Department of Chemistry, Graduate School of Science, Chiba University, 1-33 Yayoi-cho, Inage, Chiba 263-8522, Japan.
Nano Life Science Institute (WPI NanoLSI), Kanazawa University, Kakuma-machi, Kanazawa 920-1192, Japan.
Sci Adv. 2019 Jan 30;5(1):eaau8149. doi: 10.1126/sciadv.aau8149. eCollection 2019 Jan.
V-ATPase is an ATP-driven rotary motor that is composed of a ring-shaped AB complex and a central DF shaft. The nucleotide-free AB complex of , composed of three identical AB heterodimers, showed a unique asymmetrical structure, probably due to the strong binding of the N-terminal barrel domain, which forms a crown structure. Here, we mutated the barrel region to weaken the crown, and performed structural analyses using high-speed atomic force microscopy and x-ray crystallography of the mutant AB. The nucleotide-free mutant AB complex had a more symmetrical open structure than the wild type. Binding of nucleotides produced a closely packed spiral-like structure with a disrupted crown. These findings suggest that wild-type AB forms a metastable (stressed) asymmetric structure composed of unstable AB conformers due to the strong constraint of the crown. The results further the understanding of the principle of the cooperative transition mechanism of rotary motors.
V-ATPase 是一种由环形 AB 复合物和中央 DF 轴组成的 ATP 驱动的旋转马达。无核苷酸的 AB 复合物由三个相同的 AB 异二聚体组成，显示出独特的不对称结构，可能是由于 N 端桶状结构域的强结合，形成了冠结构。在这里，我们突变了桶状区域以削弱冠，并使用高速原子力显微镜和突变体 AB 的 X 射线晶体学进行了结构分析。无核苷酸的突变体 AB 复合物具有比野生型更对称的开放结构。核苷酸结合产生了一个紧密堆积的螺旋状结构，冠被破坏。这些发现表明，由于冠的强烈限制，野生型 AB 形成了一种由不稳定的 AB 构象组成的亚稳态（应激）不对称结构。该结果进一步加深了对旋转马达协同跃迁机制原理的理解。
