Kindlin-2 is a focal adhesion protein essential for integrin activation and linkage to the actin cytoskeleton, yet the structural basis of its direct interaction with F-actin remains poorly understood. As a member of the FERM-domain family, kindlin-2 contains F0-F3 subdomains that serve as potential interfaces for cytoskeletal and membrane binding. Here, we combined computational docking, molecular dynamics simulations, binding free energy calculations, and coimmunoprecipitation assays to dissect the molecular interfaces of the kindlin-2-actin complex. Our analysis revealed a previously unrecognized actin-binding site within the F3 domain, in addition to the established binding site in the F0 domain. The F3 domain engages actin through extensive electrostatic and hydrophobic contacts, with hydrophobic residues overlapping those that interact with the cytoplasmic tail of integrin β1, suggesting F3 as a shared docking hub for both actin and integrins. Functional validation using domain truncations confirmed the critical role of the F3 domain, while excluding alternative interfaces predicted from docking models. Integrating these findings, we propose a structural model for a dimeric kindlin-2-actin complex, in which one protomer forms a stable F3-actin interface and the other adopts a more flexible conformation with an unbound F0 domain. This asymmetric configuration provides a mechanistic framework for how kindlin-2 simultaneously couples integrins to actin and coordinates recruitment of adhesion-associated proteins. Our study establishes the F3 domain as a central element in kindlin-mediated integrin-actin coupling and highlights its broader significance in adhesion signaling.
Yan et al. (Wed,) studied this question.