Departments of Chemistry and Biochemistry , The University of Western Ontario , London , Ontario N6A 5B7 , Canada.
J Am Chem Soc. 2018 Nov 7;140(44):14860-14869. doi: 10.1021/jacs.8b08692. Epub 2018 Oct 26.
F-ATPase uses ATP hydrolysis to drive rotation of the γ subunit. The γ C-terminal helix constitutes the rotor tip that is seated in an apical bearing formed by αβ. It remains uncertain to what extent the γ conformation during rotation differs from that seen in rigid crystal structures. Existing models assume that the entire γ subunit participates in every rotation. Here we interrogated E. coli F-ATPase by hydrogen-deuterium exchange (HDX) mass spectrometry. Rotation of γ caused greatly enhanced deuteration in the γ C-terminal helix. The HDX kinetics implied that most F complexes operate with an intact rotor at any given time, but that the rotor tip is prone to occasional unfolding. A molecular dynamics (MD) strategy was developed to model the off-axis forces acting on γ. MD runs showed stalling of the rotor tip and unfolding of the γ C-terminal helix. MD-predicted H-bond opening events coincided with experimental HDX patterns. Our data suggest that in vitro operation of F-ATPase is associated with significant rotational resistance in the apical bearing. These conditions cause the γ C-terminal helix to get "stuck" (and unfold) sporadically while the remainder of γ continues to rotate. This scenario contrasts the traditional "greasy bearing" model that envisions smooth rotation of the γ C-terminal helix. The fragility of the apical rotor tip in F-ATPase is attributed to the absence of a c ring that stabilizes the rotation axis in intact FF. Overall, the MD/HDX strategy introduced here appears well suited for interrogating the inner workings of molecular motors.
F-ATP 酶利用 ATP 水解来驱动 γ 亚基的旋转。γ C 端螺旋构成了位于 αβ 形成的顶端轴承中的转子尖端。γ 在旋转过程中的构象与刚性晶体结构中观察到的构象有何不同，目前仍不确定。现有的模型假设整个 γ 亚基都参与了每一次旋转。在这里，我们通过氘代氢交换（HDX）质谱法研究了大肠杆菌 F-ATP 酶。γ 的旋转导致 γ C 端螺旋的氘代程度大大增强。HDX 动力学表明，大多数 F 复合物在任何给定时间都以完整的转子运行，但转子尖端容易偶尔展开。开发了一种分子动力学（MD）策略来模拟作用在γ上的非轴力。MD 运行表明，转子尖端停止旋转，γ C 端螺旋展开。MD 预测的氢键打开事件与实验 HDX 模式吻合。我们的数据表明，F-ATP 酶在体外的运作与顶端轴承中显著的旋转阻力有关。这些条件导致 γ C 端螺旋偶尔“卡住”（展开），而γ 的其余部分继续旋转。这种情况与传统的“油腻轴承”模型形成对比，后者设想γ C 端螺旋的平滑旋转。F-ATP 酶中顶端转子尖端的脆弱性归因于缺乏 c 环，该环在完整的 FF 中稳定旋转轴。总的来说，这里引入的 MD/HDX 策略似乎非常适合研究分子马达的内部工作原理。
