School of Computational Sciences, Korea Institute for Advanced Study, 85 Hoegiro Dongdaemun-gu, Seoul 02455, Republic of Korea.
Bioinformatics Institute, Agency for Science, Technology and Research (A*STAR), 30 Biopolis Str., #07-01 Matrix, 138671, Singapore.
Biochim Biophys Acta Biomembr. 2019 May 1;1861(5):1004-1010. doi: 10.1016/j.bbamem.2019.02.010. Epub 2019 Mar 1.
V-type ATPases are multi-protein complexes, which acidify cellular compartments in eukaryotes. They pump protons against an ion gradient, driven by a mechano-chemical framework that exploits ATP hydrolysis as an energy source. This process drives the rotation of the so-called c-ring, a membrane embedded complex in the V-domain of the V-type ATPase, resulting in translocation of protons across the membrane. One way in which the enzyme is regulated is by disassembly and reassembly of the V-domain with the V-domain, which inactivates and reactivates the enzyme, respectively. Recently, structural data for the isolated V-domain from S. cerevisiae in an inactivated state were reported, suggesting the location of previously unobserved proton access pathways within the cytoplasmic and luminal compartments of the stator subunit a in V. However, the structural rationale for this inactivation remained unclear. In this study, the water accessibility pathway at the cytoplasmic side is confirmed, and novel insights into the role of the luminal channel with respect to the inactivation mechanism are obtained, using atomic-resolution molecular dynamics simulations. The results show that protonation of the key-glutamate, located in the c-ring of the V-domain, and facing the luminal compartment is preserved, when residing in the V-depleted state. Maintaining the protonation of this essential glutamate is necessary to lock the luminal channel in the inactive, solvent-free state. Based on these theoretical observations and previous experimental results, a model of the proton translocation mechanism in the V-domain from V-type ATPases is proposed.
V 型 ATP 酶是多蛋白复合物，在真核生物中使细胞区室酸化。它们在机械化学框架的驱动下逆质子浓度梯度泵出质子，该框架利用 ATP 水解作为能量来源。这一过程驱动所谓的 c 环旋转，c 环是 V 型 ATP 酶 V 结构域中嵌入的膜复合物，导致质子跨膜转运。酶的一种调节方式是 V 结构域与 V 结构域的解体和组装，分别使酶失活和重新激活。最近，报道了来自酿酒酵母的分离 V 结构域在失活状态下的结构数据，这表明在定子亚基 a 的细胞质和腔室隔间内存在以前未观察到的质子进入途径。然而，这种失活的结构原理仍不清楚。在这项研究中，使用原子分辨率的分子动力学模拟，在细胞质侧确认了水可及性途径，并获得了关于腔内通道在失活机制中的作用的新见解。结果表明，当位于 V 结构域耗尽状态时，位于 V 结构域的 c 环中并朝向腔室隔间的关键谷氨酸的质子化得以保留。保持这种必需谷氨酸的质子化对于将腔内通道锁定在非活性、无溶剂状态是必要的。基于这些理论观察和以前的实验结果，提出了 V 型 ATP 酶 V 结构域中质子转运机制的模型。
