Mechanism of Phosphate Release from Actin Filaments

After ATP-actin monomers assemble filaments, the [Formula: see text]-phosphate is hydrolyzed from ATP within seconds and dissociates from the filament over several minutes. We used all-atom 2-dimensional well-tempered metadynamics computer simulations to sample the release of phosphate from filaments with unbiased molecular dynamics simulations to 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 recognized 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 yet been documented in cryo-EM reconstructions. (150 words)