Calcium directs actin assembly via allosteric activation of formin INF2

Abstract Calcium signals spatiotemporally orchestrate cytoskeletal dynamics, typically through Rho GTPase-mediated signaling cascades, yet the direct molecular transducers that convert local calcium transients into spatially controlled actin assembly have remained elusive. Here, we uncover a structure-based mechanism by which calcium-bound calmodulin (Ca2+-CaM) directly activates formin INF2 to drive actin assembly. We demonstrate that Ca2+-CaM binds to the diaphanous inhibitory domain (DID) of INF2 with nanomolar affinity, inducing allosteric conformational changes that sterically disrupt INF2 autoinhibition. The unexpected bipartite Ca2+-CaM binding interface on INF2 enables ultrasensitive decoding of local calcium microdomains, such as ER-mitochondrial contacts, where activated INF2 promotes actin polymerization to facilitate mitochondrial fission. We further show that the Charcot-Marie-Tooth disease–associated INF2 R91G mutation enhances Ca2+-CaM binding via optimized interfacial dynamics, suggesting a gain-of-function disease mechanism. Our work establishes the Ca2+-CaM–INF2 axis as a direct molecular transducer linking spatial calcium signals to actin-dependent organelle dynamics, defining a Rho-GTPase-independent paradigm for calcium–cytoskeleton communication, with broad implications for INF2-linked pathologies. Competing Interest Statement The authors have declared no competing interest. Data availability The atomic coordinates of apo INF2 DID, the INF2 DID_WT–Ca2+-CaM complex and the INF2 DID_R91G–Ca2+-CaM complex have been deposited to the Protein Data Bank under the accession codes 9W3X, 9W46, and 9W4G, respectively. All other data are available from the corresponding author upon reasonable request.