Mechanism of Phosphate Release from Actin Filaments

Abstract

AbstractAfter ATP-actin monomers assemble filaments, the γ-phosphate is hydrolyzed from ATP within seconds and dissociates from the filament over several minutes. We used all-atom well-tempered metadynamics molecular dynamics simulations to sample the release of phosphate from filaments along with unbiased molecular dynamics simulations to study residues that gate release. Dissociation of phosphate from Mg2+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. These occluded states have not been documented in cryo-EM reconstructions.Significance StatementThe protein actin assembles into filaments that participate in muscle contraction and other cellular movements. An ATP bound to the actin monomer is hydrolyzed rapidly during filament assembly, but the γ-phosphate dissociates very slowly from the filament. We identified the dissociation of phosphate from Mg2+as the rate-limiting step in phosphate release from actin based on an energy barrier that aligns with the experimentally determined release rate. Release of phosphate from the protein requires opening a gate in the actin molecule formed by interaction of the sidechains of arginine 177 and asparagine 111. Surprisingly, the 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 the low temperature cryo-EM structures.