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Title: Synergism Between Halide Binding and Proton Transport in a CLC-type Exchanger

Abstract

The Cl{sup -}/H{sup +} exchange-transporter CLC-ec1 mediates stoichiometric transmembrane exchange of two Cl{sup -} ions for one proton. A conserved tyrosine residue, Y445, coordinates one of the bound Cl{sup -} ions visible in the structure of this protein and is located near the intersection of the Cl{sup -} and H{sup +} pathways. Mutants of this tyrosine were scrutinized for effects on the coupled transport of Cl{sup -} and H{sup +} determined electrophysiologically and on protein structure determined crystallographically. Despite the strong conservation of Y445 in the CLC family, substitution of F or W at this position preserves wild-type transport behavior. Substitution by A, E, or H, however, produces uncoupled proteins with robust Cl{sup -} transport but greatly impaired movement of H{sup +}+. The obligatory 2 Cl{sup -}/1 H{sup +} stoichiometry is thus lost in these mutants. The structures of all the mutants are essentially identical to wild-type, but apparent anion occupancy in the Cl{sup -} binding region correlates with functional H{sup +} coupling. In particular, as determined by anomalous diffraction in crystals grown in Br{sup -}, an electrophysiologically competent Cl{sup -} analogue, the well-coupled transporters show strong Br{sup -} electron density at the 'inner' and 'central' Cl{sup -} binding sites.more » However, in the uncoupled mutants, Br{sup -} density is absent at the central site, while still present at the inner site. An additional mutant, Y445L, is intermediate in both functional and structural features. This mutant clearly exchanges H{sup +} for Cl{sup -}, but at a reduced H{sup +}-to-Cl{sup -} ratio; likewise, both the central and inner sites are occupied by Br{sup -}, but the central site shows lower Br{sup -} density than in wild-type (or in Y445F,W). The correlation between proton coupling and central-site occupancy argues that halide binding to the central transport site somehow facilitates movement of H{sup +}, a synergism that is not readily understood in terms of alternating-site antiport schemes.« less

Authors:
; ; ; ;
Publication Date:
Research Org.:
Brookhaven National Laboratory (BNL) National Synchrotron Light Source
Sponsoring Org.:
Doe - Office Of Science
OSTI Identifier:
930095
Report Number(s):
BNL-80733-2008-JA
Journal ID: ISSN 0022-2836; JMOBAK; TRN: US0806703
DOE Contract Number:  
DE-AC02-98CH10886
Resource Type:
Journal Article
Resource Relation:
Journal Name: Journal of Molecular Biology; Journal Volume: 362; Journal Issue: 4
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 72 PHYSICS OF ELEMENTARY PARTICLES AND FIELDS; ANIONS; COORDINATES; CORRELATIONS; COUPLING; CRYSTALS; DENSITY; DIFFRACTION; ELECTRON DENSITY; FUNCTIONALS; HALIDES; IONS; MUTANTS; PROTEIN STRUCTURE; PROTEINS; PROTON TRANSPORT; PROTONS; STOICHIOMETRY; SYNERGISM; TRANSPORT; TYROSINE; national synchrotron light source

Citation Formats

Accardi,A., Lobet, S., Williams, C., Miller, C., and Dutzler, R. Synergism Between Halide Binding and Proton Transport in a CLC-type Exchanger. United States: N. p., 2006. Web. doi:10.1016/j.jmb.2006.07.081.
Accardi,A., Lobet, S., Williams, C., Miller, C., & Dutzler, R. Synergism Between Halide Binding and Proton Transport in a CLC-type Exchanger. United States. doi:10.1016/j.jmb.2006.07.081.
Accardi,A., Lobet, S., Williams, C., Miller, C., and Dutzler, R. Sun . "Synergism Between Halide Binding and Proton Transport in a CLC-type Exchanger". United States. doi:10.1016/j.jmb.2006.07.081.
@article{osti_930095,
title = {Synergism Between Halide Binding and Proton Transport in a CLC-type Exchanger},
author = {Accardi,A. and Lobet, S. and Williams, C. and Miller, C. and Dutzler, R.},
abstractNote = {The Cl{sup -}/H{sup +} exchange-transporter CLC-ec1 mediates stoichiometric transmembrane exchange of two Cl{sup -} ions for one proton. A conserved tyrosine residue, Y445, coordinates one of the bound Cl{sup -} ions visible in the structure of this protein and is located near the intersection of the Cl{sup -} and H{sup +} pathways. Mutants of this tyrosine were scrutinized for effects on the coupled transport of Cl{sup -} and H{sup +} determined electrophysiologically and on protein structure determined crystallographically. Despite the strong conservation of Y445 in the CLC family, substitution of F or W at this position preserves wild-type transport behavior. Substitution by A, E, or H, however, produces uncoupled proteins with robust Cl{sup -} transport but greatly impaired movement of H{sup +}+. The obligatory 2 Cl{sup -}/1 H{sup +} stoichiometry is thus lost in these mutants. The structures of all the mutants are essentially identical to wild-type, but apparent anion occupancy in the Cl{sup -} binding region correlates with functional H{sup +} coupling. In particular, as determined by anomalous diffraction in crystals grown in Br{sup -}, an electrophysiologically competent Cl{sup -} analogue, the well-coupled transporters show strong Br{sup -} electron density at the 'inner' and 'central' Cl{sup -} binding sites. However, in the uncoupled mutants, Br{sup -} density is absent at the central site, while still present at the inner site. An additional mutant, Y445L, is intermediate in both functional and structural features. This mutant clearly exchanges H{sup +} for Cl{sup -}, but at a reduced H{sup +}-to-Cl{sup -} ratio; likewise, both the central and inner sites are occupied by Br{sup -}, but the central site shows lower Br{sup -} density than in wild-type (or in Y445F,W). The correlation between proton coupling and central-site occupancy argues that halide binding to the central transport site somehow facilitates movement of H{sup +}, a synergism that is not readily understood in terms of alternating-site antiport schemes.},
doi = {10.1016/j.jmb.2006.07.081},
journal = {Journal of Molecular Biology},
number = 4,
volume = 362,
place = {United States},
year = {Sun Jan 01 00:00:00 EST 2006},
month = {Sun Jan 01 00:00:00 EST 2006}
}