip link complex via multidentate interactions between harmonin and cadherin 23[END_REF] [START_REF] Faure | The C-terminal extension of human RTEL1, mutated in Hoyeraal-Hreidarsson syndrome, contains Harmonin-N-like domains[END_REF]. Harmonin is the only multi-PDZ scaffolding protein in the USH1 protein systems, which has been reported to interact with all other USH1 proteins except Protocadherin 15, either in hair cells in cochlea or photoreceptor cells in the retina through PDZ-PBM recognitions and other PPI interactions [START_REF] Reiners | Photoreceptor expression of the Usher syndrome type 1 protein protocadherin 15 (USH1F) and its interaction with the scaffold protein harmonin (USH1C)[END_REF][115] [START_REF] Yu | Myosin 7 and its adaptors link cadherins to actin[END_REF]. Harmonin mutations can also lead to autosomal recessive hearing loss (DFNB18A) [START_REF] Ouyang | Mutations in the alternatively spliced exons of USH1C cause non-syndromic recessive deafness[END_REF] [START_REF] Ahmed | Nonsyndromic recessive deafness DFNB18 and usher syndrome type IC are allelic mutations of USHIC[END_REF].

-Cadherin 23 (USH1D) USH1D is the second most prevalent form of USH1 after USH1B. The encoded protein Cadherin 23 is one of the two components forming the tip link [START_REF] Kazmierczak | Cadherin 23 and protocadherin 15 interact to form tip-link filaments in sensory hair cells[END_REF]. It belongs to a large protein family, which is responsible for cell-cell junctions and extracellular linkers formations. Cadherin 23 contains 27 extracellular cadherin (EC) repeats and forms cis parallel dimer in the tip links [START_REF] Kazmierczak | Cadherin 23 and protocadherin 15 interact to form tip-link filaments in sensory hair cells[END_REF], linked to a similar cis dimer structure of Protocadherin 15, the second component of these links. The EC domain is the main structural feature of cadherin, which is a relatively small seven-strand β-barrel consisting of approximately 110 residues. EC domains are connected by three Calcium ions bound to highly conserved negatively charged residues located between two EC repeats. Cadherin 23 is anchored to the membrane through a single α helix followed by a C-terminal cytoplasmic domain [119][120]. This cytoplasmic region of Cadherin 23 encompasses two protein-protein interaction motifs for the Harmonin recognition; an internal sequence (3181-3200) to interact with the HHD domain of Harmonin and the very C-terminal PBM with the second PDZ domain of Harmonin. These interactions are thought to anchor Cadherin 23 at the upper-tip-link density [START_REF] Pan | Assembling stable hair cell tip link complex via multidentate interactions between harmonin and cadherin 23[END_REF] [START_REF] Siemens | The Usher syndrome proteins cadherin 23 and harmonin form a complex by means of PDZdomain interactions[END_REF]. Mutations of Cadherin 23 may also result in DFNB12 [START_REF] Wagatsuma | Distribution and frequencies of CDH23 mutations in Japanese patients with non-syndromic hearing loss[END_REF], and pituitary adenomas [START_REF] Zhang | Germline Mutations in CDH23, Encoding Cadherin-Related 23, Are Associated with Both Familial and Sporadic Pituitary Adenomas[END_REF].

-Protocadherin 15 (USH1F) Protocadherin is the largest subfamily in the cadherin family with a similar structural features, also forming a cis parallel dimer in the tip links [START_REF] Kazmierczak | Cadherin 23 and protocadherin 15 interact to form tip-link filaments in sensory hair cells[END_REF]. The Protocadherin 15 consists of 11 EC repeats, a single-pass transmembrane α helix and a C-terminal cytoplasmic domain sharing a terminal PBM. In mouse, 26 isoforms of Protocadherin 15 have been reported according to the Uniprot databank (Q99PJ1), belonging to three main classes of all the isoforms that only differ by their cytoplasmic sequences. The three classes are CD1 (exon 35), CD2 (exon 38) and CD3 (exon 39), while the CD2 is the only remaining class in the mature auditory hair cells [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]. Between the N-terminus linked to Cadherin 23 and the C-terminus located in the cytoplasm, the C-terminal transmembrane helix of Protocadherin 15 interacts with the LHFPL5 (lipoma high mobility group IC fusion partner-like 5) dimer as monitored by cryo-EM [START_REF] Ge | Structure of mouse protocadherin 15 of the stereocilia tip link in complex with LHFPL5[END_REF], while the C-terminal region of the isoform class CD2 interacts also with LHFPL5, TMC and TMIE (transmembrane inner ear protein) proteins [START_REF] Xiong | TMHS is an integral component of the mechanotransduction machinery of cochlear hair cells[END_REF] [START_REF] Zhao | TMIE is an essential component of the mechanotransduction machinery of cochlear hair cells[END_REF]. Mutations of Protocadherin 15 are also responsible for DFNB23 [START_REF] Ahmed | PCDH15 is expressed in the neurosensory epithelium of the eye and ear and mutant alleles are responsible for both USH1F and DFNB23[END_REF].

-SANS (USH1G) SANS (Scaffolds Protein Containing Ankyrin Repeats and SAM Domain) is a relatively small scaffolding protein in the USH protein family with 460 amino acids, which encodes for 4 ankyrin repeats at its N-terminus, followed by a central region, a SAM (sterile alpha motif) domain and a PBM at the C-terminus. SAM is a small globular domain formed by a 5-helix bundle, a well-known PPI domain that has no specific binding pattern like PDZ. SAM can interact with various different proteins, RNA, and homo-/hetero SAM-SAM interactions have also been reported [START_REF] Kim | SAM domains: Uniform structure, diversity of function[END_REF][130] [START_REF] Wang | Specific Eph receptor-cytoplasmic effector signaling mediated by SAM-SAM domain interactions[END_REF]. In the auditory hair cells, SANS has been shown to interact with Harmonin and Myosin 7a and also to self-associate to form a large protein complex at the upper-tip-link densities. Detailed complex formation will be discussed in the next section. SANS is able to interact with all three USH2 proteins in vitro, but the knockout SANS mouse showed not effect on the distribution of USH2 proteins in hair cells [START_REF] Zou | The roles of USH1 proteins and PDZ domain-containing USH proteins in USH2 complex integrity in cochlear hair cells[END_REF]. As an USH protein, SANS is also expressed in photoreceptor cells, where it interacts with USH2 proteins Whirlin and Usherin forming a ternary protein complex [START_REF] Sorusch | Characterization of the ternary Usher syndrome SANS/ush2a/whirlin protein complex[END_REF].

-Usherin (USH2A) USH2A is the most common form of USH2 syndrome, which was the second identified USH gene in 1998, after USH1B gene encoding for Myosin 7a (1995) [START_REF] Eudy | Mutation of a gene encoding a protein with extracellular matrix motifs in Usher syndrome type IIa[END_REF]. Usherin is a long extracellular linker protein, anchored to the membrane of the stereocilia. It is potentially one of the two proteins forming ankle links. Usherin has a signal peptide at the N-terminus, an extracellular region of around 5000 residues, an unique transmembrane segment and around 140 residues for the C-terminal cytoplasmic region [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]. As shown by GST pull down and co-immuno-precipitation (co-IP) experiments, this cytoplasmic region interacts with the Cterminal MyTH4-FERM tandem of Myosin 7a [START_REF] Michalski | Molecular Characterization of the Ankle-Link Complex in Cochlear Hair Cells and Its Role in the Hair Bundle Functioning[END_REF], with Harmonin [START_REF] Reiners | Scaffold protein harmonin (USH1C) provides molecular links between Usher syndrome type 1 and type 2[END_REF], Whirlin (USH2D) [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], [START_REF] Van Wijk | The DFNB31 gene product whirlin connects to the Usher protein network in the cochlea and retina by direct association with USH2A and VLGR1[END_REF] and PDZD7 [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]; the interactions with Harmonin, Whirlin and PDZD7 involve the C-terminal PBM of Usherin and the PDZ1 of Harmonin [START_REF] Reiners | Scaffold protein harmonin (USH1C) provides molecular links between Usher syndrome type 1 and type 2[END_REF], the tandem PDZ1-PDZ2 of Whirlin [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] and the PDZ2 of PDZD7 [START_REF] Ebermann | PDZD7 is a modifier of retinal disease and a contributor to digenic Usher syndrome[END_REF] [139] [START_REF] Booth | PDZD7 and hearing loss: More than just a modifier[END_REF].

-ADGRV1/VLGR1/GPR98/MASS1 (USH2C) The USH3C gene encoded for the protein ADGRV1 (Adhesion G Protein-Coupled Receptor V1) also named VLGR1 (Very Large G-protein Receptor 1), GPR98 (G protein-coupled receptor 98) or MASS1 (monogenic audiogenic seizure susceptible 1). It belongs to the adhesion GPCR family expressing an extracellular region that contains functional domains to facilitate cell-cell or cell-membrane interactions. The extracellular region of ADGRV1 is exceptionally long with over 5000 residues, which contains, after a signal peptide sequence at the N-terminus, several calcium-binding Calx-B repeats, an EAR (epilepsy-associated repeat) motif and one pentraxin domain [START_REF] Mcmillan | Loss of the transmembrane and cytoplasmic domains of the very large G-protein-coupled receptor-1 ( VLGR1 or Mass1 ) causes audiogenic seizures in mice[END_REF]. Seven-pass transmembrane GPCR domain and 150 residues into the cytoplasm constitute the C-terminal region of ADGRV1. ADGRV1 has the same interaction partners than the ones of Usherin in the stereocilia: Myosin 7a, Harmonin, Whirlin and PDZD7 as monitored by in vitro experiments [START_REF] Reiners | Scaffold protein harmonin (USH1C) provides molecular links between Usher syndrome type 1 and type 2[END_REF] [134] [138][91][139] [START_REF] Booth | PDZD7 and hearing loss: More than just a modifier[END_REF]. Besides the scaffolding function, ADGRV1 is a GPCR that also serves as a signalling transmitter. Like other adhesion class GPCR members, ADGRV1 has a GAIN (GPCR auto-proteolysis-inducing) domain in its ectodomain closed to the C-terminal transmembrane domain, which undergoes an auto-proteolysis process, the ectodomain breaking into two parts. Then, the new N-terminal region sequence, which is a tethered ligand (or Stachel peptide), has the ability to constitutively activate the GPCR, and conducts the downstream signalling pathways. However, the downstream pathways are not fully clear. It has been found that the GPCR can activate PKA and PKC through Gαs and Gαq in response to the extracellular Ca 2+ [START_REF] Shin | Very large G protein-coupled receptor 1 regulates myelin-associated glycoprotein via Gαs/Gαq-mediated protein kinases A/C[END_REF]. By contrast, another research group found, when co-expressed with PDZD7 (DFNB57), that ADGRV1 is coupled with Gαi and decreased the forskolininduced production of cAMP [START_REF] Hu | Constitutive Gαi coupling activity of very large G protein-coupled receptor 1 (VLGR1) and its regulation by PDZD7 protein[END_REF].

-Whirlin (USH2D) Before starting the story of Whirlin, a short background about the whirler mouse is needed. In 1963, a recessive mutation in mice was described with circling behaviour and head-bobbing and -tossing, which was named whirler mouse [START_REF] Lane | Whirler mice: A recessive behavior mutation in linkage group viii[END_REF]. In 1990s, whirler mouse was gene mapped and located at the mouse chromosome 4 and human chromosome 9q32-q34 [START_REF] Pilz | Comparative mapping of mouse Chromosome 4 and human Chromosome 9: Lv, Orm, and Hxb are closely linked on mouse Chromosome 4[END_REF], [START_REF] Fleming | Linkage Analysis of the whirler Deafness Gene on Mouse Chromosome 4[END_REF]. These research teams also found the phenotypes of deafness and vestibular dysfunction in adult mouse. Later, researchers focused on the relation between whirler mouse and its dysmorphology of hair cell stereocilia [START_REF] Holme | Elongation of hair cell stereocilia is defective in the mouse mutant whirler[END_REF], and the team of P r Christine Petit identified DFNB31 gene at the locus of human chromosome 9q32-q34, which is exactly the whirler mouse mutation place [START_REF] Mustapha | DFNB31, a recessive form of sensorineural hearing loss, maps to chromosome 9q32-34[END_REF]. Before 2003, details about the protein Whirlin were still unknown. It has been named KIAA1526 after a first brain cDNA mapping [START_REF] Nagase | Prediction of the coding sequences of unidentified human genes. XX. The complete sequences of 100 new cDNA clones from brain which code for large proteins in vitro[END_REF] and then called CIP98 (CASK interacting protein 98kDa), knowing that this protein co-localized with CASK in brain nerve system [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 the same year, the team of C. Petit characterized the protein causing DFNB31 from whirler mouse, and called it Whirlin for the first time [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]. Finally, USH2D has been described in 2007 and linked to Whirlin [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]. Whirlin is a multi-tasking, multi-PDZ scaffolding protein, which is distributed at different locations in the stereocilia and has different functions in both immature and mature stereocilia. Whirlin has mainly three isoforms, which differently express in retina photoreceptor cells and auditory hair cells: the FL (full-length), N (N-terminal) and C (Cterminal) isoforms. In mouse, the most common FL isoform is 907 amino acids long (same as human Whirlin), containing 5 structured domains and a long Proline rich region (180 amino acids). From its very N-terminus, FL isoform starts with an unfolded sequence of 30 residues, then an HHD1 domain followed by a PDZ1-PDZ2 tandem, another HHD2 domain, which is 35-residue away from the PDZ2, a long unstructured region (310 residues) where lays the proline-rich region, a PDZ3 domain at the very C-terminus and ends up with a PBM (Fig. 17). Both N and C isoforms are shortened versions of FL isoform, where the N isoform lacks the proline-rich region, PDZ3 and PBM, while C isoform lacks HHD1 and PDZ1-PDZ2 tandems. HHD2 is the only common feature in both short isoforms. The N isoform expresses exclusively in photoreceptor cells, and C isoform expresses exclusively in the auditory hair cells, while FL isoform expresses in both sensory cells. More detailed interactions involving Whirlin will be discussed in the section 2.4. and PDZ3 domains. Contrary to Harmonin and Whirlin, PDZD7 does not exhibit a PBM sequence at its C-terminus. PDZD7 is essential to maintain the distribution of ADGRV1, Usherin and Whirlin in the auditory hair cells [START_REF] Zou | Deletion of PDZD7 disrupts the Usher syndrome type 2 protein complex in cochlear hair cells and causes hearing loss in mice[END_REF]. In PDZD7 knockout mice, ADGRV1 moved to the tip of stereocilia in both IHCs and OHCs, and Usherin either moved to the tip of stereocilia or diffused at the base of hair bundle in OHCs and mainly disappeared in IHCs. The localization of Whirlin at stereocilia base is also lost [START_REF] Zou | Deletion of PDZD7 disrupts the Usher syndrome type 2 protein complex in cochlear hair cells and causes hearing loss in mice[END_REF]. Furthermore, PDZD7 has also been reported to autoassociate via its PDZ2 domain [START_REF] Booth | PDZD7 and hearing loss: More than just a modifier[END_REF].

