An elastic element in the protocadherin-15 tip link of the inner ear

Abstract

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.

Mechanotransduction by PCDH15 Relies on a Novel cis-Dimeric Architecture

“…It is still unclear whether the tip link contributes to the gating spring, or if the gating spring is provided by one or more molecules connected in series to the tip link. Earlier structural studies of Cdh23 and Pcdh15 have focused on the question of tertiary structural elasticity – whether force applied to Pcdh15 will result in protein extension without unfolding (Araya-Secchi et al, 2016; Powers et al, 2017; Sotomayor et al, 2010, 2012). In these studies, Sotomayor and coworkers have reported crystal structures for monomeric EC domain pairs or triples produced in E. coli , and computationally assessed the spring properties of these cis -monomeric ectodomain fragments using steered molecular dynamics.…”

“…In silico simulation of monomeric protein fragments encompassing the EC1 and EC2 domains of Cdh23 was reported to suggest that tip links might be too stiff to function as the gating spring, a hypothesis that was initially based on the observed double-stranded nature of the tip link (Kachar et al, 2000; Sotomayor et al, 2010). However, subsequent studies suggested that only some regions of the tip link might be too stiff to act as the gating spring, whereas other regions could potentially elongate significantly in response to force (Araya-Secchi et al, 2016; Powers et al, 2017; Sotomayor et al, 2012). These conclusions were based on crystal structures of Pcdh15 ectodomain fragments EC1-2, EC3-5, EC8-10, and EC9-10 (Araya-Secchi et al, 2016; Powers et al, 2017; Sotomayor et al, 2010, 2012).…”

“…However, subsequent studies suggested that only some regions of the tip link might be too stiff to act as the gating spring, whereas other regions could potentially elongate significantly in response to force (Araya-Secchi et al, 2016; Powers et al, 2017; Sotomayor et al, 2012). These conclusions were based on crystal structures of Pcdh15 ectodomain fragments EC1-2, EC3-5, EC8-10, and EC9-10 (Araya-Secchi et al, 2016; Powers et al, 2017; Sotomayor et al, 2010, 2012). Steered molecular dynamics simulations were used to generate force extension curves, which suggested that each of these fragments were too stiff to extend significantly under forces of the magnitude experienced by the tip link.…”

“…Thus, lateral homodimerization, helix formation, and a novel trans adhesive dimer interface are specializations of Pcdh15 and Cdh23 that likely evolved for their critical function in mechanotransduction. Crystallographic studies have provided first insights into the structure of monomeric fragments of Pcdh15 (Araya-Secchi et al, 2016; Powers et al, 2017; Sotomayor et al, 2010, 2012), but we know little about the mechanisms that drive dimerization and helix formation, or that constitute the adhesive interface in the trans tetramer.…”

“…While previous structural studies of Pcdh15 utilized fragments expressed in bacteria, most of which appeared monomeric (Araya-Secchi et al, 2016; Powers et al, 2017; Sotomayor et al, 2012), we focused on proteins expressed in eukaryotic cells thus containing modifications such as glycosylations that might affect protein function. We identified two distinct interfaces that mediate homodimerization in Pcdh15: one near the trans -adhesive interface that is contained within domains EC2 and EC3, and the other adjacent to the membrane mediated by the non-cadherin PICA domain.…”