Structural insights into calcium-dependent CIB2-TMC1 interaction in hair cell mechanotransduction.

Calcium- and integrin-binding protein 2 (CIB2) plays a crucial role in mechanoelectrical transduction (MET) in cochlear hair cells, particularly in modulating the function and localization of the core components of MET channels TMC1/2. CIB2, along with its homolog CIB3, interacts with TMC1/2 through two distinct sites. Here, our study unveils CIB2/3's role as a calcium sensor in its interaction with TMC1. Utilizing X-ray crystallography, we elucidate the high-resolution structure of the mammalian CIB2-TMC1 complex. Structural analyses reveal that cation-bound CIB2 forms a negatively charged surface that aligns with a positively charged surface on the TMC1 N-terminus. Moreover, our data suggest that Ca²⁺ modulates CIB2's interaction with both the N-terminal domain and the loop 1 region of TMC1, and that Ca²⁺-bound CIB2 is capable of simultaneously binding to both regions of TMC1. Critically, we examine pathogenic variants of CIB2 associated with hearing loss, discovering that these variants have differential impacts on CIB2's interactions with TMC1's dual binding sites, displaying diminished calcium-binding affinities for several of these CIB2 mutations. These findings provide a deeper understanding of the molecular mechanisms underlying CIB2 function and its implications in hearing loss, offering potential avenues for therapeutic interventions in deafness.