Usher syndrome I (USH1) and tip links

USH1 is the most severe form of Usher syndrome; USH1 patients have congenital severe-toprofound deafness, vestibular areflexia and develop Retinitis pigmentosa (RP) in the first decade of life. RP results in the degeneration of rod and cone photoreceptors, leading to a decreased night vision, a loss of peripheral vision in early state that progressively becomes severe towards complete blindness. The five USH1 proteins are expressed and located at the tip links anchoring points. The tip link itself is formed by the handshake interaction of Cadherin 23 and Protocadherin 15 between both the N-terminal first two EC repeats as an overlapping and antiparallel (trans) hetero-tetramer (Fig. 18). Cadherin 23 has 27 EC repeats, and Protocadherin 15 has 11, and each canonical EC repeat is 4.5 nm long. So, the Cadherin 23 extracellular region is roughly 120 nm long, while Protocadherin 15 is 50 nm long; so in total the full length linker is around 170 nm long [START_REF] Sotomayor | Structure of a forceconveying cadherin bond essential for inner-ear mechanotransduction[END_REF]. The two anchoring points gather cytoplasmic proteins (Fig. 19), which form a large protein density, which is supposed to strengthen the anchoring point and to diffuse the tension applied to the membrane. On the side of Cadherin 23 C-terminal cytoplasmic region, three USH1 proteins (myosin 7a, Harmonin and SANS) form large protein condensate via liquid-liquid phase separation in vitro [START_REF] Yunyun He | Myosin VII, USH1C, and ANKS4B or USH1G Together Form Condensed Molecular Assembly via Liquid-Liquid Phase Separation[END_REF]. In the upper-tip link density, where the Cadherin 23 is anchoring at, three USH1 proteins are localized: Myosin 7a, SANS, and Harmonin. These three proteins form interactions with each other by distinct domain from other interactions. The N-terminal HHD and PDZ1 tandem of Harmonin binds strongly to SAM-PBM at the C-terminus of SANS, and the C-terminal PDZ3-PBM region of Harmonin interacts with the second MyTH4-FERM tandem of Myosin 7a [START_REF] Yu | Myosin 7 and its adaptors link cadherins to actin[END_REF] [START_REF] Yan | The structure of the harmonin/sans complex reveals an unexpected interaction mode of the two Usher syndrome proteins[END_REF]. The interaction between SANS and Myosin 7a requires the central region of SANS and the first MyTH4-FERM tandem of Myosin 7a [START_REF] Wu | Structure of myTH4-FERM domains in myosin VIIa tail bound to cargo[END_REF]. There is no overlapping interaction site, and all three USH1 self-associate to form oligomers [START_REF] Yu | Myosin 7 and its adaptors link cadherins to actin[END_REF][155] [START_REF] Bahloul | Conformational switch of harmonin, a submembrane scaffold protein of the hair cell mechanoelectrical transduction machinery[END_REF] [START_REF] Sakai | Cargo binding activates myosin VIIA motor function in cells[END_REF]. Thus, this scaffolding architecture is not necessarily formed with only supermolecule heterotrimers, but rather like a large protein-protein crosslinking matrix [START_REF] Yunyun He | Myosin VII, USH1C, and ANKS4B or USH1G Together Form Condensed Molecular Assembly via Liquid-Liquid Phase Separation[END_REF]. As mentioned before, Harmonin has two binding sites to interact with the cytoplasmic tail of Cadherin 23; the HHD domain of Harmonin binds to an internal sequence of Cadherin 23 in its C-terminal region and the PDZ2 of Harmonin recognizes, via a canonical PDZ-PBM interaction, the Cadherin 23 C-terminal PBM [START_REF] Pan | Assembling stable hair cell tip link complex via multidentate interactions between harmonin and cadherin 23[END_REF]. On the other side of the anchoring point of Protocadherin 15, the transmembrane helical dimer tightly bundled with the LHFPL5 transmembrane protein dimer [START_REF] Ge | Structure of mouse protocadherin 15 of the stereocilia tip link in complex with LHFPL5[END_REF]. The membrane Figure 23: The distribution of whirlin isoforms in wildtype mouse auditory hair cells, vestibular cells and photoreceptor cells at different stages of development. A: the distribution of Whirlin isoforms in the IHC and OHC of auditory hair cells in immature hair cells. B: the distribution of Whirlin isoforms in vestibular hair cells C: the distribution of Whirlin isoforms in the mature hair cells. D: the distribution of Whirlin isoforms in photoreceptor cells. PMC: periciliary membrane complex. Adapted from [START_REF] Mathur | Usher syndrome and non-syndromic deafness : Functions of different whirlin isoforms in the cochlea , vestibular organs , and retina[END_REF]

mutations and diseases

The mutations located in the region upstream HHD2 cause USH2 syndrome and those downstream C-terminal region are responsible for DFNB31 (Fig. 24). Mutation types found in Whirlin are stop codon mutations and frame shift inducing early stop codon. Up to date, there is no point mutation identified [START_REF] Mathur | Usher syndrome and non-syndromic deafness : Functions of different whirlin isoforms in the cochlea , vestibular organs , and retina[END_REF]. In the auditory hair cells, the whirlin immuno-fluorescence signal can be detected at the ankle links and tip of the tallest row of stereocilia, where the signal is much higher [START_REF] Ebrahim | Alternative Splice Forms Influence Functions of Whirlin in Mechanosensory Hair Cell Stereocilia[END_REF]. Several proteins co-localize at the tip of stereocilia; -Whirlin: Whirlin displays self-association. It has been shown by yeast two-hybrid test and pull down assay, that the three PDZ domains are able to form self-association. FL-isoform and C-isoform are also able to interact with themselves [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] [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]. But, our group previously confirmed that both PDZ1 and PDZ2 are mainly monomeric by measuring NMR correlation time (τc) [START_REF] Delhommel | Structural Characterization of Whirlin Reveals an Unexpected and Dynamic Supramodule Conformation of Its PDZ Tandem[END_REF]. I will give a detailed domain-by-domain test of auto-association in the results chapter.

-Myosin 15a: The unconventional actin-based motor myosin 15a is found located in the tip of the three rows of stereocilia. The differential elongation of the stereocilia and tip location of Whirlin requires the complex formation of myosin 15a and Whirlin. Their direct interaction has been shown and confirmed as a canonical PDZ-PBM interaction between the PDZ3 of Whirlin and the PBM of Myosin 15a [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]. However, the details of this binding are still unknown; I will present the structure of Whirlin PDZ3 complexed to the PBM of Myosin 15a in my result chapter.

-EPS8: the actin filaments barbed-end capping protein EPS8 (epidermal growth factor receptor pathway substrate 8) is localized at the tip of the tallest row of stereocilia. It is important to generate the actin filaments propulsion force at the tip of stereocilia [START_REF] Disanza | Eps8 controls actin-based motility by capping the barbed ends of actin filaments[END_REF]. The Myosin 15a, EPS8 and Whirlin has been proposed to interact with each others at the tip of the stereocilia [START_REF] Manor | Regulation of stereocilia length by myosin XVa and whirlin depends on the actin-regulatory protein Eps8[END_REF] [START_REF] Ebrahim | Alternative Splice Forms Influence Functions of Whirlin in Mechanosensory Hair Cell Stereocilia[END_REF]. The regions involve in the interaction between Whirlin and EPS8 are not identified; I will show the interaction tests in my results chapter.

-CASK: The MAGUK (membrane-associated guanylate kinase) family protein CASK (Calcium-calmodulin serine kinase) localizes at the tip of stereocilia. Whirlin has another name, CIP98, which was the one of the early name of Whirlin meaning CASK Interacting protein 98kDa, to be related to the interaction with CASK in the brain tissue and involving the PDZ3 region of Whirlin [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]. CASK contains an internal PBM [START_REF] Mburu | Whirlin complexes with p55 at the stereocilia tip during hair cell development[END_REF]. Interactions between internal PBM and PDZ are rarely identified, and the detail is unknown. The details of the interaction between CASK PBM and Whirlin will be discussed in results part.

-MPP1: MPP1 (membrane palmitoylated protein 1), another MAGUK family protein, has a similar distribution and interaction pattern with Whirlin as the ones of CASK in auditory hair cells stereocilia [START_REF] Mburu | Whirlin complexes with p55 at the stereocilia tip during hair cell development[END_REF]. The last 10 residues of its C-terminus are identical to the sequence of CASK, which has also an internal PBM. The PDZ-PBM binding model has been predicted with the Whirlin PDZ3 domain [START_REF] Gosens | MPP1 links the Usher protein network and the Crumbs protein complex in the retina[END_REF], suggesting that the interaction between Whirlin PDZ3 and CASK might be similar.

-P4.1: Protein 4.1 is a multi functional scaffolding protein essential for the cytoskeleton shape and membrane mechanical properties. Four isoforms of P4.1 have been identified: 4.1B, 4.1G, 4.1R and 4.1N [START_REF] Baines | The Protein 4.1 family: Hub proteins in animals for organizing membrane proteins[END_REF]. 4.1N and 4.1R interact with CASK and MPP1, respectively [START_REF] Biederer | CASK and Protein 4.1 Support F-actin Nucleation on Neurexins[END_REF][184] [START_REF] Diakowski | Protein 4.1, a component of the erythrocyte membrane skeleton and its related homologue proteins forming the protein 4.1/FERM superfamily[END_REF], and mutations of Whirlin or Myosin 15a affect the distribution of 4.1R in the stereocilia [START_REF] Mburu | Whirlin complexes with p55 at the stereocilia tip during hair cell development[END_REF]. The two other isoforms 4.1B and 4.1G are also reported to be expressed in the stereocilia, and 4.1B is suggested to be a member of the Myosin 15a-Whirlin complex [START_REF] Okumura | Protein 4.1 expression in the developing hair cells of the mouse inner ear[END_REF].

-GPSM2: GPSM2 (G-protein signalling modulator 2) is a mitotic spindle-orienting protein [START_REF] Mochizuki | Identification and cDNA cloning of a novel human mosaic protein, LGN, based on interaction with G(αi2)[END_REF]. GPSM2 is essential for hearing [START_REF] Bhonker | The GPSM2 / LGN GoLoco motifs are essential for hearing[END_REF], is expressed at the tip of the tallest row of stereocilia [START_REF] Tadenev | GPSM2-GNAI Specifies the Tallest Stereocilia and Defines Hair Bundle Row Identity[END_REF], and is involved in the formation of Myosin 15a-Whirlin complex. The interaction between GPSM2 and Whirlin involves the GPSM2 N-terminal TPR region and the proline rich region of Whirlin [START_REF] Mauriac | Defective Gpsm2/Gαi3signalling disrupts stereocilia development and growth cone actin dynamics in Chudley-McCullough syndrome[END_REF].

Ankle links area

As mentioned in section 2.3.3, Whirlin co-localizes with PDZD7, Usherin, ADGRV1 and Myosin 7a. By high-throughput phage display screening, a new binding partner Taperin (DFNB79), which is a stereocilia rootlet protein [191][192] [START_REF] Pacentine | Stereocilia Rootlets : Actin-Based Structures That Are Essential for Structural Stability of the Hair Bundle[END_REF] has been shown to interact with PDZ3 of Whirlin (Susanne Lüchow & Ylva Yvarsson, Uppsala University, Sweden). Taperin also co-localizes with GRXCR2 at the base of stereocilia; in the model of GRXCR2 knockout mice, Taperin proteins accumulate at the tip of stereocilia [START_REF] Liu | GRXCR2 Regulates Taperin Localization Critical for Stereocilia Morphology and Hearing Article GRXCR2 Regulates Taperin Localization Critical for Stereocilia Morphology and Hearing[END_REF]. In my results chapter, I will present the high-resolution structure of the Whirlin PDZ3/Taperin complex.

