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Journal of the American Chemical Society





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F1-ATPase uses ATP hydrolysis to drive rotation of the γ subunit. The γ C-terminal helix constitutes the rotor tip that is seated in an apical bearing formed by α3β3. It remains uncertain to what extent the γ conformation during rotation differs from that seen in rigid crystal structures. Existing models assume that the entire γ subunit participates in every rotation. Here we interrogated E. coli F1-ATPase by hydrogen-deuterium exchange (HDX) mass spectrometry. Rotation of γ caused greatly enhanced deuteration in the γ C-terminal helix. The HDX kinetics implied that most F1 complexes operate with an intact rotor at any given time, but that the rotor tip is prone to occasional unfolding. A molecular dynamics (MD) strategy was developed to model the off-axis forces acting on γ. MD runs showed stalling of the rotor tip and unfolding of the γ C-terminal helix. MD-predicted H-bond opening events coincided with experimental HDX patterns. Our data suggest that in vitro operation of F1-ATPase is associated with significant rotational resistance in the apical bearing. These conditions cause the γ C-terminal helix to get "stuck" (and unfold) sporadically while the remainder of γ continues to rotate. This scenario contrasts the traditional "greasy bearing" model that envisions smooth rotation of the γ C-terminal helix. The fragility of the apical rotor tip in F1-ATPase is attributed to the absence of a c10 ring that stabilizes the rotation axis in intact FoF1. Overall, the MD/HDX strategy introduced here appears well suited for interrogating the inner workings of molecular motors.

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