Intracellular proton access in a Cl(-)/H(+) antiporter.
Intracellular proton access in a Cl(-)/H(+) antiporter.
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DOI：
10.1371/journal.pbio.1001441
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发表时间：
2012
期刊：
影响因子：
9.8
通讯作者：
Miller C
中科院分区：
文献类型：
--
作者：
Lim HH;Shane T;Miller C
诱变、功能分析和晶体结构确定了一个水样通道，质子通过该通道进入肠道细菌的Cl−/H+反端口蛋白内部。CLC家族的氯离子转运膜蛋白以两种不同的机制形式出现：H+门控Cl -通道和Cl - /H+反转运蛋白。跨膜H+运动是两种CLC的基本特征。CLC反转运蛋白的x射线晶体结构显示了Cl -离子通过这些蛋白质的途径，但H+途径仅通过两个保守的谷氨酸残基推断，这两个残基作为H+通过蛋白质的途径的中转站。在转运过程中，面向细胞外的H+转移谷氨酸直接暴露在水溶液中，而细胞内的谷氨酸E203 （Gluin）则被埋在蛋白质中。这里研究了细菌反转运蛋白CLC-ec1细胞内表面的两个区域，即“极性”和“界面”，作为细胞内水质子获得胶粘蛋白的可能途径。极性区多个残基的突变对反转运率影响不大。相反，E202（一种位于蛋白质-水界面的保守谷氨酸）的突变导致Cl−/H+反转运速率严重减慢。E202Y的x射线晶体结构是这些取代中受抑制最强烈的，显示出一个水通道，导致胶粘蛋白被交叉亚基相互作用物理阻断；此外，这种突变对单体CLC变异的影响很小，后者必然缺乏这种相互作用。提出的几条实验线表明，E202作为一种水组织者，创造了一个质子管道，将细胞内溶剂与胶质连接起来。“CLC”超家族的氯质子反端口蛋白是一种跨膜蛋白，可形成同型二聚体，并用于无数生理目的，所有这些都需要Cl -阴离子和H+阳离子在生物膜上以相反的方向协调运动。虽然Cl -离子通过CLC反转运蛋白的途径是已知的，但我们目前只间接地瞥见了质子如何通过这些膜嵌入的蛋白质。通过结合机制和结构方法，我们在细菌Cl−/H+反转运蛋白中发现了一条质子进入途径，该途径允许细胞内质子进入蛋白质内部并参与耦合的反转运机制。我们得出结论，E202是一种高度保守的谷氨酸残基，它可以组织水分子并将质子引导到邻近的谷氨酸E203（称为“Gluin”），这是反港口机制的关键残基。
Mutagenesis, functional analysis, and crystal structures identify a watery tunnel through which protons enter the interior of a Cl−/H+ antiport protein involved in acid resistance of enteric bacteria. Chloride-transporting membrane proteins of the CLC family appear in two distinct mechanistic flavors: H+-gated Cl− channels and Cl−/H+ antiporters. Transmembrane H+ movement is an essential feature of both types of CLC. X-ray crystal structures of CLC antiporters show the Cl− ion pathway through these proteins, but the H+ pathway is known only inferentially by two conserved glutamate residues that act as way-stations for H+ in its path through the protein. The extracellular-facing H+ transfer glutamate becomes directly exposed to aqueous solution during the transport cycle, but the intracellular glutamate E203, Gluin, is buried within the protein. Two regions, denoted “polar” and “interfacial,” at the intracellular surface of the bacterial antiporter CLC-ec1 are examined here as possible pathways by which intracellular aqueous protons gain access to Gluin. Mutations at multiple residues of the polar region have little effect on antiport rates. In contrast, mutation of E202, a conserved glutamate at the protein–water boundary of the interfacial region, leads to severe slowing of the Cl−/H+ antiport rate. An X-ray crystal structure of E202Y, the most strongly inhibited of these substitutions, shows an aqueous portal leading to Gluin physically blocked by cross-subunit interactions; moreover, this mutation has only minimal effect on a monomeric CLC variant, which necessarily lacks such interactions. The several lines of experiments presented argue that E202 acts as a water-organizer that creates a proton conduit connecting intracellular solvent with Gluin. Chloride-proton antiport proteins of the “CLC” superfamily are transmembrane proteins that form homodimers and are used for myriad physiological purposes, all requiring the coordinated movements of Cl− anions and H+ cations in opposite directions across biological membranes. While the pathway for Cl− ions through CLC antiporters is known, we currently have only indirect glimpses of how protons navigate their way through these membrane-embedded proteins. By combining mechanistic and structural approaches, we identify a proton-access pathway in a bacterial Cl−/H+ antiporter that allows intracellular protons to enter the protein interior and engage in the coupled antiport mechanism. We conclude that E202, a highly conserved glutamate residue, serves to organize water molecules and guide protons to the adjacent glutamate E203 (known as “Gluin”), a critical residue for the antiport mechanism.
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影响因子：
3.8
作者：
Accardi, Alessio;Walden, Michael;Nguitragool, Wang;Jayaram, Hariharan;Williams, Carole;Miller, Christopher
通讯作者：
Miller, Christopher
CLC Cl-/H+ 交换器中的细胞内质子转移突变体。
DOI：
10.1085/jgp.200810112
发表时间：
2009-02
期刊：
The Journal of general physiology
影响因子：
--
作者：
Lim HH;Miller C
通讯作者：
Miller C
影响因子：
3.4
作者：
Phillips, Sabrina;Brammer, Ashley E.;Matulef, Kimberly
通讯作者：
Matulef, Kimberly
影响因子：
56.9
作者：
Dutzler, R;Campbell, EB;MacKinnon, R
通讯作者：
MacKinnon, R
影响因子：
2.9
作者：
MIDDLETON, RE;PHEASANT, DJ;MILLER, C
通讯作者：
MILLER, C