Structure and Stability of Tip Link Cadherin-23 Fragments Involved in Hearing and Deafness

Abstract

Tip links, core components of vertebrate hearing, are thought to directly pull on mechanotransduction channels to transform sound into electrical signals. Cadherin-23 (cdh23) and protocadherin-15 (pcdh15), the two proteins that form the tip link, belong to the cadherin superfamily of calcium-dependent adhesion molecules that feature extracellular cadherin (EC) repeats. These EC repeats are ∼ 100 residues long and are similar but not identical to each other in terms of fold and sequence. Cdh23 features twenty-seven EC repeats and pcdh15 eleven, with calcium-binding sites located at the linkers between them. Calcium and highly conserved calcium-binding residues are crucial for the elasticity and function of the tip link. These same residues are often mutated in inherited deafness. Here, we present the first crystal structures of cdh23 EC19-21, the least conserved cdh23 fragment among different species, in wild type form and carrying disease-related mutations (R2029W and D2148N). In addition, we use thermal melting and calcium-binding assays to characterize various wild type and mutant cdh23 fragments, including cdh23 EC12-13, EC13-15, EC19-21, and EC21-23. These experiments show how deafness mutations and natural sequence variations throughout the enormous cdh23 extracellular domain affect folding, affinity for calcium, stability, and function. Overall, our structures and biophysical studies provide insight into cdh23 and tip link function in mechanosensation.