ATPase Active-Site Electrostatic Interactions Control the Global Conformation of the 100 kDa SecA Translocase
ATPase Active-Site Electrostatic Interactions Control the Global Conformation of the 100 kDa SecA Translocase
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DOI：
10.1021/ja306361q
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发表时间：
2013-02-27
影响因子：
15
通讯作者：
Hunt, John F.
中科院分区：
文献类型：
--
作者：
Kim, Dorothy M.;Zheng, Haiyan;Hunt, John F.
SECA是一种被广泛研究的机械酶，它通过真细菌细胞膜上的蛋白质传导通道，利用三磷酸腺苷的水解酶来驱动分泌蛋白质的排泄。SecA的ATPase马达与DEAD-box RNA解旋酶的ATPase马达有很强的同源性。目前尚不清楚其ATPase活性位点上的局部化学事件如何控制一个类似100 kDa的多结构域酶的整体构象，并驱动蛋白质的运输。在本文中，我们利用生物物理方法建立了ATPase活性位点上的单个静电电荷控制着SecA的整体构象。该酶经历了ATP调节的吸热构象转变(ECT)，据信涉及与蛋白质运输反应相似的结构力学。我们已经表征了催化碱基中等构型谷氨酸到谷氨酰胺的突变的影响，这种突变模仿了活性位点上ATP水解的直接静电后果。量热研究表明，该突变促进了大肠杆菌SecA中的ECT，并在枯草芽孢杆菌SecA中完全触发了ECT。与在ECT过程中观察到的熵的大幅增加一致，氢-重离子交换质谱仪表明，它增加了远离ATPase活性位点的结构域界面上的蛋白质骨架动力学。催化谷氨酸是SECA中类似于250个带电氨基酸之一，但其侧链电荷的中和足以引发这个100 kDa酶的全球有序-无序转变。介导这一效应的复杂的结构相互作用网络将ATP水解过程中的局部静电变化与SecA的整体构象和动态变化结合在一起。这个网络构成了变构机械力化学的基础，它有效地利用储存在ATP中的化学能来驱动复杂的机械过程。
SecA is an intensively studied mechanoenzyme that uses ATP hydrolysis to drive processive extrusion of secreted proteins through a protein-conducting channel in the cytoplasmic membrane of eubacteria. The ATPase motor of SecA is strongly homologous to that in DEAD-box RNA helicases. It remains unclear how local chemical events in its ATPase active site control the overall conformation of an similar to 100 kDa multidomain enzyme and drive protein transport. In this paper, we use biophysical methods to establish that a single electrostatic charge in the ATPase active site controls the global conformation of SecA. The enzyme undergoes an ATP-modulated endothermic conformational transition (ECT) believed to involve similar structural mechanics to the protein transport reaction. We have characterized the effects of an isosteric glutamate-to-glutamine mutation in the catalytic base, a mutation which mimics the immediate electrostatic consequences of ATP hydrolysis in the active site. Calorimetric studies demonstrate that this mutation facilitates the ECT in Escherichia coli SecA and triggers it completely in Bacillus subtilis SecA. Consistent with the substantial increase in entropy observed in the course of the ECT, hydrogen-deuterium exchange mass spectrometry demonstrates that it increases protein backbone dynamics in domain-domain interfaces at remote locations from the ATPase active site. The catalytic glutamate is one of similar to 250 charged amino acids in SecA, and yet neutralization of its side chain charge is sufficient to trigger a global order-disorder transition in this 100 kDa enzyme. The intricate network of structural interactions mediating this effect couples local electrostatic changes during ATP hydrolysis to global conformational and dynamic changes in SecA. This network forms the foundation of the allosteric mechanochemistry that efficiently harnesses the chemical energy stored in ATP to drive complex mechanical processes.