Conformational dynamics of actin filaments crosslinked with alpha-actinin and their roles in suppressing cofilin-induced helical shortening and cluster formation

Actin is a conserved cytoskeletal protein essential for processes such as morphogenesis, motility, and division. Its versatility stems from filament assembly and regulation by actin-binding proteins (ABPs). Among these, alpha-actinin organizes actin filaments into bipolar bundles or unipolar networks, while cofilin preferentially binds to ADP-actin regions, clustering and shortening the half helical pitch (HHP). How alpha-actinin alters or stabilizes filament and protomer conformations and affects cofilin binding remains unsolved. Using high-speed AFM, all-atom MD simulations, and principal component analysis (PCA), we show that alpha-actinin stabilizes actin filaments in the canonical helical state more effectively than bare filaments, thereby preventing cofilin-induced helical shortening required for cooperative binding and clustering. Our results further reveal stabilized local fluctuations without significant changes in protomer spacing or subdomain geometry, maintaining a flattened protomer conformation that restricts the twisting necessary for cofilin cooperativity. We decipher that cofilin binding and clustering proceed through initial attachment to shortened ADP-bound HHP, followed by protomer twisting and further HHP shortening with fewer protomers, leading to enhanced cooperative binding. Conversely, the absence of these steps inhibits cooperative binding. Together, these findings reveal a previously unrecognized atomic mechanism by which alpha-actinin modulates actin filament dynamics and structure-dependent interactions with other ABPs essential for spatiotemporal cytoskeletal organization.