Other Whirlin interactions in stereocilia.

There is no evidence to confirm the localization of Whirlin at the upper-tip link densities, which mainly contain the USH1 protein Harmonin, Cadherin 23, SANS and Myosin 7a. Usher proteins without co-localization observed in hair cells, can be found co-localized together like USH1 with USH2 protein in the photoreceptor cells [132][194][172]. As an example, we identified that the PBMs of Cadherin 23 and Harmonin a1 bind in vitro to the PDZ3 of Whirlin using our high-throughput Hold-up assay [START_REF] Vincentelli | Quantifying domain-ligand affinities and specificities by high-throughput holdup assay[END_REF]. Some other proteins reported to be able to interact with Whirlin in stereocilia are listed below.

-CIB2: CIB2 has been previously reported as a deafness protein (DFNB48) and Usher protein (USH1J) [START_REF] Riazuddin | Alterations of the CIB2 calcium-and integrin-binding protein cause Usher syndrome type 1J and nonsyndromic deafness DFNB48[END_REF]. However, the fact that mutations of CIB2 could cause the Usher syndrome has been questioned [START_REF] Booth | Variants in CIB2 cause DFNB48 and not USH1J[END_REF]. CIB2 has been found to interact with Whirlin and Myosin 7a by Co-IP [START_REF] Riazuddin | Alterations of the CIB2 calcium-and integrin-binding protein cause Usher syndrome type 1J and nonsyndromic deafness DFNB48[END_REF].

-Lrcc4c/NGL1: By using P2-P6 inner ear cDNA library generated from dissected vestibular sensory epithelia of 292 mice, Lrcc4c (Leucine-rich repeat-containing protein 4C) was shown to interact with Whirlin by yeast-two-hybrid system. The membrane protein Lrcc4c also interacts with PDZ1-PDZ2 of Whirlin via its cytoplasmic fragment [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].

-Espin: The actin binding and bundling protein espin is an important protein in maintaining the actin filaments structure in the stereocilia. It has been reported that actin-free espin is able to bind to Whirlin in yeast-two-hybrid screening [START_REF] Wang | Whirlin interacts with espin and modulates its actin-regulatory function: An insight into the mechanism of Usher syndrome type II[END_REF]. By Co-IP, espin could also interact with the N-terminal part of Whirlin (1-693) [START_REF] Wang | Identification of whirlin domains interacting with espin: A study of the mechanism of Usher syndrome type II[END_REF].

Whirlin outside inner ear

Whirlin is a deafness protein and an Usher syndrome protein, related to the auditory and vision systems. However, the in situ hybridization analysis shows that Whirlin is expressed in many other tissues, such as the heart, brain, spleen, lung, liver, skeletal muscle, kidney and testis [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].

-Retina Whirlin is distributed at the PMC and connecting cilium [START_REF] Yang | Ablation of whirlin long isoform disrupts the USH2 protein complex and causes vision and hearing loss[END_REF] [START_REF] Maerker | A novel Usher protein network at the periciliary reloading point between molecular transport machineries in vertebrate photoreceptor cells[END_REF]. In the PMC, Whirlin colocalizes with ADGRV1, Usherin, and PDZD7 [198][137]. Whirlin interacts with RPGR orf15 (Retinitis Pigmentosa GTPase Regulator) isoform and SANS at the connecting cilium, and also forms a ternary complex with SANS and Usherin [START_REF] Wright | Rpgr ORF15 connects to the usher protein network through direct interactions with multiple whirlin isoforms[END_REF] [START_REF] Sorusch | Characterization of the ternary Usher syndrome SANS/ush2a/whirlin protein complex[END_REF]. In vitro complex formation tests have been performed between SANS SAM-PBM region and Whirlin N-terminal HHD1-PDZ1 construct, as reported in my results chapter.

-Other tissues In the Central Nervous System, Whirlin has been detected in the thalamus and cortex neurons layer III-V, where Whirlin showed connections to CASK, as monitored by yeast two-hybrid system [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]. Both isoform 4.1B of protein 4.1 and Whirlin cooperatively maintain the integrity of Purkinje neurons myelinated axons in the cerebellum [START_REF] Saifetiarova | Ablation of cytoskeletal scaffolding proteins , Band 4 . 1B and Whirlin , leads to cerebellar purkinje axon pathology and motor dysfunction[END_REF]. Whirlin also facilitates the mechanosensory signalling in the skeleton muscle proprioceptive sensory neurons [START_REF] De Nooij | The PDZ-Domain Protein Whirlin Facilitates Mechanosensory Signaling in Mammalian Proprioceptors[END_REF].

Chapter 3: PDZ Domain

PDZ domain

In 1992, Cho and their colleagues [START_REF] Cho | The rat brain postsynaptic density fraction contains a homolog of the drosophila discs-large tumor suppressor protein[END_REF] discovered that PSD-95 (postsynaptic density-95), the Drosophila tumour suppressor protein DLG (discs large) and P55 (protein 55kDa/MPP1), have a fragment of approximately 90 amino acid in common, they called it 'GLGF repeat' due to the high conservation of a short sequence in between proteins. In 1995, to avoid scientific world to miss address the 'GLGF repeat' as DHR (DLG Homology Domain) [START_REF] Ponting | DHR domains in syntrophins, neuronal NO synthases and other intracellular proteins[END_REF], and find a better name to represent this domain, Kennedy and their colleagues [START_REF] Kennedy | Origin of PDZ (DHR, GLGF) domains[END_REF] named it from the three proteins PDS-95, DLG and the tight junction protein ZO-1 (zonula occludens 1) to become PDZ. PDZ is a protein-protein interaction module wildly found in all metazoans, with 266 PDZ domains identified in 152 proteins [206] [START_REF] Luck | The emerging contribution of sequence context to the specificity of protein interactions mediated by PDZ domains[END_REF] in human genome. Although PDZ domain is one of most common and studied domain for protein-protein interaction, it is not as abundant as others such as immunoglobin family domain, WD40 or SH3 [START_REF] Christian Stirnimann | WD40 proteins propel cellular networks[END_REF]. PDZ is well studied for its interaction with the final three residues of C-terminus of target proteins, called the PDZ Binding Motif. Most PDZ-containing proteins are involved in developing and maintaining cell-cell tight junctions [START_REF] Hurd | Direct interaction of two polarity complexes implicated in epthelial tight junction assembly[END_REF][210], signalling pathways [START_REF] Borg | ERBIN: A basolateral PDZ protein that interacts with the mammalian ERBB2/HER2 receptor[END_REF][212] and cell polarity [START_REF] Hildebrand | Shroom, a PDZ domain-containing actin-binding protein, is required for neural tube morphogenesis in mice[END_REF]. The neuron cells, for example, contain a large amount of various PDZ containing proteins, especially in the synapse region. PDZ domains appear in proteins in one copy or as multiple repeats [START_REF] Harris | Mechanism and role of PDZ domains in signaling complex assembly[END_REF]. PDZ containing proteins are mostly found in scaffolding proteins, which are famous for the 'beads-on-a-string' structures. PDZ domains can be associated to other globular domains; the MAGUK family (membrane-associated guanylate kinase) is an example for such protein, which contains a highly conserved triple-domain structure encompassing a PDZ, a SH3 (SRC Homology 3) and a GK (guanylate kinase) domain. PDZ domain can also be combined with other structured domains like LRR, L27, LIM, PTP, WW and so on [START_REF] Javier | Emerging Theme: Cellular PDZ Proteins as Common Targets of Pathogenic Viruses[END_REF] (Fig. 25).

PDZ-PBM interaction. B. All human 266 PDZ domains sequences alignment illustrated by a LOGO (http://weblogo.threeplusone.com/) representation, which showed highly conserved residues in 266 PDZ domains with 8 hydrophobic residues, one Asp and one Asn. C. Conserved residues location mapping on the model of PSD-95 PDZ3. C1. An overview of localization of 10 conserved residues coloured in orange and blue, and GLGF loop in red. C2, conserved Asp side chain on β4 lined with side chain of Arg on β1 strand. C3. Asn in the loop between α2 and β4 stabilizes the α2 and β5 by forming H-bonds to link both sides of the residue. C4. The highly conserved Ala at the beginning of α1 helix is important to stabilize GLGF loop by forming a H-bond with Leu. While all other residues coloured in orange contributed to form the hydrophobic core of PDZ.

PDZ extensions

N-terminal upstream and C-terminal downstream extension sequences of the PDZ domain can influence the stability of PDZ domains as well as their protein-protein interaction properties [START_REF] Wang | Extensions of PDZ domains as important structural and functional elements[END_REF]. In some cases, a wrong delimitation of domain, can cause misfolding, low solubility, low expression yield or even cause protein aggregation and precipitation. Hence N-or Cterminal extension sequences are important for a good domain delimitation [START_REF] Luck | The emerging contribution of sequence context to the specificity of protein interactions mediated by PDZ domains[END_REF]. The extension can also form an extra α-helix or β-sheet, that could be important for the function and folding of PDZ (Fig. 27). N-terminal extension has been reported in Shank3 (SH3 and multiple ankyrin repeat domains 3) PDZ to form an extra β hairpin, increasing the binding specificity and affinity with SAPAP (synapse-associated protein 90/postsynaptic density-95associated protein) [START_REF] Zeng | A binding site outside the canonical PDZ domain determines the specific interaction between Shank and SAPAP and their function[END_REF]. C-terminal extension can also form a mini structured domain with a small β hairpin followed by an α helix as found in Harmonin, a multi domain scaffolding protein expressed in the inner hear. This C-terminal extension after the first PDZ domain of Harmonin increases the binding to SANS (scaffold protein containing ankyrin repeats and SAM domain) [START_REF] Yan | The structure of the harmonin/sans complex reveals an unexpected interaction mode of the two Usher syndrome proteins[END_REF]. However, not only folded extension is helpful for binding; as an example, unstructured extension of the PDZ3 in PSD-95 has an allosteric conformational change upon binding of target protein PBM, and this binding induces a conformational transition from the flexible part to an α helix and thus promotes the multi-domain coupling [START_REF] Zeng | PDZ Ligand Binding-Induced Conformational Coupling of the PDZ-SH3-GK Tandems in PSD-95 Family MAGUKs[END_REF][223] [START_REF] Gautier | Addressing the role of the α-helical extension in the folding of the third PDZ domain from PSD-95[END_REF]. Apart from the enhancement of target proteins binding, extension can also be a PDZ binding target by it-self, [START_REF] Hillier | Unexpected modes of PDZ domain scaffolding revealed by structure of nNOS-syntrophin complex[END_REF] as reported with a β hairpin C-terminal extension of nNOS (neuronal Nitric oxide synthase) PDZ domain that can be recognized by syntrophin PDZ. The PDZ structured extension can also interact with another PDZ extension and reinforce a PDZ-PDZ supramodule, for example the PDZ1-hairpin and PDZ2-hairpin in protein Whirlin [START_REF] Delhommel | Structural Characterization of Whirlin Reveals an Unexpected and Dynamic Supramodule Conformation of Its PDZ Tandem[END_REF].

PDZ1-PDZ2 tandem with Fras1 (Fraser extracellular matrix complex subunit 1) C-terminal PBM peptide (PDB entry 2QT5). C: INADL PDZ8-PDZ9 with PLCβ4 C-terminal CC-PBM (PDB entry 6IRD). D: INADL PDZ4-PDZ5 with NORPA CC-PBM (PDB entry 6IRE

). E: X11 PDZ1-PDZ2 tandem, with auto-inhibition C-terminal residues at PDZ1 binding pocket, while PDZ2 is free to interact (PDB entry 1U3B). F: hetero-bidomain of Harmonin HHD and PDZ1 in interaction with the SAM domain of SANS (PDB entry 3K1R).

Apart from the PDZ-PDZ supramodules, PDZ is known to form supramodules with other globular domains. In Harmonin, as described before, the N-terminal region HHD and PDZ1 domains, and the HHD domain forms a compact supramodule with the PDZ1 domain via its C-terminal extension mini domain; this heterotypic PDZ supramodule stabilised the functional PDZ domain in binding to SANS SAM (Sterile alpha motif) domain [START_REF] Yan | The structure of the harmonin/sans complex reveals an unexpected interaction mode of the two Usher syndrome proteins[END_REF]. Another well-known example is the compact bidomain supramodule made of SH3 and GK domain of PSD-95 [START_REF] Mcgee | Structure of the SH3-guanylate kinase module from PSD-95 suggests a mechanism for regulated assembly of MAGUK scaffolding proteins[END_REF]; the ligand binding induced the formation of a triple-domain supramodule of PDZ-SH3-GK, its structure has been modelled in 2013 [START_REF] Zhang | Supertertiary structure of the MAGUK core from PSD-95[END_REF] and experimentally confirmed in 2018 [START_REF] Zeng | Reconstituted Postsynaptic Density as a Molecular Platform for Understanding Synapse Formation and Plasticity[END_REF].

