Mechanistic basis for calmodulin‑dependent allosteric activation of the neuropathy-associated formin INF2

Calcium signaling regulates the atypical formin INF2 to drive actin assembly in diverse cellular processes, yet the underlying molecular mechanism remains elusive. Here, we uncover a direct, nanomolar-affinity interaction between calcium-bound calmodulin (Ca²⁺-CaM) and the diaphanous inhibitory domain (DID) of INF2. The high-resolution crystal structure of the Ca²⁺-CaM-INF2 DID complex reveals a unique allosteric activation mechanism distinct from canonical Rho GTPase-mediated formin regulation. Ca²⁺-CaM binding induces conformational changes that disrupt the autoinhibitory DID-DAD interaction, triggering its actin assembly activity. We show that Ca²⁺-CaM activates ER-bound INF2, thereby promoting mitochondrial fission. We further demonstrate that a Charcot-Marie-Tooth neuropathy-associated INF2 mutation enhances Ca²⁺-CaM binding through optimized interfacial dynamics, revealing a gain-of-function disease mechanism. Our findings provide a mechanistic framework for Ca²⁺-CaM-dependent activation of INF2, establishing the CaM-INF2 axis as a direct activator of actin-dependent organelle dynamics, with implications for INF2-linked pathologies.