Tip link filaments convey force and gate inner-ear hair-cell transduction channels to mediate perception of sound and head movements. Cadherin-23 and protocadherin-15 form tip links through a calcium-dependent interaction of their extracellular domains made of multiple extracellular cadherin (EC) repeats. These repeats are structurally similar, but not identical in sequence, often featuring linkers with conserved calcium-binding sites that confer mechanical strength to them. Here we present the X-ray crystal structures of human protocadherin-15 EC8–EC10 and mouse EC9–EC10, which show an EC8–9 canonical-like calcium-binding linker, and an EC9–10 calcium-free linker that alters the linear arrangement of EC repeats. Molecular dynamics simulations and small-angle X-ray scattering experiments support this non-linear conformation. Simulations also suggest that unbending of EC9–10 confers some elasticity to otherwise rigid tip links. The new structure provides a first view of protocadherin-15's non-canonical EC linkers and suggests how they may function in inner-ear mechanotransduction, with implications for other cadherins.

The vertebrate inner ear, responsible for hearing and balance, is able to sense minute mechanical stimuli originating from an extraordinarily broad range of sound frequencies and intensities or from head movements. Integral to these processes is the tip-link protein complex, which conveys force to open the inner-ear transduction channels that mediate sensory perception. Protocadherin-15 and cadherin-23, two atypically large cadherins with 11 and 27 extracellular cadherin (EC) repeats, are involved in deafness and balance disorders and assemble as parallel homodimers that interact to form the tip link. Here we report the X-ray crystal structure of a protocadherin-15 + cadherin-23 heterotetrameric complex at 2.9-Å resolution, depicting a parallel homodimer of protocadherin-15 EC1-3 molecules forming an antiparallel complex with two cadherin-23 EC1-2 molecules. In addition, we report structures for 10 protocadherin-15 fragments used to build complete high-resolution models of the monomeric protocadherin-15 ectodomain. Molecular dynamics simulations and validated crystal contacts are used to propose models for the complete extracellular protocadherin-15 parallel homodimer and the tip-link bond. Steered molecular dynamics simulations of these models suggest conditions in which a structurally diverse and multimodal protocadherin-15 ectodomain can act as a stiff or soft gating spring. These results reveal the structural determinants of tip-link–mediated inner-ear sensory perception and elucidate protocadherin-15’s structural and adhesive properties relevant in disease.

156 Words) 2 , an atypical member of the cadherin superfamily, is essential for vertebrate hearing 3 and its dysfunction has been associated with deafness and progressive blindness. The PCDH15 ectodomain, 4 made of eleven extracellular cadherin (EC1-11) repeats and a membrane adjacent domain (MAD12), assembles 5 as a parallel homodimer that interacts with cadherin-23 (CDH23) to form the tip link, a fine filament necessary 6 for inner-ear mechanotransduction. Here we report X-ray crystal structures of a PCDH15 + CDH23 7 heterotetrameric complex and ten PCDH15 fragments that were used to build complete high-resolution models 8 of the monomeric PCDH15 ectodomain. Using molecular dynamics (MD) simulations and validated crystal 9 contacts we propose models for complete PCDH15 parallel homodimers and the tip-link bond. Steered MD 10 simulations of these models predict their strength and suggest conditions in which a multimodal PCDH15 11 ectodomain can act as a stiff or soft gating spring. These results provide a detailed view of the first molecular 12 steps in inner-ear sensory transduction.

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