4.8 Article

Phosphate release coupled to rotary motion of F1-ATPase

Publisher

NATL ACAD SCIENCES
DOI: 10.1073/pnas.1305497110

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Funding

  1. Intramural Research Program of the National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health
  2. Max Planck Society
  3. Japan Society for the Promotion of Science

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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.

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