Protons find a path Adenosine triphosphate (ATP) synthases are dynamos that interconvert rotational and chemical energy. Capturing the complete structure of these multisubunit membrane-bound complexes has been hindered by their inherent ability to adopt multiple conformations. Srivastava et al. used protein engineering to freeze mitochondrial ATP synthase from yeast in a single conformation and obtained a structure with the inhibitor oligomycin, which binds to the rotating c-ring within the membrane. Hahn et al. show that chloroplast ATP synthase contains a built-in inhibitor triggered by oxidizing conditions in the dark chloroplast. The mechanisms by which these machines are powered are remarkably similar: Protons are shuttled through a channel to the membrane-embedded c-ring, where they drive nearly a full rotation of the rotor before exiting through another channel on the opposite side of the membrane (see the Perspective by Kane). Science, this issue p. eaas9699, p. eaat4318; see also p. 600 The structure of an intact ATP synthase provides insight into how the motor and catalytic components are coupled. INTRODUCTION The mitochondrial adenosine triphosphate (ATP) synthase is the enzyme responsible for the synthesis of more than 90% of the ATP produced by mammalian cells under aerobic conditions. The chemiosmotic mechanism, proposed by Peter Mitchell, states that the enzyme transduces the energy of a proton gradient, generated by the electron transport chain, into the major energy currency of the cell, ATP. The enzyme is a large (about 600,000 Da, in the monomer state) multisubunit complex, with a water soluble complex (F1) that contains three active sites and a membrane complex (Fo) that contains the proton translocation pathway, principally comprised of the a subunit and a ring of 10 c subunits, the c10-ring (10 in yeast, 8 in mammals). F1 has a central rotor that, at one end, is within the core of F1 and, at the other end, is connected to the c10-ring of Fo. During ATP synthesis, the c10-ring rotates, driven by the movement of protons from the cytosol to the mitochondrion, and in turn, the rotor rotates within F1 in steps of 120o. The rotation of the rotor causes conformational changes in the catalytic sites, which provides the energy for the phosphorylation of adenosine diphosphate (ADP), as first proposed in the binding-change hypothesis by Paul Boyer. The peripheral stalk acts as a stator connecting F1 with Fo and prevents the futile rotation of F1 as the rotor spins within it. RATIONALE Structural studies of the ATP synthase have made steady progress since the structure of the F1 complex was described in pioneering work by John Walker. However, obtaining a high-resolution structure of the intact ATP synthase is challenging because it is inherently dynamic. To overcome this conformational heterogeneity, we locked the yeast mitochondrial rotor in a single conformation by fusing a subunit of the stator with a subunit of the rotor, also called the central stalk. The engineered ATP synthase was expressed in yeast and reconstituted into nanodiscs. This facilitated structure determination by cryo–electron microscopy (cryo-EM) under near native conditions. RESULTS Single-particle cryo-EM enabled us to determine the structures of the membrane-embedded monomeric yeast ATP synthase in the presence and absence of the inhibitor oligomycin at 3.8- and 3.6-Å resolution, respectively. The fusion between the rotor and stator caused a twisting of the rotor and a 9° rotation of the c10-ring, in the direction of ATP synthesis, relative to the putative resting state. This twisted conformation likely represents an intermediate state in the ATP synthesis reaction cycle. The structure also shows two proton half-channels formed largely by the a subunit that abut the c10-ring and suggests a mechanism that couples transmembrane proton movement to c10-ring rotation. The cryo-EM density map indicates that oligomycin is bound to at least four sites on the surface of the Fo c10-ring that is exposed to the lipid bilayer; this is supported by binding free-energy molecular dynamics calculations. The sites of oligomycin-resistant mutations in the a subunit suggest that changes in the side-chain configuration of the c subunits at the a-c subunit interface are transmitted through the entire c10-ring. CONCLUSION Our results provide a high-resolution structure of the complete monomeric form of the mitochondrial ATP synthase. The structure provides an understanding of the mechanism of inhibition by oligomycin and suggests how extragenic mutations can cause resistance to this inhibitor. The approach presented in this study paves the way for structural characterization of other functional states of the ATP synthase, which is essential for understanding its functions in physiology and disease. Structure of the monomeric yeast ATP synthase, as determined by cryo-EM, shown as a ribbon diagram. The subunits are shown in different colors. The F1 complex is located at the top center and is composed of six subunits forming a nearly spherical structure and three subunits comprising the central stalk, or rotor. The Fo complex is located at the bottom, with the identity of the c10-ring clearly seen. The peripheral stalk, or stator, is on the left, and the rotor is in the center of the molecule, extending into F1. Mitochondrial adenosine triphosphate (ATP) synthase comprises a membrane embedded Fo motor that rotates to drive ATP synthesis in the F1 subunit. We used single-particle cryo–electron microscopy (cryo-EM) to obtain structures of the full complex in a lipid bilayer in the absence or presence of the inhibitor oligomycin at 3.6- and 3.8-angstrom resolution, respectively. To limit conformational heterogeneity, we locked the rotor in a single conformation by fusing the F6 subunit of the stator with the δ subunit of the rotor. Assembly of the enzyme with the F6-δ fusion caused a twisting of the rotor and a 9° rotation of the Fo c10-ring in the direction of ATP synthesis, relative to the structure of isolated Fo. Our cryo-EM structures show how F1 and Fo are coupled, give insight into the proton translocation pathway, and show how oligomycin blocks ATP synthesis.

