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Mutation of external glutamate residue reveals a new intermediate transport state and anion binding site in a CLC Cl-/H+ antiporter

Title
Mutation of external glutamate residue reveals a new intermediate transport state and anion binding site in a CLC Cl-/H+ antiporter
Authors
Park, KunwoongLee, Byoung-CheolIm, Hyun Ho
Issue Date
2019-08
Citation
Proceedings of the National Academy of Sciences of the United States of America, 116(35), 17345-17354
Type
Article
Article Type
Article
Author Keywords
chloride channelantiporteranion bindingcrystal structure
Keywords
CHLORIDE CHANNELSSUBSTRATE-BINDINGPROTON TRANSPORTPORESTOICHIOMETRYSELECTIVITYMECHANISMPATHWAYSRESISTANCEPHYSIOLOGY
ISSN
0027-8424
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, Gluex, could explain the transport cycle of CLC-type Cl-/H+ antiporters. However, the presence of multiple conformations (Up, Middle, and Down) of the Gluex has been suggested from combined structural snapshots of 2 different CLC antiporters: CLCec1 from Escherichia coli and cmCLC from a thermophilic red alga, Cyanidioschyzon merolae. Thus, we aimed to investigate further the heterogeneity of Gluex-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 Gluex 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. © 2019 National Academy of Sciences. All rights reserved.
URI
http://hdl.handle.net/20.500.11750/10659
DOI
10.1073/pnas.1901822116
Publisher
National Academy of Sciences
Files:
There are no files associated with this item.
Collection:
ETC1. Journal Articles


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