PDZ mediated interactions

Protein-protein interactions are essential for all of living cells, and among them, the recognition mediated by globular domains, including PDZ domains, and SLiMs (a short linear motifs) are mostly found in cell signalling pathways. The SLiMs recognized by PDZ are called PBMs, which attach to the PDZ binding groove [START_REF] Davey | Discovery of short linear motif-mediated interactions through phage display of intrinsically disordered regions of the human proteome[END_REF]. In signalling complexes, PDZ domain is an important component in those scaffolding proteins, which drives the localization of essential enzymes and their substrates. PDZ containing proteins can also attach to cell membranes to locate and fix the protein complexes, and auto-associate or interact with other scaffolding proteins to form large protein complexes as cell-cell linker anchors or to reshape the cell structures. The PDZ-PBM interactions have an affinity in the micromolar range (mainly around 1-100 μM) [START_REF] Harris | Mechanism and role of PDZ domains in signaling complex assembly[END_REF], [START_REF] Luck | Putting into practice domain-linear motif interaction predictions for exploration of protein networks[END_REF], [START_REF] Stiffler | PDZ Domain Binding Selectivity Is Optimized Across the Mouse Proteome[END_REF].

Canonical PBM binding

Most PDZ binding patterns are based on PDZ-PBM recognition, where PBM are normally short SLiMs at the C-termini of target proteins (at least 3 residues) [START_REF] Sheng | Pdz d o s c[END_REF][214] [START_REF] Zhang | Organization of signaling complexes by PDZ-domain scaffold proteins[END_REF]. The last C-terminal residues of PDZ partners forming the PBM are numbered from the last position as position 0 (P0), and going towards N-terminal as P(-1), P(-2), P(-3) and so on. In the PBM sequence, P(0) and P(-2) are hallmarks for binding and contribute the most to the binding, while P(-1) and P(-3) have less effect [START_REF] Luck | The emerging contribution of sequence context to the specificity of protein interactions mediated by PDZ domains[END_REF]; more detailed binding position will be discussed below. Upon binding, PBMs adopt an anti-parallel β strand conformation contacting the β2 strand of the PDZ domain with several backbone H-bonds formed. The PDZ binding pocket is formed by a peptide-C-terminal carboxylate binding loop known as the 'GLGF' loop [START_REF] Harris | Mechanism and role of PDZ domains in signaling complex assembly[END_REF], β2 strand, β3 strand, β2-β3 loop, and α2 helix [START_REF] Doyle | Crystal structures of a complexed and peptide-free membrane protein-binding domain: Molecular basis of peptide recognition by PDZ[END_REF]. The overall PDZ backbone shows only small change of conformation during PBM binding, with a backbone carbon α RMSD value ranging from 0.42 to 3.51 Å, between apo and holo PDZ [START_REF] Gerek | Identification of specificity and promiscuity of PDZ domain interactions through their dynamic behavior[END_REF].

Based on the PBM sequences, PDZ domains have mainly three recognition patterns. Class I PDZ recognizes PBM with the final three residues as a motif pattern -[Ser/Thr]-X-ΦCOOH, Class II PBM as -Φ-X-ΦCOOH, and Class III as -[Asp/Glu]-X-ΦCOOH, where X means an unspecified residue and Φ is a hydrophobic residue [242][217] [START_REF] Ernst | A structural portrait of the PDZ domain family[END_REF]. The C-terminal carboxylate group forms hydrogen bonds with the backbone of the 'GLGF' loop and the hydrophobic side chain of P(0) inserts into the PDZ hydrophobic binding pocket, which is one of the main features of PDZ-PBM canonical binding pattern. Due to the lack of salt-bridges formation between carboxylate amino acid and carboxylate binding 'GLGF' loop, the recognition of PBM is more of a spatial recognition rather than carboxylate recognition. This indirect binding phenomena may have two purposes: first, to remain of moderate binding affinity, and second to avoid other free carboxylated amino acid to bind [START_REF] Harris | Mechanism and role of PDZ domains in signaling complex assembly[END_REF] (Fig. 29). As mentioned before, the PBMs bind to the PDZ binding pocket in an anti-parallel manner with the side chain of the last residue facing inside of the hydrophobic core of the PDZ, the side chain of P(-1) being exposed to the solvent and P(-2) side chain facing the α2 helix [START_REF] Doyle | Crystal structures of a complexed and peptide-free membrane protein-binding domain: Molecular basis of peptide recognition by PDZ[END_REF]. At P(-1), the amino acid can be very variable, which depends on the chemical environment of the PDZ. The side chain of P(-1) can form extra contacts with residues on the β2 strand, β3 strand or α2 helix by forming polar interactions. If the PDZ's β2 and β3 strands form an hydrophobic surface, the P(-1) can also be a hydrophobic residue, its side chain laying on the hydrophobic surface created by β2 and β3 strand. The P(-1) residue is generally not going to disrupt the PBM binding even with unfavourable amino acids [START_REF] Songyang | Recognition of unique carboxyl-terminal motifs by distinct PDZ domains[END_REF] [START_REF] Fournane | Surface plasmon resonance analysis of the binding of high-risk mucosal HPV E6 oncoproteins to the PDZ1 domain of the tight junction protein MAGI-1[END_REF]. However, it can modulate the PBM binding; it has been reported that the PDZ3 of PSD-95 binds to -Q-T-S-VCOOH [START_REF] Doyle | Crystal structures of a complexed and peptide-free membrane protein-binding domain: Molecular basis of peptide recognition by PDZ[END_REF], but the PBM sequence changed to -Q-T-D-VCOOH [START_REF] Niethammer | CRIPT, a novel postsynaptic protein that binds to the third PDZ domain of PSD-95/SAP90[END_REF] prefers to interact with the PDZ2 of PSD-95 instead of PDZ3. The special case of proline at P(-1) has been found in the PBM of p38γ with -ETPL, a partner of the unique PDZ of PTPN3 (protein tyrosine phosphatase non-receptor type 3) and PTPN4, where the proline does not contribute to any hydrophobic or electrostatic interactions [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] [START_REF] Hou | PTPH1 Dephosphorylatesates and Cooperates with p38y MAPK to Increase Ras Oncogenesis through PDZ-Mediated Interaction[END_REF]. By contrast, the recognition of P(-2) has been described as the key for all three classes of PDZ-PBM binding [START_REF] Harris | Mechanism and role of PDZ domains in signaling complex assembly[END_REF]. In the first PDZ crystal solved by [START_REF] Doyle | Crystal structures of a complexed and peptide-free membrane protein-binding domain: Molecular basis of peptide recognition by PDZ[END_REF], the peptide recognition by the PDZ3 of PSD-95 is based on Class I PDZ binding model. The final 4 residues are -Q-T-S-VCOOH, in which a Thr at P(-2) forms a salt bridge with the His side chain at the N-terminus of α2 helix. It has been reported that this Histidine on PDZ is important for the Class I PBM, and then researchers grouped the PDZ domain as Class I according to the PBM classification [START_REF] Songyang | Recognition of unique carboxyl-terminal motifs by distinct PDZ domains[END_REF]. The Class II PDZ has a hydrophobic residue at the same position as the His residue on the α2 helix, and this residue also recognizes the P(-2) as a hydrophobic amino acid, meaning the Class II PBM pattern is the -Φ-X-ΦCOOH [START_REF] Songyang | Recognition of unique carboxyl-terminal motifs by distinct PDZ domains[END_REF]. The Class III PDZ was described by [242] via a peptide library screening. The Class III PDZs recognize the PBM with pattern -[Asp/Glu]-X-ΦCOOH. In place of the Histidine on the α2 helix, the Class III nNOS PDZ has a Tyrosine, which recognizes the Asp at P(-2) of a synthetic peptide [START_REF] Tochio | Solution structure of the extended neuronal nitric oxide synthase PDZ domain complexed with an associated peptide[END_REF].

interaction with PSD-95's second PDZ is disrupted, consequently decreasing the expression of NMDA at the membrane [START_REF] Hee | Regulation of the NMDA receptor complex and trafficking by activity-dependent phosphorylation of the NR2B subunit PDZ ligand[END_REF]. A similar example is the PBM of AMPA (α-amino-3hydroxy-5-methyl-4-isoxazolepropionic acid) receptor, where the phosphorylation of the Thr at P(-2) abrogated the interaction with the first PDZ of PSD-95 [START_REF] Chetkovich | Phosphorylation of the postsynaptic density-95 (PSD-95)/discs large/zona occludens-1 binding site of stargazin regulates binding to PSD-95 and synaptic targeting of AMPA receptors[END_REF]. As mentioned before, the phosphorylation can also strengthen the interaction. The phosphorylated RPS6KA2 (ribosomal protein S6 kinase A2) PBM at P(-3) has a five-fold higher affinity with Scribble PDZ than its counterpart, while the phosphorylation of the MCC (mutated in colorectal cancers) PBM P(-1) increases the affinity by three-fold [START_REF] Sundell | Proteome-wide analysis of phospho-regulated PDZ domain interactions[END_REF]. However, it has also been reported that the phosphorylated P(-1) of MCC PBM and Scribble PDZ didn't change the binding affinity, which it is not in agreement with the previous report [START_REF] Caria | Structural analysis of phosphorylation-associated interactions of human MCC with Scribble PDZ domains[END_REF]. Phosphorylations can also occur on the PDZ domain and regulate its binding properties. The phosphorylation at Ser(232) (right next to the GLGF loop at the N-terminal upstream) of the PDZ-containing protein SAP97 (Synapse-associated protein 97) abolished the binding to the PBM of NMDA receptor NR2A subunit [START_REF] Mauceri | Dual role of CaMKII-dependent SAP97 phosphorylation in mediating trafficking and insertion of NMDA receptor subunit NR2A[END_REF]. A positive regulation has been reported in the protein PTEN (phosphatase, tensin homologue), where the phosphorylation at its C-terminus PBM Thr(401) improved the binding to the target PDZ [START_REF] Adey | Threonine phosphorylation of the MMAC1/PTEN PDZ binding domain both inhibits and stimulates PDZ binding[END_REF]. PDZ-PBM recognition is mainly classified via the C-terminal last three amino acids of the PBM, but the N-terminal upstream residues at position (-3), (-4) or even further to (-11) [START_REF] Terrien | Interference with the PTEN-MAST2 interaction by a viral protein leads to cellular relocalization of PTEN[END_REF] on PBM have been shown to participate in the PDZ-PBM binding [START_REF] Tonikian | A specificity map for the PDZ domain family[END_REF][217][244] [START_REF] Luck | The emerging contribution of sequence context to the specificity of protein interactions mediated by PDZ domains[END_REF]. Nterminal upstream residues could be phosphorylation targets, affecting the binding affinity with PDZ. For example, the phosphorylation at Ser P(-8) of vascular endothelial cadherin (VE-Cad) increased the affinity of binding to the third PDZ of Par-3 (Partitioning defective) [START_REF] Tyler | Distal interactions within the par3 -VEcadherin complex[END_REF], while the Tyr (-7) phosphorylation decreased the affinity of binding between the ErbB2 (Receptor tyrosine-protein kinase erbB-2) PBM and the PDZ of Erbin (Erbb2 Interacting Protein) [START_REF] Birrane | Novel mode of ligand recognition by the Erbin PDZ domain[END_REF]. These extra interaction sites indicate that a potential fine-tuned regulation could take place in the PDZ-PBM recognition pattern.