中文翻译：

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脂膜中酵母 ATP 合酶的高分辨率冷冻电镜分析

质子找到一条路径 三磷酸腺苷 (ATP) 合酶是旋转能和化学能相互转换的发电机。这些多亚基膜结合复合物的完整结构因其固有的采用多种构象的能力而受到阻碍。斯里瓦斯塔瓦等人。利用蛋白质工程将酵母中的线粒体 ATP 合酶冷冻成单一构象，并获得了含有抑制剂寡霉素的结构，该结构与膜内的旋转 C 环结合。哈恩等人。表明叶绿体 ATP 合酶含有由黑暗叶绿体中的氧化条件触发的内置抑制剂。这些机器的供电机制非常相似：质子通过通道穿梭到嵌入膜的 C 形环，在那里它们驱动转子几乎完整旋转，然后通过膜另一侧的另一个通道离开（参见凯恩的观点）。科学，本期第 14 页。eaas9699，p。eaat4318; 另见 p. 600 完整 ATP 合酶的结构提供了对马达和催化组件如何耦合的深入了解。简介 线粒体三磷酸腺苷 (ATP) 合酶负责合成哺乳动物细胞在有氧条件下产生的 ATP 的 90% 以上。Peter Mitchell 提出的化学渗透机制指出，酶将电子传输链产生的质子梯度能量转换为细胞的主要能量货币 ATP。该酶是一种大型（单体状态下约 600,000 Da）多亚基复合物，具有包含三个活性位点的水溶性复合物 (F1) 和包含质子易位途径的膜复合物 (Fo)，主要由亚基和 10 c 亚基环，即 c10 环（酵母中有 10 个，哺乳动物中有 8 个）。F1 有一个中心转子，其一端位于 F1 的核心内，另一端连接到 Fo 的 c10 环。在 ATP 合成过程中，c10 环在质子从细胞质到线粒体的运动驱动下旋转，反过来，转子在 F1 内以 120o 的步长旋转。转子的旋转会引起催化位点的构象变化，从而为二磷酸腺苷 (ADP) 的磷酸化提供能量，正如 Paul Boyer 在结合变化假说中首次提出的那样。外围轴充当连接 F1 和 Fo 的定子，并防止当转子在其中旋转时 F1 徒劳地旋转。基本原理 自从 John Walker 在开创性工作中描述了 F1 复合物的结构以来，ATP 合酶的结构研究取得了稳步进展。然而，获得完整 ATP 合酶的高分辨率结构具有挑战性，因为它本质上是动态的。为了克服这种构象异质性，我们通过将定子的亚基与转子的亚基融合，将酵母线粒体转子锁定在单一构象中，也称为中央柄。工程化的 ATP 合酶在酵母中表达并重构为纳米圆盘。这有助于在接近自然条件下通过冷冻电子显微镜（cryo-EM）进行结构测定。结果单颗粒冷冻电镜使我们能够分别以 3.8 和 3.6 Å 的分辨率确定在存在和不存在抑制剂寡霉素的情况下膜嵌入的单体酵母 ATP 合酶的结构。转子和定子之间的融合引起转子的扭转和 c10 环相对于假定的静止状态沿 ATP 合成方向旋转 9°。这种扭曲的构象可能代表 ATP 合成反应循环中的中间状态。该结构还显示了两个质子半通道，主要由邻接 c10 环的 a 亚基形成，并提出了一种将跨膜质子运动与 c10 环旋转耦合的机制。冷冻电镜密度图表明寡霉素与暴露于脂质双层的 Fo c10 环表面上的至少四个位点结合；结合自由能分子动力学计算支持了这一点。a 亚基中的寡霉素抗性突变位点表明，ac 亚基界面处的 c 亚基侧链构型的变化是通过整个 c10 环传递的。结论 我们的结果提供了线粒体 ATP 合酶完整单体形式的高分辨率结构。该结构提供了对寡霉素抑制机制的理解，并表明外源突变如何导致对该抑制剂的抗性。本研究中提出的方法为 ATP 合酶其他功能状态的结构表征铺平了道路，这对于理解其在生理学和疾病中的功能至关重要。通过冷冻电镜测定的单体酵母 ATP 合酶的结构，显示为带状图。子单元以不同的颜色显示。F1复合体位于顶部中心，由形成近球形结构的六个亚基和构成中心柄或转子的三个亚基组成。Fo复合物位于底部，c10环的身份清晰可见。外围的茎（或定子）位于左侧，转子位于分子的中心，延伸到 F1 中。线粒体三磷酸腺苷 (ATP) 合酶包含嵌入膜的 Fo 马达，该马达旋转以驱动 F1 亚基中的 ATP 合成。我们使用单颗粒冷冻电子显微镜 (cryo-EM) 分别以 3.6 埃和 3.8 埃的分辨率获得了在不存在或存在抑制剂寡霉素的情况下脂质双层中完整复合物的结构。为了限制构象异质性，我们通过将定子的 F6 亚基与转子的 δ 亚基融合，将转子锁定在单一构象中。相对于分离的 Fo 的结构，具有 F6-δ 融合体的酶的组装引起转子的扭转和 Fo c10 环在 ATP 合成方向上的 9° 旋转。我们的冷冻电镜结构展示了 F1 和 Fo 是如何耦合的，深入了解质子易位途径，并展示了寡霉素如何阻断 ATP 合成。