Department of Biochemistry & Molecular Biology, SUNY Upstate Medical University, 750 E Adams St, Syracuse, NY 13210, USA.
Biochim Biophys Acta Bioenerg. 2020 Jul 1;1861(7):148189. doi: 10.1016/j.bbabio.2020.148189. Epub 2020 Mar 17.
ATP synthases are important energy-coupling, rotary motor enzymes in all kingdoms of life. In all F-type ATP synthases, the central rotor of the catalytic F complex is composed of the γ subunit and the N-terminal domain (NTD) of the ε subunit. In the enzymes of diverse bacteria, the C-terminal domain of ε (εCTD) can undergo a dramatic conformational change to trap the enzyme in a transiently inactive state. This inhibitory mechanism is absent in the mitochondrial enzyme, so the εCTD could provide a means to selectively target ATP synthases of pathogenic bacteria for antibiotic development. For Escherichia coli and other bacterial model systems, it has been difficult to dissect the relationship between ε inhibition and a MgADP-inhibited state that is ubiquitous for FF from bacteria and eukaryotes. A prior study with the isolated catalytic complex from E. coli, EcF, showed that these two modes of inhibition are mutually exclusive, but it has long been known that interactions of F with the membrane-embedded F complex modulate inhibition by the εCTD. Here, we study membranes containing EcFF with wild-type ε, ε lacking the full εCTD, or ε with a small deletion at the C-terminus. By using compounds with distinct activating effects on F-ATP-ase activity, we confirm that εCTD inhibition and ubiquitous MgADP inhibition are mutually exclusive for membrane-bound E. coli F-ATP-ase. We determine that most of the enzyme complexes in wild-type membranes are in the ε-inhibited state (>50%) or in the MgADP-inhibited state (30%).
ATP 合酶是所有生命领域中重要的能量偶联、旋转马达酶。在所有 F 型 ATP 合酶中，催化 F 复合物的中央转子由 γ 亚基和 ε 亚基的 N 端结构域 (NTD) 组成。在不同细菌的酶中，ε 的 C 端结构域 (εCTD) 可以发生剧烈的构象变化，将酶捕获在短暂的无活性状态。这种抑制机制在线粒体酶中不存在，因此 εCTD 可以提供一种选择性靶向致病性细菌 ATP 合酶的方法，用于抗生素的开发。对于大肠杆菌和其他细菌模型系统，很难剖析 ε 抑制与普遍存在于细菌和真核生物 FF 中的 MgADP 抑制状态之间的关系。先前对大肠杆菌分离的催化复合物 EcF 的研究表明，这两种抑制模式是相互排斥的，但长期以来人们都知道 F 与膜嵌入的 F 复合物的相互作用调节了 εCTD 的抑制作用。在这里，我们研究了含有野生型 ε 的 EcFF 膜、缺乏完整 εCTD 的 ε 膜或 ε 膜 C 端有小缺失的膜。通过使用对 F-ATP-ase 活性具有不同激活作用的化合物，我们证实了 εCTD 抑制和普遍存在的 MgADP 抑制对膜结合的大肠杆菌 F-ATP-ase 是相互排斥的。我们确定野生型膜中大多数酶复合物处于 ε 抑制状态（>50%）或 MgADP 抑制状态（30%）。
