Charge Transport in the ClC-type Chloride-Proton Anti-porter from Escherichia coli
Charge Transport in the ClC-type Chloride-Proton Anti-porter from Escherichia coli
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DOI：
10.1074/jbc.m110.163246
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发表时间：
2011-01-28
影响因子：
4.8
通讯作者：
Knapp, Ernst-Walter
中科院分区：
文献类型：
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作者：
Kieseritzky, Gernot;Knapp, Ernst-Walter
在C1 C氯离子通道超家族中鉴定的第一个氯离子转运蛋白来自大肠杆菌（Escherichia coli，EC 1C）（Accardi，A.，和米勒，C.（2004）Nature 427，803-807）。质子和氯离子转运及其耦合的途径、能量学和机制目前尚不清楚。为了弥合质子运输的疏水间隙，我们将四个稳定的埋藏沃茨建模为WT ECIC结构的两个亚基。它们一起形成了一条连接Glu-203与中心位点的氯化物的“水线”，后者又连接到Glu-148，即假设的质子出口位点。假设在EClC的中心氯离子结合位点中瞬时产生盐酸盐，则水线可以建立从Glu-203开始一直下游到Glu-148的跨膜质子转运途径。我们证明，通过静电和量子化学计算，质子化的中央氯离子是积极可行的。我们的特点是所有的氯占据和质子化状态可能相关的质子-氯离子运输循环ECLC，并构建了一个工作模型。因此，ECIC通过涉及多达两个过量质子和一到三个氯化物的状态演变，这是满足实验观察到的2：1化学计量所需的。我们发现ECLC的Y 445 F和E203 H突变体可以类似地工作，从而解释了为什么它们表现出几乎WT活性水平。耦合氯质子运输ECLC中的拟议机制与现有的实验数据是一致的，并允许预测特定氨基酸的重要性，这可能是由突变实验探测。
The first chloride transporter identified in the superfamily of ClC chloride channels was from Escherichia coli (EClC) (Accardi, A., and Miller, C. (2004) Nature 427, 803-807). Pathways, energetics, and mechanism of proton and chloride translocation and their coupling are up to now unclear. To bridge the hydrophobic gap of proton transport, we modeled four stable buried waters into both subunits of the WT EClC structure. Together they form a "water wire" connecting Glu-203 with the chloride at the central site, which in turn connects to Glu-148, the hypothetical proton exit site. Assuming the transient production of hydrochloride in the central chloride binding site of EClC, the water wire could establish a transmembrane proton transport pathway starting from Glu-203 all the way downstream onto Glu-148. We demonstrated by electrostatic and quantum chemical computations that protonation of the central chloride is energetically feasible. We characterized all chloride occupancies and protonation states possibly relevant for the proton-chloride transport cycle in EClC and constructed a working model. Accordingly, EClC evolves through states involving up to two excess protons and between one and three chlorides, which was required to fulfill the experimentally observed 2: 1 stoichiometry. We show that the Y445F and E203H mutants of EClC can operate similarly, thus explaining why they exhibit almost WT activity levels. The proposed mechanism of coupled chloride-proton transport in EClC is consistent with available experimental data and allows predictions on the importance of specific amino acids, which may be probed by mutation experiments.