Phosphate release coupled to rotary motion of F1-ATPase.
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 αβ-dimers creates torque on its central γ-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 γ-subunit in the hydrolysis direction, the nucleotide-free αβE 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 γ-rotation tightens the ATP-bound αβTP interface, as required for hydrolysis. The calculated rate for the outward release of doubly charged Pi from the αβE interface 120° after ATP hydrolysis closely matches the ~1-ms functional timescale. Conversely, Pi release from the ADP-bound αβDP 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.
Department of Theoretical Biophysics, Max Planck Institute of Biophysics, 60438 Frankfurt am Main, Germany.
Kei-ichi
K
Okazaki
Gerhard
G
Hummer
D000255:Adenosine Triphosphate / Q000378:metabolism; D006868:Hydrolysis; D008956:Models, Chemical; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D020409:Molecular Motor Proteins / Q000737:chemistry* / Q000378:metabolism*; D010710:Phosphates / Q000378:metabolism*; D011487:Protein Conformation; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000378:metabolism*; D015003:Yeasts
D020409:Molecular Motor Proteins; D010710:Phosphates; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases