Structure of a force-conveying cadherin bond essential for inner-ear mechanotransduction

Abstract

Hearing and balance use hair cells in the inner ear to transform mechanical stimuli into electrical signals1. Mechanical force from sound waves or head movements is conveyed to hair-cell transduction channels by tip links2,3, fine filaments formed by two atypical cadherins: protocadherin-15 and cadherin-234,5. These two proteins are products of deafness genes6–10 and feature long extracellular domains that interact tip-to-tip5,11 in a Ca2+-dependent manner. However, the molecular architecture of the complex is unknown. Here we combine crystallography, molecular dynamics simulations, and binding experiments to characterize the cadherin-23 and protocadherin-15 bond. We find a unique cadherin interaction mechanism, with the two most N-terminal cadherin repeats (EC1+2) of each protein interacting to form an overlapped, antiparallel heterodimer. Simulations predict that this tip-link bond is mechanically strong enough to resist forces in hair cells. In addition, the complex becomes unstable upon Ca2+ removal due to increased flexure of Ca2+-free cadherin repeats. Finally, we use structures and biochemical measurements to understand molecular mechanisms by which deafness mutations disrupt tip-link function. Overall, our results shed light on the molecular mechanics of hair-cell sensory transduction and on new interaction mechanisms for cadherins, a large protein family implicated in tissue and organ morphogenesis12,13, neural connectivity14, and cancer15.

Usher proteins in inner ear structure and function

“…Based on immunohistochemical studies cadherin 23 and protocadherin 15, two known USH proteins, are localized to the upper and lower parts of tip links, respectively (1,21). Crystallographic structural evaluation revealed that the first and second ectodomains of cadherin 23 and protocadherin 15 interact with each other in a "handshake" mode to form the tip-link filaments (31). The current hypothesis is that the mature tip links are formed by protocadherin 15 and cadherin 23 (21), although during regeneration the transient but functional tip links are made of two protocadherin 15 isoforms (18).…”

Structure and Function of Cadherin Extracellular Regions

“…This structure, which is required for hearing, is composed of a complex between the very large proteins cadherin-23 (27 EC domains) and protocadherin-15 (11 EC domains) (Ahmed et al 2006;Kazmierczak et al 2007;Elledge et al 2010;Sotomayor et al 2010). There is binding at the tips between these two large cadherins that extend from adjacent stereocilia (Sotomayor et al 2012), however, the cable-like structure they produce is tuned to sensing vibration and transmitting it to the hair cells via associated ion channels to produce neural representations of sound (Kazmierczak et al 2007). Similar proteins are found in other sensory systems as well, but their functions remain unknown (Seiler et al 2005).…”

Cadherins in the Auditory Sensory Organ

“…13.4). Crystallographic studies have shown that the two aminoterminal cadherin repeats (extracellular cadherin (EC) repeats 1 and 2) of protocadherin-15 and cadherin-23 interact in an 'extended handshake' manner to form an overlapping antiparallel heterodimer (Sotomayor et al 2012). Molecular dynamics simulations and binding experiments have indicated that the bond formed by protocadherin-15 and cadherin-23 is mechanically strong enough to withstand the forces applied to the tip-link during hair bundle deflection (Sotomayor et al 2012).…”

“…Crystallographic studies have shown that the two aminoterminal cadherin repeats (extracellular cadherin (EC) repeats 1 and 2) of protocadherin-15 and cadherin-23 interact in an 'extended handshake' manner to form an overlapping antiparallel heterodimer (Sotomayor et al 2012). Molecular dynamics simulations and binding experiments have indicated that the bond formed by protocadherin-15 and cadherin-23 is mechanically strong enough to withstand the forces applied to the tip-link during hair bundle deflection (Sotomayor et al 2012). The three-dimensional (3D) structures of the cadherin-23 and protocadherin-15 EC1 domains are similar to those of other cadherins (three Ca 2þ -binding sites (1, 2, and 3) at the linker between EC1 and EC2), but with several unusual features, such as an elongated N-terminus stabilised at the tip by Ca 2þ -binding site 0 (Elledge et al 2010;Sotomayor et al 2010 , about 20-40 μM, in the endolymph (Bosher and Warren 1978).…”