Structural determinants of protocadherin-15 mechanics and function in hearing and balance perception

Abstract

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.

Single-molecule force spectroscopy reveals the dynamic strength of the hair-cell tip-link connection

“…However, further biochemical studies have shown that full-length CDH23 and PCDH15 preferentially form dimers in vitro 10 , both by themselves and when bound together. Interestingly, although these studies show that tip-link filaments form a helical structure, recent molecular dynamics simulations show that helix unwinding of full-length dimeric PCDH15 bound to CDH23 EC1-2 during stretching does not significantly twist the PCDH15 EC1-2 binding domains, likely due to strong cis-dimerization at EC3 52 .…”

“…What can account for this? Recent studies using optical tweezers and molecular dynamics simulations suggest that tip link proteins are far more elastic than initially thought 51,52 . Elasticity would allow unbound domains in the B1 state ( Fig.…”

“…1g and 4c) 11,19 . Molecular dynamics simulations suggest that as the concentration of Ca 2+ bathing the tip link decreases and there is decreased occupancy of Ca 2+ -binding sites, the junctions between each EC-domain become more flexible and the series elasticity of EC domains is increased 52,58 . Because protein elasticity is important in determining the extent of avidity under force (Figs.…”

“…However, the concentration of free tip-link dimer ends is likely much higher thañ 0.3 µM. From a structural point of view, CDH23 and PCDH15 are relatively stiff at endolymphatic Ca 2+ concentration 5,52,58 , are complexed with membrane and intracellular proteins 6,43,86 , and their insertion points appear to be spatially constrained even when broken 14,77,79 . These observations make it likely that free dimer ends occupy a much smaller space than a 210 nm sphere and are consequently at higher than 0.3 µM concentration.…”

Collective Mechanical Responses of Cadherin-Based Adhesive Junctions as Predicted by Simulations

“…Independent virtual springs (stiffness k s = 1 kcal mol -1 Å -2 ) were attached to each C-terminal C α atom, and the free ends of these springs were moved at a constant velocity along the x-axis and away from the protein (tensile mode). Unlike slab schemes (123)(124)(125), forces are not redistributed as bonds rupture. Additional harmonic constraints in the y and z directions were applied to guide stretching along the x-axis only.…”