V-ATPases are ubiquitous proton-transporting ATPases of eukaryotic and prokaryotic membranes that utilize energy from ATP hydrolysis. The hydrophilic catalytic part called V(1)ATPase is composed of a ringshaped hexametric A(3)B(3) complex and a central DF shaft. We previously proposed a rotation mechanism of the Enterococcus hirae V(1)ATPase based on the crystal structures of the V-1 and A(3)B(3) complexes. However, the driving force that induces the conformational changes of A(3)B(3) and rotation of the DF shaft remains unclear. In this study, we investigated the binding affinity changes between subunits of V(1)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 A(1)B(1) tight conformation. Furthermore, the DF shaft bound to the reconstituted A(1)B(1) complex with high affinity, suggesting that the tight A(1)B(1) complex is a major binding unit of the shaft in the A(3)B(3) ring complex. Based on these results, we propose that rotation of the V-1-ATPase is driven by affinity changes between each subunit via thermal fluctuations. (C) 2020 Elsevier Inc. All rights reserved.