Mechanism of Phosphate Release from Actin Filaments

Abstract After ATP-actin monomers assemble filaments, the ATP’s γ-phosphate is hydrolyzed within seconds and dissociates over minutes. We used all-atom molecular dynamics simulations to sample the release of phosphate from filaments and study residues that gate release. Dissociation of phosphate from Mg 2+ is rate limiting and associated with an energy barrier of 20 kcal/mol, consistent with experimental rates of phosphate release. Phosphate then diffuses in an internal cavity toward a gate formed by R177 suggested in prior computational studies and cryo-EM structures. The gate is closed when R177 hydrogen bonds with N111 and is open when R177 forms a salt bridge with D179. Most of the time interactions of R177 with other residues occludes the phosphate release pathway. Machine learning analysis reveals that the occluding interactions fluctuate rapidly, underscoring the secondary role of backdoor gate opening in P i release, in contrast with the previous hypothesis that gate opening is the primary event. Significance Statement The protein actin assembles into filaments that participate in muscle contraction and cellular movements. An ATP bound to the actin monomer is hydrolyzed rapidly during filament assembly, but the γ-phosphate dissociates slowly from the filament. We identified phosphate dissociation from Mg 2+ as the rate-limiting step in phosphate release from actin based on an energy barrier that aligns with the experimentally determined release rate. The release of phosphate from the protein requires opening a gate in the actin molecule formed by the interaction between sidechains of arginine 177 and asparagine 111. Surprisingly, simulations revealed other interactions of the sidechain of arginine 177 that occlude the release pathway most of the time but have not been observed in low-temperature cryo-EM structures.