Rotation-Direction-Dependent Mechanism of the Inhibitor Protein IF₁ for Mitochondrial ATP Synthase from Atomistic...

ATPase inhibitory factor 1 (IF₁) is an endogenous regulatory protein for mitochondrial F_oF₁-ATP synthase. It blocks the catalysis and rotation of the F₁ 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 IF₁, rescuing F₁ from the IF₁-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 IF₁-bound F₁ 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 IF₁-bound αβ was only allowed in the synthesis direction. Also, the 120° rotation in the synthesis direction disrupted its contacts with IF₁, destabilizing the short helix of IF₁. After additional rotation up to the synthetic 240° state, the closed-to-open conformational change of the IF₁-bound β subunit pulled IF₁ outwardly, deforming the long helix of IF₁. These stepwise destabilizations of the IF₁ helices should be crucial for IF₁ ejection. Our simulations also provided insight into the nullification mechanism of the hydrolytic rotation, highlighting the steric clash between F22 of IF₁ and the β_TP subunit. Finally, we discuss a sufficient proton motive force to rescue F_oF₁-ATP synthase from the IF₁-inhibited state.