Exploratory Research Center on Life and Living Systems (ExCELLS), National Institutes of Natural Sciences, 38 Nishigonaka, Myodaiji, Okazaki, Aichi, 444-8585, Japan.
National Institute for Physiological Sciences, National Institutes of Natural Sciences, 38 Nishigonaka, Myodaiji, Okazaki, Aichi, 444-8585, Japan.
Commun Biol. 2023 Jul 28;6(1):755. doi: 10.1038/s42003-023-05110-8.
The vacuolar-type ATPase from Enterococcus hirae (EhV-ATPase) is a thus-far unique adaptation of V-ATPases, as it performs Na transport and demonstrates an off-axis rotor assembly. Recent single molecule studies of the isolated V domain have indicated that there are subpauses within the three major states of the pseudo three-fold symmetric rotary enzyme. However, there was no structural evidence for these. Herein we activate the EhV-ATPase complex with ATP and identified multiple structures consisting of a total of six states of this complex by using cryo-electron microscopy. The orientations of the rotor complex during turnover, especially in the intermediates, are not as perfectly uniform as expected. The densities in the nucleotide binding pockets in the V domain indicate the different catalytic conditions for the six conformations. The off-axis rotor and its' interactions with the stator a-subunit during rotation suggests that this non-uniform rotor rotation is performed through the entire complex.
从肠球菌（EhV-ATPase）中提取的液泡型 ATP 酶是 V-ATPase 的独特适应性，因为它可以进行 Na 转运，并表现出非轴性转子组件。最近对分离的 V 结构域的单分子研究表明，在伪三重复旋转酶的三个主要状态内存在亚暂停。然而，目前还没有结构证据来证明这一点。在这里，我们使用 cryo-electron microscopy 通过用 ATP 激活 EhV-ATPase 复合物，并鉴定了总共包含六个状态的该复合物的多个结构。在周转过程中，转子复合物的取向，尤其是在中间体中的取向，并不像预期的那样完全均匀。V 结构域中核苷酸结合口袋的密度表明六个构象的不同催化条件。非轴性转子及其在旋转过程中与定子 a 亚基的相互作用表明，这种非均匀的转子旋转是通过整个复合物完成的。
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