In:
Proceedings of the National Academy of Sciences, Proceedings of the National Academy of Sciences, Vol. 116, No. 35 ( 2019-08-27), p. 17345-17354
Abstract:
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.
Type of Medium:
Online Resource
ISSN:
0027-8424
,
1091-6490
DOI:
10.1073/pnas.1901822116
Language:
English
Publisher:
Proceedings of the National Academy of Sciences
Publication Date:
2019
detail.hit.zdb_id:
209104-5
detail.hit.zdb_id:
1461794-8
SSG:
11
SSG:
12
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