Internal PBM binding

Besides the well-established pattern of canonical C-terminal PBM binding, some PDZs accommodate other types of binding modes. One of the earliest PDZ internal PBM binding is found in the nNOS signalling pathway involved in muscle contractions [START_REF] Constantin | Dystrophin complex functions as a scaffold for signalling proteins[END_REF], where the complex between nNOS and the syntrophin PDZ has been described. The nNOS PDZ domain has a C-terminal β hairpin extension, which inserts into the syntrophin PDZ [START_REF] Hillier | Unexpected modes of PDZ domain scaffolding revealed by structure of nNOS-syntrophin complex[END_REF]. In this internal PBM binding model, the PDZ of syntrophin recognizes the Class I PBM in the β hairpin -T-T-F-. A similar structure was also reported from Dishevelled PDZ domain, which forms a homodimer, where the internal PBM is -S-N-E-located in the sharp turn between the β2 and β3 is inserted into the other PDZ (PDB code: 3FY5). This is not a canonical Class I PBM as the Glu is not hydrophobic, but this Glu side chain mimics the C-terminal carboxylate binding to the GLGF loop of the other PDZ. The protein Dishevelled has a Class III PBM (except in Drosophila Dishevelled that has a Class II PBM), and auto-inhibition was reported vital for Xenopus embryo development [START_REF] Lee | Conformational change of dishevelled plays a key regulatory role in the wnt signaling pathways[END_REF] involving its own PDZ and PBM sequences. However, the Dishevelled PDZ has also been reported to be able to recognize Class II PBMs, as well as internal Class II PBMs [START_REF] Zhang | Inhibition of Wnt signaling by Dishevelled PDZ peptides[END_REF]. Interestingly, all internal PBM sequences mentioned in this research have an Asp at binding (+1) which has its side chain 3.6.2. Signalling pathways Due to their scaffolding property, PDZ-containing proteins are also important partners in several signalling pathways via PDZ mediated protein recruiting. Three examples are described below. In the canonical Wnt signalling pathway, once extracellular Wnt binds to the LRP5/LRP6/Frizzled co-receptor group, the PDZ-containing protein Dishevelled and other downstream proteins assemble to the membrane. Then, β-catenin starts to accumulate and passes through nuclear pores to participate to gene regulation. Dishevelled PDZ is a key regulator here, responsible for the canonical and non-canonical Wnt signalling pathways via its PDZ domain [START_REF] Moon | Signalling polarity[END_REF] [266] (Fig. 33A). In non-canonical Wnt pathway, Dishevelled is critical in controlling the orientation of polarized cell, and cell convergent extension (CE) [START_REF] Tae | Subcellular localization and signaling properties of Dishevelled in developing vertebrate embryos[END_REF][286] [START_REF] Schwarz-Romond | The DIX domain of Dishevelled confers Wnt signaling by dynamic polymerization[END_REF]. Syntrophin was first identified from the post-synaptic membrane of the electric organ of the Torpedo fish [START_REF] Wagner | The 87K postsynaptic membrane protein from torpedo is a protein-tyrosine kinase substrate homologous to dystrophin[END_REF]. As a multi-domain scaffolding protein, syntrophin is involved in proteinlipid and protein-protein interactions with several signalling molecules and kinases, like the Dystrophin complex mediated signalling pathway, which is critical for muscle contraction. One of the most well known PDZ-internal PBM binding structure is coming from the interaction between syntrophin and nNOS; the syntrophin knockout mice lost the function of nNOS [START_REF] Adams | Syntrophin binds directly to multiple spectrin-like repeats in dystrophin and mediates binding of nNOS to repeats 16-17[END_REF][225] [START_REF] Constantin | Dystrophin complex functions as a scaffold for signalling proteins[END_REF][290], [START_REF] Ivarsson | Plasticity of PDZ domains in ligand recognition and signaling[END_REF] (Fig 33B). Syntenin is a 32 kDa multi-PDZ protein, first identified as a binding partner of syndecans [START_REF] Grootjans | Syntenin, a PDZ protein that binds syndecan cytoplasmic domains[END_REF]. It is a multi-tasking signalling related protein involved in membrane structure, virus infection, cell motility, etc [START_REF] Shimada | Syntenin : PDZ Protein Regulating Signaling Pathways and Cellular Functions[END_REF]. For instance, Syntenin interacts with membrane-bound proteins and modulates the trafficking of its binding partners, and was described as having PDZ-lipid binding properties. In the secretory pathways, where the correct transformation of G protein-coupled coreceptor syndecan's to cell surface depends on Syntenin [START_REF] Beekman | The ins and outs of syntenin, a multifunctional intracellular adaptor protein[END_REF]. However, the Syntenin deficient mice show an increase of intestinal immunoglobulins, immature spines formation of neurons and memory impairment [START_REF] Tamura | Increased production of intestinal immunoglobulins in Syntenin-1-deficient mice[END_REF][295] (Fig. 33C). regulation of Rab5 activity with the assistance of SynGAP during the early stage of endosome. g. Anti-apoptotic protective autophagy induced by Syntenin activated signalling pathways. Adapted from [START_REF] Shimada | Syntenin : PDZ Protein Regulating Signaling Pathways and Cellular Functions[END_REF].

3.6.3. Cell junctions, cell polarities and phase separations ZO-1 was one of the first three proteins found with PDZ domain, which is a key participant in the tight-junction formation of epithelial cells, a type of cell with strong cell polarity. PDZcontaining proteins partners are mainly involved in these two functions: tight-junction formation and cell polarity control. Both processes are linked to PDZ protein associated phase separation phenomenon. In epithelial tight-junction cells, ZO-1, ZO-2 and ZO-3 are found enriched at the junctional fractions [START_REF] Furuse | Occludin: A novel integral membrane protein localizing at tight junctions[END_REF][298], along with the three cell polarity complexes PAR-6/PAR-3/aPKC, Crumbs/PATJ/PALS1 and LGL1/2/Scribble/DLG1. Most of these key partners are PDZ containing proteins, which organize the protein trafficking and localizations in the tight junction regions and sustain the cellular polarity [START_REF] Heinemann | Structural features of tight-junction proteins[END_REF]. Recently, ZO proteins have been shown to cause the formation of liquid-liquid phase separation, which is critical for raising the local functional protein concentration and increasing the tight-junction regions rigidness [START_REF] Beutel | Phase Separation of Zonula Occludens Proteins Drives Formation of Tight Junctions[END_REF] (Fig. 34A). In post-synaptic densities (PSD), the PDZ-SH3-GK multi-domain of MAGUK family proteins PSD-95 can form a supramodule upon SynGAP (Synaptic GTPase-Activating Protein) binding, and thus triggers the inter-molecule oligomerization. It finally causes the phase separation phenomenon, which could be important for the formation of bell-shaped PSD [START_REF] Zeng | PDZ Ligand Binding-Induced Conformational Coupling of the PDZ-SH3-GK Tandems in PSD-95 Family MAGUKs[END_REF] [START_REF] Zeng | Phase Transition in Postsynaptic Densities Underlies Formation of Synaptic Complexes and Synaptic Article Phase Transition in Postsynaptic Densities Underlies Formation of Synaptic Complexes and Synaptic Plasticity[END_REF]. PDZ is important for the supramodule and supermolecule formation, the PDZ binding coupled with the change of PDZ extension secondary structure trigger the autoassociation (Fig. 34B). The similar liquid-liquid phase separation has been found in auditory hair cells as well. The PDZ containing protein Harmonin establishes local interactions with hearing proteins Myosin 7A and SANS. This complex forms a phase separation in hair cells from the inner hear, which is important to spread the high tension applied to the stereocilia by the acoustic waves; and maintain other functional proteins and mechano-transduction ion channel well connected [START_REF] Yunyun He | Myosin VII, USH1C, and ANKS4B or USH1G Together Form Condensed Molecular Assembly via Liquid-Liquid Phase Separation[END_REF].

PDZ as a hijacking target of pathogenic virus

Numerous viral proteins contain PBM at the C-termini of envelope proteins, which have been shown to interact with the PDZ domain of proteins in infected cells. Investigating the infection mechanisms, viral PBMs mainly target proteins involved in the developing of tightjunction, cell polarity and apoptosis, which appears to be a common strategy of the viral cell hijacking. The perturbation of the host PDZ protein function will facilitate the viral infection and dissemination [START_REF] Javier | Emerging Theme: Cellular PDZ Proteins as Common Targets of Pathogenic Viruses[END_REF]. Rabies virus causes acute encephalitis, and exclusively infects neuronal cells. Various virulent strains affect the homeostasis of infected cells by the glycoprotein (G protein) trimer on the envelope during infection. The virulent and attenuated strains have opposite strategies upon infection; while virulent strains keep neurons surviving, attenuated strains trigger the apoptosis of the cells. Upon infection, the death and survival balance is controlled by one single-point mutation in the C-terminus PBM of the G protein, which displays a Q(-3)E substitution from the virulent virus strain to the attenuated ones. This single mutation changes the pattern of host protein recognition; for the virulent strain, the last 4 residues are -QTRL and bind to the PDZ domain of the human MAST2 (microtubule-associated serine and threonine kinase 2), but the -ETRL PBM of attenuated strain targets not only MAST2 but also tyrosine-phosphatase PTPN4, which is known to work as an apoptosis inhibitor [START_REF] Caillet-Saguy | Strategies to interfere with PDZ-mediated interactions in neurons: What we can learn from the rabies virus[END_REF] (Fig. 35A). Apart from PTPN4, its homolog PTPN3 is another example of a cellular target of the oncovirus human papillomavirus (HPV) E6, based on the same principle. PTPN3 is responsible for maintaining the homeostasis and cell growth control, but dysfunction or viral infection targeting PTPN3 leads to various cancer types [START_REF] Hendriks | Non-transmembrane PTPs in Cancer[END_REF]. However, HPV E6 not only hijacks PTPN3, but a large variety of PDZ containing proteins, which are related to the regulation of cell proliferation and polarity [START_REF] Thomas | Human papillomaviruses, cervical cancer and cell polarity[END_REF] [START_REF] Vincentelli | Quantifying domain-ligand affinities and specificities by high-throughput holdup assay[END_REF](Fig. 35B).

the protein-protein interactions, PDZ1 was identified as a lipid binder due to a large positively charged patch detected onto its surface [START_REF] Ivarsson | Prevalence , Specificity and Determinants of Lipid-Interacting PDZ Domains from an In-Cell Screen and In Vitro Binding Experiments[END_REF]. We addressed the questions: -Do the HHD1-PDZ1-PDZ2 domains of Whirlin form a supramodule? If so, what is the structure of such supramodule organization? -What is the function of the HHD1, PDZ1 and PDZ2 of Whirlin? What is the detail of Protein-Protein and Protein-Lipid interactions of PDZ1 and HHD1-PDZ1-PDZ2 module?

Results

Secondments

Delimitations of Whirlin, USH proteins and other Whirlin interacting proteins

Knowing the high-throughput capacity of the NZYtech company to clone and test the expression of proteins in Escherichia coli, we decided to design a large number of constructs from putative and identified partners of Whirlin in stereocilia. Some of them have been used for my project, some others have been planned for further perspectives and side projects.

Details of Whirlin constructs

Based on previous researches, published or from our team, the interactions that involved Whirlin are listed below (table). Including Whirlin, there are 12 Mus musculus proteins I subcloned into prokaryotic overexpression vectors. In the first secondment, I planned to make 40 constructs from 10 proteins that have been found to interact with Whirlin or in related protein networks (Table. 1). Taking into account the aborted synthesis of Myosin 15a, I subcloned 34 constructs from 9 different proteins: PDZD7, Usherin, ADGRV1, CASK, MPP1, CIB2, NGL1/Lrrc4c, SANS, Myosin 7a and Myosin 15a. The synthesised genes were subcloned into pHTP (plasmid High-Throughput) vector with different tags based on homologue recombination approach similar to GATEWAY ® cloning method. The genes were then subcloned with three affinity-purification-tag vectors: pHTP1 (HIS), pHTP11 (GST) and pHTP6 (MBP). In addition, the Whirlin (UniprotKB number Q80VW5-4), EPS8 (Q08509-1) and Myosin 15a (Q9QZZ4-1) genes were cloned at the Institut Pasteur (Paris) in the pGST//2, a GST tagged prokaryotic expression vector. A TEV protease cleavage site has been systematically introduced between the tag and the targeted gene for all constructs; thus five We performed SAXS experiments to estimate the overall shapes of the EPS8 constructs. SAXS is a structural method allowing to detect the protein in solution and report an overall structure at low resolution, giving access to the shape of the molecule. This technique is therefore sensitive to distinguish unstructured regions from compact modules. As predicted, the PTB and SH3 showed a compact form, while the predicted unfolded domain adopted an elongated structure with extra large Dmax (maximal distance distribution) and MW (Table. 4). However, the PTB-VHS construct exhibits a highly compact organization, which was unexpected.

Table 4: SAXS data of five purified constructs of Eps8. Rg (Radius of gyration), Dmax (maximal distance distribution), MW (Molecular Weight) predicted by http://saxs.ifsc.usp.br/ from the SAXS scatting data.

In order to know which region in the N-terminal region of ESP8 can interact with Whirlin, I performed series of biophysical and biochemical tests (Fig. 14). For Whirlin, we used HHD2, Proline-Rich, Proline-Rich-PDZ3ΔPBM, Proline-Rich-PDZ3, HHD2-PDZ3 and PDZ3. Analytical SEC: inject single protein samples first to get a elution chromatogram as reference and inject a 1:1 molarity mixture after to compare the peak shift by running on a superdex 200 increase 10/300 GL analytical column (GE). MST: Microscale thermophoresis (MST) is a sensitive way to detect by fluorescence the diffusion of molecules in solution, in a gradient of temperature. This allows to estimate the protein-protein interaction (PPI), because larger protein complex can diffuse differently than unbound proteins. These experiments were performed at the biophysical platform of the Institut Pasteur. Fluorescence: Due to the sensitivity of the emission light of Tryptophans depending on their environment, we can monitor variations of signal according to the concentration of ligand, thus following the PPI (provided that the complexity of the signal is sufficiently low, e.g. that the construct do not have few Trp). NMR: By detecting the isotopically labelled protein backbone N-H bond resonance signal changes upon ligand binding, NMR is one of the most sensitive method to monitor the PPI at atomic level resolution. Experiments have been done with our collaborator Florence Cordier (Structural bioinformatic unit, Institut Pasteur) . GST pulldown: We charged GST-tagged protein on the GSTrap (GE) column and run the putative partner after through the column. The eluted protein or complex will be checked by SDS-PAGE with both single form proteins as negative control.

Most interaction tests were performed by more than one technique to ensure the detection of any existing interaction. However, we did not detetected any significant interaction between Rg (Å) Dmax (Å) MW (kDa) MW (real) (

Abstract

Hearing is a mechanical and biochemical process, which occurs in the hair cells of inner ear that converts the sound vibrations into electrical signals transmitted to the brain. Understanding the molecular organization of proteins in hair cells is the key to unveil this highly specialized mechano-transduction machinery. The multi-domain scaffolding protein Whirlin plays a critical role in the formation and function of stereocilia exposed at the surface of hair cells. Among the five folded domains identified in Whirlin, three PDZ domains play a key role in the network of proteins in stereocilia. PDZs mainly bind to a short C-terminus motif PBM (PDZ Binding Motif) of their partners. While the two N-terminal PDZ domains form a supramodule involved in submembrane complexes as we found before, the third PDZ domain has not been systematically researched. So far, three proteins interacting with Whirlin PDZ3 have been reported: CASK, MPP1 and Myosin 15a.

