Phosphate release coupled to rotary motion of F1-ATPase

Abstract

F1-ATPase, the catalytic domain of ATP synthase, synthesizes most of the ATP in living organisms. Running in reverse powered by ATP hydrolysis, this hexameric ring-shaped molecular motor formed by three ?β-dimers creates torque on its central ?-subunit. This reverse operation enables detailed explorations of the mechanochemical coupling mechanisms in experiment and simulation. Here, we use molecular dynamics simulations to construct a first atomistic conformation of the intermediate state following the 40° substep of rotary motion, and to study the timing and molecular mechanism of inorganic phosphate (Pi) release coupled to the rotation. In response to torque-driven rotation of the ?-subunit in the hydrolysis direction, the nucleotide-free ?βE interface forming the "empty" E site loosens and singly charged Pi readily escapes to the P loop. By contrast, the interface stays closed with doubly charged Pi. The ?-rotation tightens the ATP-bound ?βTP interface, as required for hydrolysis. The calculated rate for the outward release of doubly charged Pi from the ?βE interface 120° after ATP hydrolysis closely matches the ?1-ms functional timescale. Conversely, Pi release from the ADP-bound ?βDP interface postulated in earlier models would occur through a kinetically infeasible inward- directed pathway. Our simulations help reconcile conflicting interpretations of single-molecule experiments and crystallographic studies by clarifying the timing of Pi exit, its pathway and kinetics, associated changes in Pi protonation, and changes of the F1-ATPase structure in the 40° substep. Important elements of the molecular mechanism of Pi release emerging from our simulations appear to be conserved in myosin despite the different functional motions.