Rotation-direction-dependent regulation of ATPase inhibitory factor 1 for mitochondrial ATP synthase from atomistic simulation

Abstract

ATPase inhibitory factor 1 (IF1) is an endogenous regulatory protein for mitochondrial FoF1-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 {gamma} subunit. In the torque-applying simulations, we first found that the core part of the {gamma} subunit rotated more in response to an external torque in the synthesis direction than in the hydrolysis direction. Further rotations of the {gamma} subunit up to 120{degrees} revealed that the conformational change of the IF1-bound {beta} was only allowed in the synthesis direction. Also, the 120{degrees} rotation in the synthesis direction disrupted its contacts with IF1, destabilizing the short helix of IF1. After additional rotation up to the synthetic 240{degrees} state, the closed-to-open conformational change of the IF1-bound {beta} 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 {beta}TP subunit. Finally, we discussed a sufficient proton motive force to rescue FoF1-ATP synthase from the IF1-inhibited state. Significance statementFoF1-ATP synthase is crucial for cellular energy production. However, its reverse reaction as an ATPase can be harmful, particularly in mammalian cells. This study reveals how ATPase inhibitory factor 1 (IF1) controls the catalysis of mitochondrial ATP synthase and provides the atomic-level description of the rotation-direction-dependent regulation using molecular dynamics simulation. ATP-synthetic {gamma} rotation dissociates IF1 from the complex, thereby recovering active ATP synthesis. However, IF1 completely suppresses the {gamma} rotation through multiple mechanisms to prevent wasteful ATP consumption in ATP-hydrolysis mode. Our analysis also reveals that sufficient torque in the synthesis direction is required to rescue FoF1-ATP synthase from the IF1-inhibited state. These findings offer valuable insights for developing inhibitors by leveraging the structural and functional similarities with IF1.