In this article, we reported at least seven stereociliary proteins that encode for PBM interacting with Whirlin PDZ3: Myosin 15a, Harmonin a1, CASK, MPP1, Protocadherin-15 CD3, Cadherin-23 and Taperin. We solved the atomic resolution structures of complexes between Whirlin PDZ3 and PBM of Myosin 15a, CASK, Harmonin a1 and Taperin, to understand how this PDZ can recognize many different proteins in stereocilia. Interestingly, the PBM of CASK is a non-canonical motif, not localized at the extreme C-terminus but shifted by one residue, which has never been reported. The Taperin recognition motif is also a rare case of internal PBM, identified here by a proteomic peptide phage display. Our highresolution structural data combining NMR and X-rays experiments have provided insights into the molecular determinants of PDZ3-PBM recognition. This large capacity to accommodate various partners such as kinases (CASK), actin-binding protein (Myosin 15a), scaffold protein (Harmonin) and deafness protein (Taperin) could be related to the distinct functions of Whirlin at different stages of the hair cell development.

Introduction

The sense of hearing is a mechanical and biochemical process that requires highly specialized sensory cells (inner and outer hair cells) in the cochlea of the inner ear. After the hair cell maturation, three rows of staircase-like actin-filled stereocilia connected to each other with tip links and multiple horizontal top and lateral connectors, are required to perform the mechanoelectrical transduction of the sound-induced vibrations [START_REF] Mathur | Usher syndrome: Hearing loss, retinal degeneration and associated abnormalities[END_REF], [START_REF] Schwander | The cell biology of hearing[END_REF], [START_REF] Siletti | Daple coordinates organ-wide and cell-intrinsic 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 formed at the stereocilia base. Both tip links and ankle links are anchored to large protein densities 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), an autosomal recessive disease that associates auditory and visual senses loss. 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], [START_REF] Mathur | Usher syndrome: Hearing loss, retinal degeneration and associated abnormalities[END_REF], [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], [START_REF] Weil | Usher syndrome type I G (USH1G) is caused by mutations in the gene encoding SANS, a protein that associates with the USH1C protein, harmonin[END_REF]. The same proteins are also found in retina photoreceptor cells. Two membrane cadherins, Protocadherin15 (USH1F) and Cadherin23 (USH1D) form the interstereociliary tip link of auditory hair cell bundles, that conveys soundevoked 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]. The submembrane scaffold proteins Harmonin (USH1C) and SANS (USH1G) anchor the upper part of this tip link to actin filaments through interactions mediated by the actin-binding protein myosin VIIa (USH1B). Usherin (USH2A) and adhesion G protein-coupled receptor V1 (ADGRV1, USH2C), two other large transmembrane proteins, compose the ankle links [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 scaffold 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] Mustapha | DFNB31, a recessive form of sensorineural hearing loss, maps to chromosome 9q32-34[END_REF], [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]. The 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 and a proline rich (PR) region and a C-terminal PBM motif (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 cells and auditory hair cells. This isoform impacts the shape of OHC (Outer Hair Cell) and the thickness of IHC (Inner Hair Cell) [START_REF] Ebrahim | Alternative Splice Forms Influence Functions of Whirlin in Mechanosensory Hair Cell Stereocilia[END_REF] and is expressed at the ankle links of both IHC and OHC, and also at the tip of the tall stereocilia of IHC in immature mice [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]. In the mature mice, the FL isoform is only found at the tip of the tall stereocilia of IHC. However, the distribution and role of the FL Whirlin are still unclear in the hair bundles of mature hair cells with discrepancies for its cellular immunolocalizations. The C-terminal isoform including the region from HHD2 to the C-terminal PBM is detected in hair cells where it plays a critical role in the polymerization and stabilization of actin filaments in stereocilia [START_REF] Ebrahim | Alternative Splice Forms Influence Functions of Whirlin in Mechanosensory Hair Cell Stereocilia[END_REF]. This C-terminal isoform is expressed at the tip of tall stereocilia in both IHC and OHC during development. It remains at the same position in IHC of mature mice but disappear in OHC [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]. Besides the Usher syndrome, the mutations in the Cterminal isoform region can lead to non-syndromic deafness (DFNB31). Finally, the Nterminal 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], [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 forming the 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 (G-protein signalling modulator 2) [START_REF] Manor | Regulation of stereocilia length by myosin XVa and whirlin depends on the actin-regulatory protein Eps8[END_REF] [START_REF] Ebrahim | Alternative Splice Forms Influence Functions of Whirlin in Mechanosensory Hair Cell Stereocilia[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 as for 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 these protein network organization. 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 Cterminus carboxylate of the motif directly contacts to the β1-β2 loop or 'TLGI' 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 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 as expressed prominently in neuron cell soma and dendrites of the central nervous system; 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 the related MPP1 (Membrane Palmitoylated Protein 1) have been shown to colocalize with Whirlin [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], [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 a 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 characterized the various modes of interaction of 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 hold-up 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. Like Whirlin, Taperin is another non-syndromic deafness protein (DFNB79) [START_REF] Li | Mutations in TPRN Cause a Progressive Form of Autosomal-Recessive Nonsyndromic Hearing Loss[END_REF]. We also characterized the PDZ mediated interactions through a series 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 FL 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 C-terminal 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 scattering curve of the PR constructs corresponding to the large inter-domain linker yields a large radius of gyration (Rg, 57.1 Å) derived from the Guinier approximation at low q value and maximum distance values (Dmax, 250 Å) estimated from the pair-distance distribution function. The same ranges of Rg and Dmax values are observed for the constructs PR, PR-PDZ3 and HHD2-PR. These SAXS data are 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. SAXS provides a sensitive means to evaluate the degree of compactness of proteins as illustrated by the dimensionless Kratky plot. This convenient representation unambiguously indicates that the PR region behaves in solution as an extended and flexible sequence with a plateau at large qvalues while HHD2 and PDZ3 adopt folded and compact conformations as represented by their bell-shaped curves (Fig. 1).

Altogether, our results demonstrated that the PDZ3 domain is independent from the other folded domain HHD2, in the larger molecular context of Whirlin.

Myosin 15a and Cadherin23 displayed significant BI values higher than 0.2, a stringent threshold used to define relevant 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, Myosin15 and Cadherin23 proteins have the capacity to bind PDZ3 in vitro in the 1-100 µM affinity range typically found for PDZ/PBM interactions. The 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. Hold-up 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 µM. Table 1: A: Holdup assay tested for the all PDZ domain constructs of Whirlin with stereociliary protein PBMs. The Binding intensity (BI) is directly linked to the binding affinity [START_REF] Vincentelli | Quantifying domain-ligand affinities and specificities by high-throughput holdup assay[END_REF]. B: Holdup assay in comparison with SPR data. 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][268] [START_REF] Hillier | Unexpected modes of PDZ domain scaffolding revealed by structure of nNOS-syntrophin complex[END_REF]. To explore the capacity of Whirlin PDZ3 to recognize internal PDZ binding motifs we used the protein as a bait in ProP-PD selections, using 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 in both the selections. 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 PDZ3 domain for various PBM sequences

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. By NMR titration, K D values of PDZ3 for 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 µM, the strongest value among the four binding partners, while Harmonin a1 peptide is the weakest binder with a K D value 45-fold lower at 132 ± 13 µM. This K D value is in the lower range of affinity commonly found for PDZ domain interactions. For Taperin, the K D 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 µM (Table . 3) was estimated for Taperin. For CASK, the slow/intermediate exchange regime observed in the spectra led to severe line broadening during the course of titration and prevented an accurate K D measurement. Therefore, we used fluorescence titration, following the emission fluorescence intensity of the unique tryptophan of the CASK peptide (-SWVY COOH ) affected by the binding to the PDZ3 domain. The K D value is 7.1 ± 1.0 µM 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 -VML COOH 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-PBM and PDZ3 deleted from the PBM of Myosin 15a. The K D values of Myosin 15a are similar for PDZ3-ΔPBM and PDZ3 with 3.4 ± 1.8 µM and 2.9 ± 1.6 µM, respectively. We concluded that the Whirlin PBM does not interfere with the binding of partner to the PDZ3 domain.

Table 3: Binding affinity of various PBMs for PDZ3 with sequences and binding pattern. The three residues that form the PBM are in bold.*: The CASK Kd value was determined using fluorescence titration.

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 by X-ray crystallography. The crystal very similar. As expected, the 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-Φ COOH , 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. However, Leu (825) of 'TLGI' loop does not directly contact 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 β2-strand, that also contacts the side chain of Ile(-3). The three residues Leu (-1), Ile (-3) and Ala (828) form with the Val(843) side chain from the β3-strand a hydrophobic patch exposed at the surface of the intermolecular β-sheet 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. 3). 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 form 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. 3). 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 to form 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 value of 1.12 S for the free form and 1.26 S for the complexed form. These S20,w values are consistent with a compact globular protein of 11kDa and 12.6kDa, for 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 . 2). 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 150 mM 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 . 2). 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 (T 1 , T 2 and { 1 H}- 15 N heteronuclear NOE). The 15 N relaxation parameters (T 1 , T 2 and { 1 H}-15 N heteronuclear NOE) were analysed in terms of internal dynamic parameters (order parameter S 2 , internal correlation time τi and exchange parameter Rex) using the Lipari-Szabo model-free approach (Fig. S3) [START_REF] Lipari | Model-Free Approach to the Interpretation of Nuclear Magnetic Resonance Relaxation in Macromolecules. 1. Theory and Range of Validity[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 S2 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. The crystal structure of PDZ3/Harmonin a1 ( NH2 PKEYDDELTFF COOH ) 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 -TFF COOH 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 the Ala(828) and Val(843) of the β-sheet. 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. S2). In addition, 3 out of 6 copies in the asymmetric unit have deformed α1 helix, and in these 3 complexes, 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 reflect 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/Harmonin a1-PBM complex

The crystal of PDZ3 complexed to the internal PBM of CASK ( NH2 TAPQWVPVSWVY COOH )

diffracted at 1.7Å resolution. The asymmetric unit only contains one copy of the complex, and the last 7 residues -VPVSWVY COOH are well defined in the electron density. In this structure, PDZ3 still recognized a Class I -SWV-as a PBM shifted of 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 C-terminus carboxyl group on 'TLGI' loop (Fig. 3). The Trp(-1) side chain is facing out of the binding groove towards the hydrophobic surface of the β2 and β3-strands. Interestingly, the indol 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. 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 Hbond 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 β2-β3 loop.

Structure of the PDZ3/Taperin-PBM complex The crystal structure of the complex formed by PDZ3 and the internal ( NH2 GLPVTFIDEVDSEEAPQ CONH2 ) peptide of Taperin was solved at 1.3 Å resolution. The N-terminal 11 residues, -GLPVTFIDEVD-, are well ordered in the crystal. PDZ3 recognises the internal PBM of Taperin peptide (-TFI-) as a Class I PBM. The 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 β3-strand 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 Myosin 15a and Harmonin a1 peptides. 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 α2helix, 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.

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 previously reported Whirlin-internal PBM binding pattern as -T-[FLV]-I-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-terminal 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 PBM 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 multitarget 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 µM). In the hair cells, Whirlin controls the stereocilia elongation and the polar orientation 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][138] [START_REF] Michalski | Molecular Characterization of the Ankle-Link Complex in Cochlear Hair Cells and Its Role in the Hair Bundle Functioning[END_REF][318] [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 light-sensitive receptor protein rhodopsin, while it is also involved in the mechanosensory signaling 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.

Here, we reported 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 multi-partner 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, 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 high-throughput chromatographic assay and SPR. Four Usher binders have been identified, Myosin 15a and Cadherin 23 with high affinity of few µM, Harmonin a1 with a moderate affinity 20 to 40-fold lower, and Protocadherin 15 CD3 with a low affinity (250 µM). 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 colocalize 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] [177] [148][180][181] [194] [132]. 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. First, Whirlin 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 (Fig. S4).

Interestingly, Phe(887) in the last C-terminal turn of α2-helix is facing the binding groove. The orientation of Phe side chain is constrained by hydrophobic contacts with with Lys(820), Leu(825) and Ile(895) on the 'TLGI' loop and the α2-β5 loop. One consequence of this sidechain 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 NH 3 + 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 entry: 1UFX). This 'Lys-Phe lock' is strictly conserved in all orthologous Whirlin sequences, the Phe being substituted by a Trp in Harmonin (Fig. S2). We propose that this stable structure of the extralong 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. In the case of the canonical PBM of Myosin 15a, only one H-bond is formed between this GLGF-like 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. 3). 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, and 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][321] [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 β3strand of PDZ3 fill-up the hydrophobic patch exposed at the surface of the β-sheet 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 Protocadh15-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 developments, 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, 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 nonsyndromic 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][325] [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 [START_REF] Liu | GRXCR2 Regulates Taperin Localization Critical for Stereocilia Morphology and Hearing Article GRXCR2 Regulates Taperin Localization Critical for Stereocilia Morphology and Hearing[END_REF]. 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 participate 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 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.

