Structural basis for alpha-actinin inhibition of cooperative cofilin binding to actin filaments

Abstract

Actin is a conserved cytoskeletal protein essential for morphogenesis, motility, and division. Its versatility arises from filament assembly and regulation by actin binding proteins. Among these, alpha-actinin organizes filaments into bipolar or unipolar networks, whereas cofilin binds preferentially to ADP-actin regions and forms clusters to shorten the half helical pitch (HHP). Here, we investigated the molecular mechanism of how alpha-actinin alters filament and protomer conformations and influences cofilin binding. Using all-atom molecular dynamics simulations, principal component analysis, and high-speed atomic force microscopy, we show that alpha-actinin crosslinking stabilizes actin filaments in the canonical helical state, thereby preventing the cofilin-induced helical shortening required for cooperative filament decoration. Stabilization occurs without significant changes in protomer twist and rise and subdomain geometry and maintains a flattened protomer conformation that restricts twisting needed for cofilin cooperative binding. By contrast, the isolated alpha-actinin-1 actin binding domain mutant (ABD-E235K), comprising two calponin homology domains (CH1-CH2), transiently binds to actin filaments and induces local transitions from the canonical double-helical filament to the single- and parallel-helical protofilament states, weakly affecting cofilin binding and cluster formation through a distinct structural and binding mechanism. Together, these findings reveal a mechanistically distinct function of full-length alpha-actinin and its isolated ABD and support a stepwise mechanism in which cooperative cofilin binding to double-helical actin filaments requires initial binding to actin regions with shortened HHP, followed by protomer twisting and further helical shortening. By stabilizing the canonical filament architecture or perturbing filament organization, alpha-actinin suppresses these structural transitions and thereby perturbs cofilin cooperativity. TeaserAlpha-actinin stabilizes actin helices, suppressing cofilin cooperative binding through distinct structural mechanisms.