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.

The accurate partitioning of Firmicute plasmid pSM19035 at cell division depends on ATP binding and hydrolysis by homodimeric ATPase δ2 (ParA) and binding of ω2 (ParB) to its cognate parS DNA. The 1.83 Å resolution crystal structure of δ2 in a complex with non-hydrolyzable ATPγS reveals a unique ParA dimer assembly that permits nucleotide exchange without requiring dissociation into monomers. In vitro, δ2 had minimal ATPase activity in the absence of ω2 and parS DNA. However, stoichiometric amounts of ω2 and parS DNA stimulated the δ2 ATPase activity and mediated plasmid pairing, whereas at high (4:1) ω2 : δ2 ratios, stimulation of the ATPase activity was reduced and δ2 polymerized onto DNA. Stimulation of the δ2 ATPase activity and its polymerization on DNA required ability of ω2 to bind parS DNA and its N-terminus. In vivo experiments showed that δ2 alone associated with the nucleoid, and in the presence of ω2 and parS DNA, δ2 oscillated between the nucleoid and the cell poles and formed spiral-like structures. Our studies indicate that the molar ω2 : δ2 ratio regulates the polymerization properties of (δ•ATP•Mg2+)2 on and depolymerization from parS DNA, thereby controlling the temporal and spatial segregation of pSM19035 before cell division.

SUMMARY The hair-cell tip link, a fine filament directly conveying force to mechanosensitive transduction channels, is composed of two proteins, protocadherin-15 and cadherin-23, whose mutation causes deafness. However, their molecular structure, elasticity, and deafness-related structural defects are unknown. We present crystal structures of the first and second extracellular cadherin repeats of cadherin-23. Overall, structures show typical cadherin folds, but reveal an elongated N-terminus that precludes classical cadherin interactions and contributes to an N-terminal Ca2+-binding site. The deafness mutation D101G, in the linker region between the repeats, causes a slight bend between repeats and decreases Ca2+ affinity. Molecular dynamics simulations suggest that cadherin-23 repeats are stiff, and that either removing Ca2+ or mutating Ca2+-binding residues reduces rigidity and unfolding strength. The structures define an uncharacterized cadherin family and, with simulations, suggest mechanisms underlying inherited deafness and how cadherin-23 may bind with itself and with protocadherin-15 to form the tip link.