Angle-gated Na⁺ binding and release in the VO motor revealed by all-atom simulations

Rotary ATPases are ubiquitous molecular motors essential for cellular ion homeostasis and energy balance. Because almost all structurally characterized rotary ATPases are proton pumps, the transported ion is difficult to visualize in structures and proton motion is challenging to capture with classical molecular dynamics (MD) simulations, leaving many mechanistic details unresolved. This limitation is addressed by our previously determined 2.2 Å cryo-EM structure of the Na⁺-transporting V _O part from Enterococcus hirae V-ATPase, in which ion transport can be tracked directly in simulations. Using this structure, we performed all-atom MD to probe the coupling between c-ring rotation and Na⁺ transport. By applying rotational torque to mimic a 36° c-ring step, we captured Na⁺ binding and release at about the same rotation angle (~ 27°), indicating an angular gating mechanism. The simulations also showed how hydration changes accompany binding and release, with coordinated dehydration–rehydration along the pathway. Throughout, electrostatic balance among Na⁺, protein residues, and water molecules was maintained, with no evidence of large energy barriers. Together, our findings define dynamic features of rotary ion transport that were beyond reach in proton systems and advance an atomistic, angle-resolved model for ion transport in rotary ATPases.