Gelsolin domains G2G3 recruit other domains and prime the actin filament for severing.

Gelsolin, a Ca2+-dependent actin filament (F-actin) severing protein, plays essential roles in apoptosis, cell motility, and the actin-scavenger system of the bloodstream. It comprises six related domains (G1 to G6) connected by variable linkers (L1 to L5), each contributing distinctly to severing. Individually, neither G1 nor G2G3 severs F-actin, whereas the combined fragment G1-G3 displays potent, Ca2+-independent severing activity, largely mediated by G1, which relies on G2G3 for recruitment to F-actin. The C-terminal fragment G4-G6 neither binds nor severs F-actin but, once recruited via G2G3, contributes to the severing efficiency of full-length gelsolin. However, it remains unclear whether the other domains rely on G2G3 solely for recruitment or also for conformational changes in F-actin that facilitate binding and severing. Here, through a series of cryo-EM structures, we show that G2G3 binds F-actin in a stepwise manner, inducing conformational changes, including a kink in the filament and rotation of a subunit at the binding site, that prime F-actin for binding by the other gelsolin domains and severing. These changes propagate beyond the G2G3 binding site, revealing how F-actin buffers the effects of kinking. Biochemically, substituting G1 with G4 within G1-G3 yields a construct, G4-G3, with substantial severing activity, albeit Ca2+-dependent and lower than that of G1-G3. This result indicates that G4 can substitute for G1 when primed by G2G3, while sequence adaptations make G1 uniquely effective at severing. Collectively, these findings define a dual role of G2G3: recruiting other gelsolin domains and priming F-actin for severing.