Rotation triggers nucleotide-independent conformational transition of the empty β subunit of F₁-ATPase.

F1-ATPase (F1) is the catalytic portion of ATP synthase, a rotary motor protein that couples proton gradients to ATP synthesis. Driven by a proton flux, the F1 asymmetric γ subunit undergoes a stepwise rotation inside the α3β3 headpiece and causes the β 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 γ and the conformations of α3β3. Using molecular dynamics simulations, we show that the nucleotide-free β subunit, initially in the open, low-affinity state, undergoes a spontaneous closing transition to the half-open state in response to the γ 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 β 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 F1 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 β. Our results also indicate that opening of the nucleotide-free β during hydrolysis is not spontaneous, as previously assumed. Rather, the fully open conformation observed in the F1 X-ray structure is enforced sterically by the γ subunit whose orientation is stabilized by interactions with the two other β subunits in the completely closed state. This finding supports the notion that γ acts by coupling the extreme conformational states of β subunits within the α3β3 hexamer and therefore is responsible for high efficiency of the coordinated catalysis.