A Partial Calcium-Free Linker Confers Flexibility to Inner-Ear Protocadherin-15

Tip links of the inner ear are protein filaments essential for hearing and balance. Two atypical cadherins, cadherin-23 and protocadherin-15, interact in a Ca²⁺-dependent manner to form tip links. The largely unknown structure and mechanics of these proteins are integral to understanding how tip links pull on ion channels to initiate sensory perception. Protocadherin-15 has 11 extracellular cadherin (EC) repeats. Its EC3-4 linker lacks several of the canonical Ca²⁺-binding residues, and contains an aspartate-to-alanine polymorphism (D414A) under positive selection in East Asian populations. We present structures of protocadherin-15 EC3-5 featuring two Ca²⁺-binding linker regions: canonical EC4-5 linker binding three Ca²⁺ ions, and non-canonical EC3-4 linker binding only two Ca²⁺ ions. Our structures and biochemical assays reveal little difference between the D414 and D414A variants. Simulations predict that the partial Ca²⁺-free EC3-4 linker exhibits increased flexural flexibility without compromised mechanical strength, providing insight into the dynamics of tip links and other atypical cadherins.