Materials & Methods

Constructs cloning, expression and purification

The constructs of Whirlin are synthesized in cDNA of Mus musculus whrn isoform 4 (UniprotKB number Q80VW5-4). It was subcloned into a pGST//2 expression vector (derived from pGEX-4T-1; Amersham) with an ampicillin resistant cassette. A TEV protease cleavage site was introduced between the N-terminal GST 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 (15NH4Cl and/or 13C-glucose) labelled M9 minimal media at 37°C. The expression was induced by 1mM IPTG (Isopropyl β-D-1thiogalactopyranoside) at O.D.600nm 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 was pelleted by centrifuge (30,000g, 1 h, 4°C). The GST tag fused proteins were purified by an affinity chromatography column: GSTrap-HP (GE Healthcare), which followed by a TEV protease cleavage at 4°C overnight. Then, the next day, a final step of a size-exclusion chromatography (SEC) by using Sephacryl S-100 HP 16/600 (GE Healthcare) with buffer Tris pH 7.5 50mM, NaCl 150mM, TCEP 0.5mM.

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 flatbottom 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 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 deepwell 96-well 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 peroxidaseconjugate anti-M13 antibody (Santa Cruz) (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 H 2 SO 4 . 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 [START_REF] Konarev | PRIMUS: A Windows PC-based system for small-angle scattering data analysis[END_REF]. The radii of gyration were evaluated using Guinier approximation [START_REF] Guinier | La diffraction des rayons X aux très petits angles : application à l'étude de phénomènes ultramicroscopiques[END_REF]. Dmax was determined from distance distribution function P(r) obtained with the program GNOM [START_REF] Svergun | Determination of the regularization parameter in indirect-transform methods using perceptual criteria[END_REF].

Fluorescence

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 are corrected according to the measured concentration. The titration curve fitting was done with software Origin 7.5.

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 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 8-hole AN50-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 [START_REF] Schuck | Size-distribution analysis of macromolecules by sedimentation velocity ultracentrifugation and Lamm equation modeling[END_REF]. 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 0.95. Pul L and Pul M partial specific volumes were 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 [START_REF] Vranken | The CCPN data model for NMR spectroscopy: Development of a software pipeline[END_REF].

Protein assignment. The backbone 1 H N , 15 N, 13 Cα, 13 Cβ and 13 CO resonance assignment of PDZ3ΔPBM 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 PDZ3ΔPBM (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 [START_REF] Barbato | Backbone Dynamics of Calmodulin Studied by 15N Relaxation Using Inverse Detected Two-Dimensional NMR Spectroscopy: The Central Helix Is Flexible?[END_REF], in an interleaved manner with a recycling time of 3 s and with 7 relaxation delays for T1 [START_REF] Brownell | Evoked mechanical responses of isolated cochlear outer hair cells[END_REF][START_REF] Li | Structural Basis of Cargo Recognition by Unconventional Myosins in Cellular Trafficking[END_REF][START_REF] Wright | Rpgr ORF15 connects to the usher protein network through direct interactions with multiple whirlin isoforms[END_REF]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 [START_REF] Dosset | Efficient analysis of macromolecular rotational diffusion from heteronuclear relaxation data[END_REF] to extract internal dynamical parameters (amplitude of the picosecond to nanosecond time scale motion S 2 , internal correlation time τi and exchange parameter Rex). An isotropic model, with a correlation time τc, was sufficient to describe the global reorientation of the protein and an anisotropic model did not improve the fit.

Peptide titration. PDZ3 (and PDZ3ΔPBM) samples initially contained about 200 mM 15N-labeled PDZ3 in 300 µL 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 of the respective complexes (Kd) were obtained by fitting the titration data (∆δavg or intensities) with a model assuming a 1:1 complex formation and with nonlinear regression. Myosin 15a and Whirlin. For Whirlin, the PDZ1 domain is responsible for both lipid and protein binding. However, the details of these complexes were still unknown. I will describe below the experiments I have performed with Whirlin PDZ1 containing constructs in interaction with both protein PBMs and lipids.

Crystallization and diffraction data collection

The Whirlin PDZ1/PIP lipid complex The PDZ1 has a pI of 10.1, and exposes a large cationic patch near the 'GLGF' loop (PDB entry 1UEZ), most likely involved in phosphoinositides (PIP) binding, especially with double or triple phosphorylated PIP (i.e. PIP2 or PIP3) [START_REF] Ivarsson | Prevalence , Specificity and Determinants of Lipid-Interacting PDZ Domains from an In-Cell Screen and In Vitro Binding Experiments[END_REF]. In our lab, we found that mutations that alter the cationic patch, like single mutation R145E, K213E or triple mutation R145E-K213E-K214E, abrogate the binding to PIP. Protein-lipid interaction between PDZ1 and PIPs has been tested by NMR titration using the head group of PIP2: the IP3 (d-Myo-inositol-1, 4, 5-triphosphate, Avanti ® ). The NMR titration showed a specific binding onto the cationic patch of the PDZ1, however with a very weak affinity (~2 mM) (Fig. 10). The chemical shift perturbations were then used as a ligand docking constraint in the HADDOCK software (Fig. 11A-D).

Molecular docking of inositol-1,4,5-trisphosphate (IP3) on PDZ1 (collaboration with Dr. Guillaume Bouvier,Unit of Structural Bioinformatic, Institut Pasteur, Paris)

The structure file was prepared using the PRODRG2 Server (http://prodrg1.dyndns.org) [PRODRG]. Then, we used the Haddock [HADDOCK] web portal [HADDOCKWEB] (https://haddock.science.uu.nl) for the docking protocol. The NMR Chemical Shift Perturbation (CSP) data were employed to define the HADDOCK active residues to specify the IP3 binding region on the PDZ1 domain. We let the default parameters of the Haddock server for the docking step, namely, 1,000 structures generated by rigid-body energy minimization followed by 200 structures obtained by semi-flexible refinement and finally 200 structures resulting from water refinement. Then, the final structures were clustered using a contact-based clustering, with a Fraction of Common Contacts sets to 0.75 and a minimum number of members in a cluster set to 4. We chose the cluster with the overall minimum energy as the final solution.

In the four lowest energy docking models, the IP3 is mainly connected by three residues: R145, K213, and K214. Single point mutagenesis of one of these three residues is enough to abrogate the lipid binding, which is confirmed by our lipid-strip binding test (Fig. 11E-F) and also match the previous paper's experiments [START_REF] Ivarsson | Prevalence , Specificity and Determinants of Lipid-Interacting PDZ Domains from an In-Cell Screen and In Vitro Binding Experiments[END_REF].

As expected, the main contribution to the binding is electrostatic interactions between the anionic phosphates of IP3 and the cationic charges of Arg and Lys residues of PDZ1. Several orientations of the ligand are consistent with our experimental constraints, but a single mutation K214E is sufficient to fully abrogate this interaction (Fig 11 We first charge the chip with the desired liposome and then run the proteins. Due to the unspecific direct binding of protein to the testing chip, after liposome charging to the plate (2 µl/min, 300 sec), we ran BSA 0.6 mg/ml for 300 seconds at speed 10 µl/min to cover the uncoated chip.

We have tested WT PDZ1, WT PDZ1hp and WT NP1 with liposomes of different compositions. We found that WT PDZ1 has a very weak interaction with liposomes, whatever their composition; WT PDZ1hp displays various affinities for liposomes depending on their content of anionic lipids (Fig. 12); weak binding is observed with neutral or weak charged liposomes, while the affinity increases with the proportion of negative charges exposed at the surface of the liposomes. An affinity of 193 +/-17 µM was calculated with POPC:POPE:PIP2 (65:25:10) liposomes. Unfortunately, we observed systematic unspecific binding of WT NP1 to the chip, preventing the estimation of affinity for this construct.

the left is supposed HHD1 and the domain on the right is PDZ1hp. D. Phyre2 model fitting in the density in three different views, where the HHD1 is supposed to form the homo-tetramer.

In parallel, we addressed the question if the full-length Whirlin also adopts a tetrameric organisation in solution. To this end, I have subcloned the full-length Whirlin (Q80VW5-3) into the expression plasmid pET20b for expression tests in E. coli. After several assays, by using overnight expression at 18°C under IPTG induction at low concentration, I managed to increase the yield of expression to around 1.4mg per litre of LB media. We collected negatively stained images of FL-Whirlin as described before for NP1 (Collaboration with Pr Catherine Vénien-Bryan). The analysis of the particles is underway and SAXS analysis is planned. We demonstrated the capacity of the N-terminal region of Whirlin to interact in vitro with membrane models and with the cytoplasmic domain of the membrane protein ADGRV1 in the Usher2 complex. We aim at identifying the sequences elements responsible for membrane targeting in a cellular environment. Thus, we analysed the cellular sub-localization of Whirlin (full-length and fragments) by classical fluorescence microscope analysis by following the GFP tagged target proteins.

In order to decipher the in cellulo expression and localization of Whirlin (GenBank: AY739114.1), but also of Harmonin (GenBank: AF228925.1), and PDZD7 (NCBI: NM_001195265.1), we synthesised all the three proteins full-length genes. For the N-terminal region of Whirlin, we designed several N-terminal sequence for subclonings (Table . 3), including truncated forms (HHD1 deletion for example), triple substitutions (cationic patch), etc. The incubation temperature, the incubation time, the cell density and the amount of transfection DNA were the four parameters to be optimized at the beginning. By Western Blotting, we checked the yield of expression of the full-length constructs of Harmonin, Whirlin and PDZD7; only Whirlin has a good expression in 24h incubation, at 37°C with 2 µg/50,000 cells in a well of 24-well plate. First, the GFP tag has been subcloned into the pMT3 vector. Second, all subcloning of Whirlin constructs were done into this pMT3-GFP vector (Table . 3).

my work, I co-crystallized and solved the structure of the PDZ1 and ADGRV1 PBM complex, and we confirmed the canonical PDZ-PBM interaction, but how does the β-hairpin and the PDZ2 domain stabilized the interaction is still a question to be answered. Our main hypothesis is that these domains could decrease the entropic cost of binding by modulating the dynamics of the PDZ1 domain.

We conclude that the N-terminal Whirlin is mainly involved in the scaffolding of USH2 proteins at the auditory hair cell ankle links as well as both USH2 and USH1 complexes in the photoreceptor cells, mediated by PDZ-PBM mediated interactions. So far, it is still not clear how does the triple domain functions as a whole in the scaffolding complexes, and if the PDZ2 only stabilizes the binding of PDZ1 interactions or is also an anchoring point for Whirlin partners. Assays to have access to the high-resolution structure of the whole Nterminal region are underway.

-Lipid interaction

As described before, the PDZ1 belongs to the family of the lipid binding PDZ domains due to its large cationic patch [START_REF] Ivarsson | Prevalence , Specificity and Determinants of Lipid-Interacting PDZ Domains from an In-Cell Screen and In Vitro Binding Experiments[END_REF]. We deeply characterised this lipid binding properties of PDZ1 through various biophysical approaches in vitro. The PDZ1 domain showed a preference for anionic lipids interaction like PIP (Phosphatidylinositol phosphates) lipids. Any alteration (single to triple substitutions) of the cationic patch exposed at the surface of the PDZ1 disrupts this interaction. We showed that the PDZ1 domain is capable to bind to isolated lipids, liposomes but also lipid monolayers, the interaction being strictly dependent on the presence of anionic lipids and of the cationic patch. As expected, the lipid/PDZ1 affinity we measured increased with the complexity of the lipid environment we have tested. We confirmed the interaction of PIP head groups (IP3) with the cationic patch of PDZ1 by NMR, and we constructed an NMR constraints based structural model showing that the lipid binding site does not overlap with the peptide binding site. However, we detected a potential interplay between lipid and peptide bindings by measuring the affinities of the PDZ1 for these molecules. However, we did not unambiguously identified membrane association of different Whirlin constructs in cell experiments. We proposed that the local PIP2 concentration in the hair cells could be higher than the one in laboratory cell lines (HEK-293 and HeLa), and the interactions with other stereocilia proteins or self-associations of Whirlin could also participate to its membrane association.

-Self-association

The self-association of Whirlin has been mentioned involving different regions of the protein, with the PDZ1, PDZ2 and PDZ3 being all able to form homodimers, but also PDZ1-PDZ2 heterodimers. However, these auto-association capacities of Whirlin have been observed by Co-IP and pull down assay, approaches well-known to produce aspecific bindings [137][162]. These conclusions are not in agreement with my experimental results obtained in vitro with quantitative biophysical methods. First, PDZ1, PDZ2 and PDZ3 are all monomers when purified as single domains, as monitored by using size-exclusion chromatography, NMR and AUC (analytical ultracentrifugation), and is independent of the protein concentrations we used. Second, the selfassociation only involves the HHD1 domain. We systematically observed auto-association process in all Whirlin constructs encompassing HHD1. This self-association is a concentration dependent multi-oligomer state. The HHD1 domain shows a poor solubility after purification, which was the main problem to solve. Unfortunately, we are unable to identify a hydrophobic patch exposed at the surface that could explain the instability of the domain. We focused on a construct (NP1) containing the HHD1-PDZ1 tandem, which is more soluble, but shows heterogeneous self-association. I tested many amphipathic molecules by chromatographic assays, and I finally used amphipol A8-35 to stabilize the NP1 construct and investigated its molecular organization by negative staining electron microscopy and SAXS experiments. Altogether, our results showed that NP1 mainly adopts a tetrameric auto-associated state through its HHD1 domain. Whirlin participates to large protein density formation at upper tip-link densities and at the tip of stereocilia, and potentially also at the ankle links. Knowing the high concentrations of Usher proteins reached in these confined areas, we assume that Whirlin itself or with other partners can form large protein complex or phase separation in stereocilia.

