Rotation-Direction-Dependent Mechanism of the Inhibitor Protein IF for Mitochondrial ATP Synthase from Atomistic Simulations.

ATPase inhibitory factor 1 (IF) is an endogenous regulatory protein for mitochondrial FF-ATP synthase. It blocks the catalysis and rotation of the F part by deeply inserting itself into the rotor-stator interface. Recent single-molecule manipulation experiments have elucidated that forcible rotations only in the ATP-synthesis direction eject IF, rescuing F from the IF-inhibited state. However, the molecular mechanism of the rotation-direction-dependent process at an atomic resolution is still elusive. Here, we have performed all-atom molecular dynamics (MD) simulations of the IF-bound F structure with a torque applied to the rotor γ subunit. In the torque-applying simulations, we first found that the core part of the γ subunit rotated more in response to an external torque in the synthesis direction than in the hydrolysis direction. Further rotations of the γ subunit up to 120° revealed that the conformational change of the IF-bound αβ was only allowed in the synthesis direction. Also, the 120° rotation in the synthesis direction disrupted its contacts with IF, destabilizing the short helix of IF. After additional rotation up to the synthetic 240° state, the closed-to-open conformational change of the IF-bound β subunit pulled IF outwardly, deforming the long helix of IF. These stepwise destabilizations of the IF helices should be crucial for IF ejection. Our simulations also provided insight into the nullification mechanism of the hydrolytic rotation, highlighting the steric clash between F22 of IF and the β subunit. Finally, we discuss a sufficient proton motive force to rescue FF-ATP synthase from the IF-inhibited state.