Load-dependent destabilization of the γ-rotor shaft in FOF1 ATP synthase revealed by hydrogen/deuterium-exchange mass spectrometry
Load-dependent destabilization of the γ-rotor shaft in FOF1 ATP synthase revealed by hydrogen/deuterium-exchange mass spectrometry
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氢/氘交换质谱揭示 FOF1 ATP 合酶中 γ 转子轴的负载依赖性不稳定
DOI：
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发表时间：
2016
影响因子：
11.1
通讯作者：
L. Konermann
中科院分区：
文献类型：
--
作者：
S. Vahidi;Y. Bi;S. Dunn;L. Konermann
FOF 1或ATP合成酶通常被称为“世界上最小的马达”。与汽车发动机类似，它采用与机械致动器相互作用的旋转轴。当在负载下操作内燃机时，轴承在曲轴上施加显著的力，从而导致增强的机械应力。在这里，我们证明了类似的负载依赖性效应发生在分子马达。当FOF 1泵送质子对抗跨膜梯度时，转子轴经历归因于其顶端轴承中的阻力的结构不稳定。如力学基本原理所预测的，当反质子泵的跨膜梯度被解偶联剂短路时，这种效应消失。我们的观察突出了宏观尺度和纳米尺度上的引擎操作之间的迷人相似之处。FoF 1是一种膜结合的分子马达，它使用质子动力（PMF）来驱动ADP和Pi合成ATP。反向操作通过ATP水解产生PMF。在任一方向上的催化涉及连接α3β3头部和膜锚定cn环的γε轴的旋转。X射线晶体学和其他技术提供了对FoF 1亚复合物的结构和功能的见解。然而，询问完整的膜结合的FoF 1在旋转催化过程中的构象动力学已被证明是困难的。在这里，我们使用氢/氘交换质谱来探测FoF 1在其自然膜结合状态下的内部工作。在水解驱动的旋转过程中，观察到γ C末端螺旋明显不稳定。该行为归因于γ中的扭转应力，该扭转应力由在顶端关节面内γ旋转期间引起阻力的γ扭转α3β3相互作用引起。有趣的是，我们发现，γ的不稳定只发生在FoF 1对PMF引起的扭矩操作时;当PMF被消除的解耦器的效果消失。这种行为类似于汽车发动机的特性，其中轴承在负载下操作时比在空转期间对曲轴施加更大的力。
Significance FOF1, or ATP synthase, is often referred to as the “world’s smallest motor.” Similar to automotive engines, it employs a rotating shaft that interacts with mechanical actuators. When operating a combustion engine under load, the bearings exert significant forces on the crankshaft, leading to enhanced mechanical stress. Here, we demonstrate that analogous load-dependent effects occur in molecular motors. When FOF1 pumps protons against a transmembrane gradient, the rotor shaft undergoes structural destabilization attributed to resistive forces in its apical bearing. The effect disappears when the transmembrane gradient opposing proton pumping is short-circuited by an uncoupler, as predicted by fundamental principles of mechanics. Our observations highlight fascinating parallels between engine operation on the macroscale and the nanoscale. FoF1 is a membrane-bound molecular motor that uses proton-motive force (PMF) to drive the synthesis of ATP from ADP and Pi. Reverse operation generates PMF via ATP hydrolysis. Catalysis in either direction involves rotation of the γε shaft that connects the α3β3 head and the membrane-anchored cn ring. X-ray crystallography and other techniques have provided insights into the structure and function of FoF1 subcomplexes. However, interrogating the conformational dynamics of intact membrane-bound FoF1 during rotational catalysis has proven to be difficult. Here, we use hydrogen/deuterium exchange mass spectrometry to probe the inner workings of FoF1 in its natural membrane-bound state. A pronounced destabilization of the γ C-terminal helix during hydrolysis-driven rotation was observed. This behavior is attributed to torsional stress in γ, arising from γ⋅⋅⋅α3β3 interactions that cause resistance during γ rotation within the apical bearing. Intriguingly, we find that destabilization of γ occurs only when FoF1 operates against a PMF-induced torque; the effect disappears when PMF is eliminated by an uncoupler. This behavior resembles the properties of automotive engines, where bearings inflict greater forces on the crankshaft when operated under load than during idling.
影响因子：
5.7
作者：
West, Graham M.;Chien, Ellen Y. T.;Katritch, Vsevolod;Gatchalian, Jovylyn;Chalmers, Michael J.;Stevens, Raymond C.;Griffin, Patrick R.
通讯作者：
Griffin, Patrick R.
影响因子：
64.8
作者：
Allegretti, Matteo;Klusch, Niklas;Davies, Karen M.
通讯作者：
Davies, Karen M.
γ R268、γ Q269 和大肠杆菌 F1-ATP 酶的 β 亚基捕获环之间的相互作用对于催化活性非常重要。
DOI：
10.1074/jbc.m309948200
发表时间：
2003
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Greene,MatthewD;Frasch,WayneD
通讯作者：
Frasch,WayneD