Phosphate release coupled to rotary motion of F1-ATPase

F-1-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 alpha beta-dimers creates torque on its central gamma-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 degrees 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 alpha beta(E) 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 P-i. The.-rotation tightens the ATP-bound alpha beta(TP) interface, as required for hydrolysis. The calculated rate for the outward release of doubly charged P-i from the alpha beta(E) interface 120 after ATP hydrolysis closely matches the similar to 1-ms functional timescale. Conversely, P-i release from the ADP-bound alpha beta(DP) interface postulated in earlier models would occur through a kinetically infeasible in-ward-directed pathway. Our simulations help reconcile conflicting interpretations of single-molecule experiments and crystallographic studies by clarifying the timing of P-i exit, its pathway and kinetics, associated changes in P-i protonation, and changes of the F-1-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.