The Whirlin C-terminal region role as an important multi-protein binder in stereocilia

So far, in absence of identified HHD2 binder, two regions in the C-terminal of Whirlin have been suggested to be part of its PPI network, the Proline rich and the very C-terminal PDZ3 domain. The Proline rich has been reported in interaction with GPSM2 to form a complex to be shipped to the tip of stereocilia [190][189]. And by our work, we confirmed that the Proline rich adopts an unfolded and elongated shape, which could have a role as a protein function separator between N-and C-terminal parts of Whirlin. As mentioned before, the C-terminal isoform participates to the growth of stereocilia and remains at the tip of the tallest row. As PDZ3 is the only identified PPI domain in the C-Whirlin isoform, it attracted researchers attention for a long time. Whirlin co-localizes and interacts with proteins at the tip locations via PDZ3 monitored interaction, like Myosin 15a, CASK and MPP1 [START_REF] Belyantseva | Myosin-XVa is required for tip localization of whirlin and differential elongation of hair-cell stereocilia[END_REF][181] [START_REF] Mburu | Whirlin complexes with p55 at the stereocilia tip during hair cell development[END_REF]. In our experiments, we used quantitative method (hold-up and SPR assays) to screen all the human PDZ domains against USH proteins C-terminal PBM, and we found some unexpected interactions that have never been reported before. For instance, we found the interactions between Whirlin PDZ3 with PBMs of Protocadherin 15-CD3, Cadherin 23 and Harmonin, USH1 proteins that are not at the same location than Whirlin in stereocilia. At the ankle links, the function of the PDZ3 is still unclear, and no partner has been reported before. With the help of our collaborators in Uppsala University, we found a new partner, Taperin, able to interact with the PDZ3 as observed by phage display. Knowing the localization of Taperin at the bottom of stereocilia, the role of this deafness protein could be related to the Usher 2 complex, as found for PDZD7 classified as a product of a modifier gene of the Usher 2 phenotype.

Noting this capacity of PDZ3 domain to recognize various partners, we addressed the determinants of specifity for this domain recognition. We chose four proteins: Myosin 15a, CASK, Harmonin and Taperin, which display quite different binding sequences and also affinity values ranging from few micromolars to more than hundreds micromolars. We solved four crystal structures at high resolution between Whirlin PDZ3 and the four PBMs. We highlighted the ability of the PDZ3 to bind to both canonical C-terminal PBMs (Myosin 15a and Harmonin) and non-canonical internal PBMs (CASK and Taperin). All the holo-PDZ3 of four complexes showed only small structural variation of the backbone compare to apo PDZ3, with RMSDs ranging from 0.917 to 1.032 Å. Despite the huge number of PBM-PDZ structures deposited in the protein data bank, this ability of a PDZ domain to bind both canonical and non-canonical PBMs has been rarely described. It illustrated first that the number of non-canonical PBMs is most likely under-estimated, and second, how the PDZ3 can act as a multi protein binder. The CASK and MPP1 are presumably containing a PBM at their C-termini and they share the same last 10 residues, so we predicted the exact same binding pattern for CASK and MPP1 to the PDZ3 of Whirlin.

  years. Especially Baptiste, who I trouble the most, I am very grateful. I thank our unit LRMN and RCN, especially Florence Cordier, who help me almost from beginning to the very end for all the NMR experiments, Marie Prevost and Nathalie Barilone, who guided me for my four-month work in their lab in eukaryotic experiments and immune-fluorescence microscope imaging. I thank the unit leaders Muriel Delepirre and Pierre-jean Corringer for their support and guide. I still own Muriel a pot of Dendrobium Nobile. I thank the help from Christine Petit's lab, especially Sylvie Nouaille for the help of cloning at the beginning of my PhD. I thank the members of my jury: Pr Lu-Yun-Lian, Dr Matthieu Sainlos, Dr Aziz el-Amraoui, Dr Françoise Ochsenbein and Pr Catherine Vénien-Bryan, who accepted my invitation and dedicated their time to correct my thesis and come to Institut Pasteur for my defense. I thank European Commission-Marie Skłodowska-Curie Actions fellowship, PDZnet programme, grant number: 675341, which supported me for this three-year PhD in Institut Pasteur. I am grateful to the chance of secondments I experienced in NZYtech and Sapienza University of Rome, where I really learnt a lot. I thank my collaborators, Susanne Lüchow and Ylva Ivarsson from Uppsala University, Dania Zuniga and Catherine Vénien-Bryan from Sorbonne University, Candice Gautier, Stefano Gianni from Sapienza University of Rome, Alexis Voegele and Alexandre Chenal from Institut Pasteur. I really appreciated to the members in the biophysical platform and crystallography platform: Betrand Raynal, Sébastien Brûlé, Patrick England, Bruno Baron, Ahmed Haouz, Ariel Mechaly, Patrick Weber, Frederick Saul and Cédric Pissis. They helped me for many many times in experiments and taught me a lot.

Figure 24 :

 24 Figure 24: Whirlin modular orgnization and identified mutations and related genetic hearing loss. HNL: Harmonin N-like domain (HHD domain). PDZ: PSD-95, Dlg1, ZO-1 domain. PR: Proline rich. PBM: PDZ Binding Motif. USH2D: Usher syndrome 2D. DFNB31: Nonsyndromic hearing loss 31. Adapted from [94].

Figure 34 :

 34 Figure 34: PDZ-containing proteins in the tight junctions and polarity post synaptic density formation. A: The core protein complexes in the tight junction, which shows the transmembrane linkers, mainly bind to ZO, PAR-6/PAR-3/aPKC, and Crumbs/PATJ/PALS1 complexes. Adapted from[299]B: A schematic view of PSD-95 and SynGAP induced autoassociation, which leads to liquid-liquid phase separation and may form the post-synaptic synapse. Adapted from [300].

My

  PhD work has been mainly conducted in the Institut Pasteur in Paris, as an MSCA-ITN (Marie Skłodowska-Curie Actions Innovative Training Networks) funded PhD student in the programme PDZnet (pdznet.eu). The PDZnet network is composed of 11 academic research groups and three industrial partners across 6 European countries. As part of this consortium, I must go through one or two secondments in other European countries. I had my first secondment in the company of biotechnology NZYtech located in Lisbon for 1.5 months in 2018 and my second secondment in the Sapienza University in Rome for 1 month in 2019. All the works in both secondments were designed to facilitate my PhD project. With the help of high-throughput robot technology developed in NZYtech, I managed to subclone 34 constructs of USH and partners proteins into three different tags prokaryotic expression vectors during my first secondment. My second secondment was done in another PDZnet host lab headed by professor Stefano Gianni, where I used stopped-flow fluorescence spectrometer to characterize the interaction between protein and liposome.

Figure S5 :

 S5 Figure S5: Backbone dynamic parameters extracted from the 15 N relaxation data using the model-free formalism of Lipari-Szabo with an isotropic global reorientation model of apo PDZ3ΔPBM (black) and in complex with Myosin 15a peptide (red).Upper panels;15 N longitudinal (T1), transversal (T2) relaxation times and heteronuclear NOEs. Lower panels; order parameters (S 2 ) and exchange contributions on the µs-ms timescale (K ex )

4. 7 .

 7 In cellulo expression test of Whirlin constructs localization (collaboration with Ms. Nathalie Barilone and Dr. Marie Prevost in our Unit)

Table 2 :

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

Table 1 . Data collection and refinement statistics.

 1

			PDZ3ΔPBM	PDZ3	PDZ3	PDZ3	PDZ3
			Myosin 15a	Myosin 15a	CASK	Taperin	Harmonin a1
	Wavelength		0.9786	0.9786	0.9786	0.9786	0.9786
	Resolution range		34.63 -1.697 (1.758 -1.697)	15.7 -1.93 (1.999 -1.93)	39.18 -1.632 (1.691 -1.632)	29.71 -1.315 (1.362 -1.315)	45.82 -3.169 (3.282 -3.169)
	Space group		P 21 21 2	R 3 :H	P 31 2 1	P 31 2 1	P 21 21 21
			54.14 112.92	85.47 85.47	50.47 50.47	50.45 50.45	76.88 81.03
	Unit cell		34.63 90 90	69.36 90 90	88.41 90 90	89.14 90 90	111.12 90 90
			90	120	120	120	90
	Total reflections	159480 (8120)	101227 (9592)	321248 (26933)	619816 (58538)	35175 (3536)
	Unique reflections		22938 (1615) 14149 (1403)	16757 (1614)	31851 (3089)	10622 (1085)
	Multiplicity		7.0 (5.0)	7.2 (6.8)	19.2 (16.7)	19.5 (19.0)	3.3 (3.3)
	Completeness (%)		94.46 (68.16) 99.38 (97.76)	99.73 (97.86)	99.61 (97.58)	86.25 (88.14)
	Mean I/sigma(I)	15.14 (1.84)	13.01 (1.18) 26.04 (3.07) 33.87 (3.11)	6.20 (0.93)
	Wilson B-factor	23.25	43.30	21.06	18.23	88.26
	R-merge		0.082 (0.8149)	0.09581 (1.71)	0.07435 (0.7445)	0.04225 (0.9054)	0.1794 (1.173)
	R-meas		0.08849 (0.9036)	0.1034 (1.85)	0.07639 (0.7692)	0.04343 (0.9309)	0.213 (1.39)
	R-pim		0.03242 (0.3731)	0.03853 (0.6961)	0.01735 (0.1903)	0.009935 (0.2126)	0.1121 (0.7268)
	CC1/2		0.998 (0.735) 0.998 (0.659)	0.999 (0.945)	1 (0.975) 0.987 (0.373)
	CC*		0.999 (0.92)	1 (0.891)	1 (0.986)	1 (0.994) 0.997 (0.737)
	Reflections used refinement	in	22938 (1614) 14149 (1394)	16757 (1601)	31851 (3065)	10622 (1085)
	Reflections used for R-free	1146 (80)	705 (70)	838 (80)	1587 (153)	532 (55)
	R-work		0.2072 (0.3396)	0.1906 (0.4727)	0.2073 (0.5965)	0.2041 (0.2816)	0.2456 (0.4134)
	R-free		0.2307	0.2162	0.2286	0.2370	0.2770

  ). We used Series S Sensor Chip L1 for Biacore SPR machine. We originally prepared six constructs for the liposome binding test: PDZ1, PDZ1-hairpin and NP1 in their wild-type forms and also the same three constructs sharing the mutation pattern R145E-K213E-K214E, namely MUT PDZ1, MUT PDZ1-hairpin and MUT NP1. SPR experiments (collaboration. Dr. Patrick England, Platform of Biophysics, Institut Pasteur, Paris)

	4.3. N-terminal constructs in lipid binding tests.
	Biophysical experiments have then been performed with liposomes to mimic a more
	physiological membrane environment. We generated several different types of SUV (Small
	Unilamellar Vesicle) liposomes with and without PIP (collaboration with the team of
	Alexandre Chenal, Institut Pasteur,Paris).
	We prepared: POPC:POPE (8:2), POPC:PI (9:1), POPC : PI4P (9:1), POPC:POPE:PI(4,5)P2
	(65:25:10), to test.

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 Régional 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. The authors are grateful to Sylvie Nouaille for providing the initial plasmid encoding for PDZ3 of whirlin. We acknowledge SOLEIL for provision of synchrotron radiation facilities, and we would like to thank the staff of the SWING and PROXIMA beamlines for assistance during the SAXS and X-ray diffractions measurements. We thank Amel Bahloul, Elise Peppermans and Christine Petit (Génétique et Physiologie de l'Audition, Institut Pasteur) for helpful discussions.

Crystallization screening trials were performed at the Crystallography Platform of the Institut Pasteur [START_REF] Weber | High-throughput crystallization pipeline at the crystallography core facility of the institut pasteur[END_REF]. 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 and PROXIMA 2a (SOLEIL synchrotron, St. Auban, France) and processed with XDS [START_REF] Kabsch | XDS[END_REF] and Aimless [START_REF] Evans | How good are my data and what is the resolution?[END_REF].

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 entry 1UFX) as search model with Phaser [START_REF] Mccoy | Phaser crystallographic software[END_REF]. Final models of the complexes were obtained through interactive cycles of manual model building with Coot [START_REF] Emsley | Coot: Model-building tools for molecular graphics[END_REF] and reciprocal space refinement with Buster [START_REF] Bricogne | BUSTER version 2.11.1[END_REF] and phenix.refine [START_REF] Afonine | Towards automated crystallographic structure refinement with phenix.refine[END_REF]. X-ray diffraction data collection and model refinement statistics are summarized in Table 1. Figures showing the crystallographic models were generated with Pymol (Schrodinger, LLC).

Supplementary figures