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Hair cells in the inner ear convert the mechanical forces from sound into electrical signals that the brain can assimilate through a process known as mechanotransduction. Long protein filaments called tip links are essential components of this process. When hair cells are stimulated by mechanical sound waves, tip links found on the apical end of these hair cells get stretched and convey force to directly trigger the opening of an ion channel producing the required electrical signal. Defects in tip links cause varying degrees of hearing loss including profound deafness. The tip link is formed by two non-classical cadherins, cadherin-23 (CDH23) and protocadherin-15 (PCDH15), which interact through their N-terminal domains in a calcium-dependent manner. Their N-terminal domains are made of multiple extracellular cadherin (EC) repeats that are ~100 amino acids long. CDH23 has 27 of such repeats making up nearly 3/4th of the tip link. Each pair of EC repeats coordinate three calcium ions between them through highly conserved calcium-binding motif residues (N-xEx-DxD-DxE-xDx-DxNDN-C) ubiquitous in all cadherins. These bound calcium ions dictate the mechanics of EC repeats and are critical for tip-link function. It is therefore not surprising that these calcium-binding residues are often targets of mutation in inherited deafness. There are ~116 missense mutations in the CDH23 extracellular domain that cause deafness. The purpose of our study is to a) elucidate the atomic structure of the complete CDH23 ectodomain through X-ray crystallography, b) to create a comprehensive map of deafness mutations and analyze their functional effects on CDH23, and c) to study the mechanical properties of CDH23 EC repeats using molecular dynamics simulations. We used X-ray crystallography to solve the structures of several CDH23 fragments that show 18 of the 27 EC repeats of CDH23, as well as of two deafness-causing variants of EC19-21 that occur at calcium-binding motif residues in the EC19-20 linker. Thermal stability assays were used to measure altered protein folding stability due to deafness related mutations and to assess changes in calcium-dependent stability brought upon by mutations present at conserved calcium-binding motif residues. Finally, the nanomechanics of CDH23 was predicted through all-atom molecular dynamics simulations. These results taken together reveal determinants of CDH23 function in hearing and possible molecular mechanisms of inherited deafness due to missense mutations. |
