Department of Biological Science and Technology, Tokyo University of Science, 6-3-1 Niijuku Katsushika-ku, Tokyo 125-8585, Japan; Department of Chemistry, Graduate School of Science, Chiba University, 1-33 Yayoi-cho Inage, Chiba, 263-8522, Japan.
Department of Chemistry, Graduate School of Science, Chiba University, 1-33 Yayoi-cho Inage, Chiba, 263-8522, Japan.
Biochem Biophys Res Commun. 2020 Dec 17;533(4):1413-1418. doi: 10.1016/j.bbrc.2020.10.033. Epub 2020 Oct 21.
V-ATPases are ubiquitous proton-transporting ATPases of eukaryotic and prokaryotic membranes that utilize energy from ATP hydrolysis. The hydrophilic catalytic part called V-ATPase is composed of a ring-shaped hexametric AB complex and a central DF shaft. We previously proposed a rotation mechanism of the Enterococcus hirae V-ATPase based on the crystal structures of the V and AB complexes. However, the driving force that induces the conformational changes of AB and rotation of the DF shaft remains unclear. In this study, we investigated the binding affinity changes between subunits of V-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 AB tight conformation. Furthermore, the DF shaft bound to the reconstituted AB complex with high affinity, suggesting that the tight AB complex is a major binding unit of the shaft in the AB ring complex. Based on these results, we propose that rotation of the V-ATPase is driven by affinity changes between each subunit via thermal fluctuations.
V-ATPases 是真核和原核细胞膜中普遍存在的质子转运 ATP 酶，利用 ATP 水解产生的能量。亲水性催化部分称为 V-ATPase，由环形六聚体 AB 复合物和中央 DF 轴组成。我们之前基于 Enterococcus hirae V-ATPase 的晶体结构提出了一种旋转机制。然而，诱导 AB 构象变化和 DF 轴旋转的驱动力仍不清楚。在这项研究中，我们通过表面等离子体共振分析研究了 V-ATPase 亚基之间结合亲和力的变化。发现 ATP 与亚基 A 的结合极大地增加了 A 和 B 亚基之间的亲和力，从而 ATP 结合有助于形成 AB 紧密构象。此外，DF 轴与重新组装的 AB 复合物以高亲和力结合，表明紧密的 AB 复合物是 AB 环复合物中轴的主要结合单元。基于这些结果，我们提出 V-ATPase 的旋转是由每个亚基通过热波动引起的亲和力变化驱动的。
