BPS2026 – Molecular insights into calcium-regulated CIB2-TMC1/2 interactions and their implications for inner-ear mechanotransduction

Inner-ear mechano-electrical transduction is mediated by the transducer channels TMC1 and TMC2, whose normal function depends on interactions with calcium and integrin-binding protein 2 (CIB2). While it has been established that CIB2 binds to TMC1/2 via at least two of their cytoplasmic domains, the N-terminal (NT) and intracellular linker-1 (IL1) domains, the mechanisms regulating channel activity remain unclear. Here, we combine isothermal titration calorimetry (ITC) and molecular dynamics (MD) simulations to investigate the molecular basis of CIB2-TMC1/2 interactions and propose potential mechanisms for channel activation. Our ITC experiments demonstrate that CIB2 binds to TMC2-NT with tight nanomolar affinity under calcium-containing buffer conditions that mimic physiological conditions. This interaction is enthalpically driven and aligns with recent findings on CIB2-TMC1 interactions (2025, Li; 2025, Wu). MD simulations of AlphaFold models along with structural data from other groups reveal that CIB2 interactions with TMC1/2-IL1 is predominantly hydrophobic, whereas its binding to TMC1/2-NT involves a combination of hydrophobic interactions, hydrogen bonding, and electrostatic interactions. Furthermore, we reveal changes in CIB2 dynamics depending on calcium binding, suggesting a potential role in regulating inner-ear hair cell adaptation. Together, our findings elucidate the molecular details of CIB2-TMC1/2 interactions and offer new insights into the mechanisms underlying inner-ear mechanotransduction.