The mechanism of rotatory catalysis in ATP-hydrolyzing molecular motors remains an unresolved puzzle in biological energy transfer. Notwithstanding the wealth of available biochemical and structural information inferred from years of experiments, knowledge on how the coupling between the chemical and mechanical steps within motors enforces directional rotatory movements remains fragmentary. Even more contentious is to pinpoint the rate-limiting step of a multistep rotation process. Here, using vacuolar or V₁-type hexameric ATPase as an exemplary rotational motor, we present a model of the complete 4-step conformational cycle involved in rotatory catalysis. First, using X-ray crystallography, a new intermediate or “dwell” is identified, which enables the release of an inorganic phosphate (or P_i) after ATP hydrolysis. Using molecular dynamics simulations, this new dwell is placed in a sequence with three other crystal structures to derive a putative cyclic rotation path. Free-energy simulations are employed to estimate the rate of the hexameric protein transformations and delineate allosteric effects that allow new reactant ATP entry only after hydrolysis product exit. An analysis of transfer entropy brings to light how the side-chain-level interactions transcend into larger-scale reorganizations, highlighting the role of the ubiquitous arginine-finger residues in coupling chemical and mechanical information. An inspection of all known rates encompassing the 4-step rotation mechanism implicates the overcoming of the ADP interactions with V₁-ATPase to be the rate-limiting step of motor action.

中文翻译：

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通过 X 射线晶体学和分子模拟揭示旋转催化的隐藏中间体

ATP水解分子马达中的旋转催化机制仍然是生物能量转移中未解决的难题。尽管从多年的实验中推断出大量可用的生化和结构信息，但关于电机内化学和机械步骤之间的耦合如何强制定向旋转运动的知识仍然是零碎的。更有争议的是确定多步旋转过程的限速步骤。在这里，使用液泡或 V ₁型六聚体 ATP 酶作为示例性旋转马达，我们提出了一个涉及旋转催化的完整 4 步构象循环的模型。首先，使用 X 射线晶体学，确定了一种新的中间体或“驻留”，它可以释放无机磷酸盐（或 P _i) ATP 水解后。使用分子动力学模拟，这个新的停留被放置在一个具有其他三个晶体结构的序列中，以推导出一个假定的循环旋转路径。使用自由能模拟来估计六聚体蛋白质转化的速率并描绘仅在水解产物退出后才允许新反应物 ATP 进入的变构效应。对转移熵的分析揭示了侧链水平的相互作用如何超越更大规模的重组，突出了无处不在的精氨酸-指残基在耦合化学和机械信息中的作用。对包含 4 步旋转机制的所有已知速率的检查表明，ADP 与 V ₁ -ATPase 的相互作用是运动动作的限速步骤。