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Abstract

F-1-ATPase (F-1) is the catalytic portion of ATP synthase, a rotary motor protein that couples proton gradients to ATP synthesis. Driven by a proton flux, the F, asymmetric gamma subunit undergoes a stepwise rotation inside the alpha(3)beta(3) headpiece and causes the beta subunits' binding sites to cycle between states of different affinity for nucleotides. These concerted transitions drive the synthesis of ATP from ADP and phosphate. Here, we study the coupling between the mechanical progression of gamma and the conformations of alpha(3)beta(3). Using molecular dynamics simulations, we show that the nucleotide-free beta subunit, initially in the open, low-affinity state, undergoes a spontaneous closing transition to the half-open state in response to the gamma rotation in the synthesis direction. We estimate the kinetics of this spontaneous conformational change and analyze its mechanism and driving forces. By computing free energy profiles, we find that the isolated empty beta subunit preferentially adopts the half-open conformation and that the transition to this conformation from the fully open state is accompanied by well-defined changes in the structure and interactions of the active site region. These results suggest that ADP binding to F, occurs via conformational selection and is preceded by the transition of the active site to the half-open conformation, driven by the intrinsic elasticity of beta. Our results also indicate that opening of the nucleotide-free beta during hydrolysis is not spontaneous, as previously assumed. Rather, the fully open conformation observed in the F-1 X-ray structure is enforced sterically by the gamma subunit whose orientation is stabilized by interactions with the two other beta subunits in the completely closed state. This finding supports the notion that gamma acts by coupling the extreme conformational states of beta subunits within the alpha(3)beta(3) hexamer and therefore is responsible for high efficiency of the coordinated catalysis.