Mutation of external glutamate residue reveals a new intermediate transport state and anion binding site in a CLC Cl-/H+ antiporter
Mutation of external glutamate residue reveals a new intermediate transport state and anion binding site in a CLC Cl-/H+ antiporter
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DOI：
10.1073/pnas.1901822116
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发表时间：
2019-08-27
影响因子：
11.1
通讯作者：
Lim, Hyun-Ho
中科院分区：
文献类型：
--
作者：
Park, Kunwoong;Lee, Byoung-Cheol;Lim, Hyun-Ho
CLC蛋白家族参与多种生理过程来控制细胞氯离子浓度。两种不同的CLC蛋白，Cl-通道和Cl-/H+反转运蛋白，在过去的几十年里已经在功能和结构上进行了研究。先前的研究表明，关键谷氨酸残基Glu（ex）的构象异质性可以解释clc型Cl-/H+反转运蛋白的转运周期。然而，两种不同的CLC反转运蛋白的结构快照表明，Glu（ex）存在多种构象（上、中、下）：来自大肠杆菌的CLC-ec1和来自嗜热红藻Cyanidioschyzon merolae的cmCLC。因此，我们的目的是在功能和结构水平上进一步研究CLC-ec1中Glu(ex)-构象的异质性，CLC-ec1是研究最深入的CLC反转运蛋白。在这里，我们发现Glu（ex）突变体E148D和野生型CLC-ec1具有不同阴离子浓度的晶体结构表明结构中间，即“Midlow”构象。我们还发现，当阴离子浓度较高时，E148D突变体的外部Cl-结合位点上方会出现一个额外的阴离子。此外，我们观察到，溶液中的羧酸盐可以利用异常可检测的短羧酸溴乙酸占据未封闭的E148A突变体的外部或中央Cl-结合位点。这些结果为以下观点提供了可信度：在单个CLC反转运蛋白的转运周期中，Gluex至少可以呈现3种不同的构象状态。
The CLC family of proteins are involved in a variety of physiological processes to control cellular chloride concentration. Two distinct classes of CLC proteins, Cl- channels and Cl-/H+ antiporters, have been functionally and structurally investigated over the last several decades. Previous studies have suggested that the conformational heterogeneity of the critical glutamate residue, Glu(ex), could explain the transport cycle of CLC-type Cl-/H+ antiporters. However, the presence of multiple conformations ( Up, Middle, and Down) of the Glu(ex) has been suggested from combined structural snapshots of 2 different CLC antiporters: CLC-ec1 from Escherichia coli and cmCLC from a thermophilic red alga, Cyanidioschyzon merolae. Thus, we aimed to investigate further the heterogeneity of Glu(ex)-conformations in CLC-ec1, the most deeply studied CLC antiporter, at both functional and structural levels. Here, we show that the crystal structures of the Glu(ex) mutant E148D and wild-type CLC-ec1 with varying anion concentrations suggest a structural intermediate, the "Midlow" conformation. We also found that an extra anion can be located above the external Cl--binding site in the E148D mutant when the anion concentration is high. Moreover, we observed that a carboxylate in solution can occupy either the external or central Cl--binding site in the ungated E148A mutant using an anomalously detectable short carboxylic acid, bromoacetate. These results lend credibility to the idea that the Gluex can take at least 3 distinct conformational states during the transport cycle of a single CLC antiporter.