文章基本信息

标题：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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作者：Kunwoong Park ; Kunwoong Park ; Byoung-Cheol Lee 等
期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2019
卷号：116
期号：35
页码：17345-17354
DOI：10.1073/pnas.1901822116
出版社：The National Academy of Sciences of the United States of America
摘要：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 Glu ex can take at least 3 distinct conformational states during the transport cycle of a single CLC antiporter..
关键词：chloride channel ; antiporter ; anion binding ; crystal structure