BPS2026 – In silico hierarchical and calcium-dependent mechanics of inner-ear tip links under resting tension

Tip links, formed by cadherin-23 (CDH23) and protocadherin-15 (PCDH15), convey mechanical forces to pull open inner-ear mechanotransduction channels. We constructed two full atomic models of the tip-link ectodomain using X-ray structures and AlphaFold prediction, incorporating all extracellular cadherin (EC) repeats and the membrane-adjacent domains (MAD) in monomeric or dimeric form. These models were subjected to all-atom molecular dynamics (MD) simulations to probe calcium-dependent dynamics and mechanics with and without resting tension. In equilibrium simulations, tip-link models fully saturated with calcium adopted conformations seen in electron microscopy images and showed subdomain twisting, hotspots of flexibility, and fluctuations at non-canonical EC linkers. In contrast, reduced calcium systems showed high flexibility at all linker regions. Constant-velocity stretching simulations starting from equilibrium conformations showed biphasic mechanics with soft (∼1 mN/m) and stiff components (up to 30 mN/m) at 0.1 nm/ns in the presence of calcium, while reduced calcium systems yielded significantly softer biphasic phases. Constant-force simulations mimicking resting tension at 10–40 pN showed tip-link extensions of 5–10 nm with effective spring constants of 1.2–2.8 mN/m, and revealed a novel ladder-like dimerization of CDH23 EC25-EC27 and MAD28. Constant-velocity simulations of pre-stretched tip links with 40 pN resting tension at 0.1 nm/ns showed only a stiff elastic response. Overall, stretching trajectories revealed a reproducible hierarchy of mechanical events across systems: (i) straightening and extensions of flexible linkers, (ii) unrolling and unfolding of CDH23 MAD28s, and (iii) subsequent unrolling and unfolding of PCDH15 MAD12s. Our all-atom simulations of the entire tip link ectodomain show the non-linear and calcium-dependent mechanical response of the tip-link ectodomains with and without resting tension, supporting tip links as good candidates for the inner-ear mechanotransduction soft gating spring under low resting tension.