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.

V-ATP酶是真核膜和原核膜中普遍存在的质子转运ATP酶，其利用来自ATP水解的能量。称为V ₁ -ATPase的亲水性催化部分由环状六价A ₃ B ₃络合物和中心DF轴组成。我们以前基于V ₁和A ₃ B ₃配合物的晶体结构提出了平肠肠球菌V ₁ -ATPase的旋转机制。但是，引起A ₃ B ₃构象变化的驱动力DF轴的旋转仍不清楚。在这项研究中，我们通过表面等离子体共振分析研究了V ₁ -ATPase亚基之间的结合亲和力变化。发现ATP与亚基A的结合大大增加了A和B亚基之间的亲和力，因此ATP结合有助于形成A ₁ B ₁紧密构象。此外，DF轴以高亲和力结合到重构的A ₁ B ₁络合物上，这表明紧密的A ₁ B ₁络合物是A ₃ B ₃环络合物中轴的主要结合单元。根据这些结果，我们建议V的旋转₁ -ATP酶由每个亚基之间经由热波动的亲和力变化驱动。