The distortion-push mechanism for the γ subunit rotation in F₁-ATPase.

Abstract

F₁-ATPase comprises the stator ring consisting of α₃β₃ subunits and the rotor γ subunit. The γ subunit rotation mechanism has been extensively investigated by biochemical analyses, structural studies, single-molecule measurements, and computational studies. Recent cryoelectron microscopy (cryo-EM) structures of F₁-ATPase from the thermophilic bacterium <i>Bacillus</i> PS3 (TF₁) provide us with further possibilities for a better understanding of the γ-rotation mechanisms. Using cryo-EM structures having the γ-rotation angles close to the binding dwell and catalytic dwell states, we investigate the relationships between the γ subunit rotation, conformational changes of the stator α₃β₃ subunits, and the nucleotide-binding and release. We performed targeted molecular dynamics (MD) simulations with external forces on the α₃β₃ subunits and observed 80° substep rotations of the γ subunit. Then, we optimized the most probable transition pathway through the mean-force string method simulations with 64 images. Finally, using umbrella sampling, we calculated the potential of mean forces along the minimum free energy pathway during the 80° substep rotation. Our MD simulations suggest that 80° substep rotation is divided into the first rotation, resting, and the second rotation. Notably, the first rotation is driven by the distortion of the stator α₃β₃ subunits, and the second rotation is induced mainly by direct β/γ subunit interactions. This model, which we call the distortion-push mechanism, is consistent with the residue-level experimental analysis on F₁-ATPase and the atomic structures determined by X-ray crystallography and cryo-EM.