An affinity change model to elucidate the rotation mechanism of V₁-ATPase

V-ATPases are ubiquitous proton-transporting ATPases of eukaryotic and prokaryotic membranes that utilize energy from ATP hydrolysis. The hydrophilic catalytic part called V₁-ATPase is composed of a ring-shaped hexametric A₃B₃ complex and a central DF shaft. We previously proposed a rotation mechanism of the Enterococcus hirae V₁-ATPase based on the crystal structures of the V₁ and A₃B₃ complexes. However, the driving force that induces the conformational changes of A₃B₃ and rotation of the DF shaft remains unclear. In this study, we investigated the binding affinity changes between subunits of V₁-ATPase by surface plasmon resonance analysis. The binding of ATP to subunit A was found to considerably increase the affinity between the A and B subunits, and thereby ATP binding contributes to forming the A₁B₁ tight conformation. Furthermore, the DF shaft bound to the reconstituted A₁B₁ complex with high affinity, suggesting that the tight A₁B₁ complex is a major binding unit of the shaft in the A₃B₃ ring complex. Based on these results, we propose that rotation of the V₁-ATPase is driven by affinity changes between each subunit via thermal fluctuations.