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Title: CLC CI-/H+ Transporters Constrained by Covalent Cross-Linking

Abstract

CLC Cl-/H+ exchangers are homodimers with Cl--binding and H+-coupling residues contained within each subunit. It is not known whether the transport mechanism requires conformational rearrangement between subunits or whether each subunit operates as a separate exchanger. We designed various cysteine substitution mutants on a cysteine-less background of CLC-ec1, a bacterial CLC exchanger of known structure, with the aim of covalently linking the subunits. The constructs were cross-linked in air or with exogenous oxidant, and the cross-linked proteins were reconstituted to assess their function. In addition to conventional disulfides, a cysteine-lysine cross-bridge was formed with I2 as an oxidant. The constructs, all of which contained one, two, or four cross-bridges, were functionally active and kinetically competent with respect to Cl- turnover rate, Cl-/H+ exchange stoichiometry, and H+ pumping driven by a Cl- gradient. These results imply that large quaternary rearrangements, such as those known to occur for 'common gating' in CLC channels, are not necessary for the ion transport cycle and that it is therefore likely that the transport mechanism is carried out by the subunits working individually, as with 'fast gating' of the CLC channels.

Authors:
;
Publication Date:
Research Org.:
Brookhaven National Laboratory (BNL) National Synchrotron Light Source
Sponsoring Org.:
Doe - Office Of Science
OSTI Identifier:
959489
Report Number(s):
BNL-82475-2009-JA
Journal ID: ISSN 0027-8424; PNASA6; TRN: US201016%%633
DOE Contract Number:  
DE-AC02-98CH10886
Resource Type:
Journal Article
Resource Relation:
Journal Name: Proceedings of the National Academy of Sciences of the USA; Journal Volume: 104; Journal Issue: 52
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 99 GENERAL AND MISCELLANEOUS//MATHEMATICS, COMPUTING, AND INFORMATION SCIENCE; AIR; CROSS-LINKING; CYSTEINE; DISULFIDES; MUTANTS; PROTEINS; PUMPING; RESIDUES; STOICHIOMETRY; TRANSPORT; national synchrotron light source

Citation Formats

Nguitragool,W., and Miller, C. CLC CI-/H+ Transporters Constrained by Covalent Cross-Linking. United States: N. p., 2007. Web. doi:10.1073/pnas.0708639104.
Nguitragool,W., & Miller, C. CLC CI-/H+ Transporters Constrained by Covalent Cross-Linking. United States. doi:10.1073/pnas.0708639104.
Nguitragool,W., and Miller, C. Mon . "CLC CI-/H+ Transporters Constrained by Covalent Cross-Linking". United States. doi:10.1073/pnas.0708639104.
@article{osti_959489,
title = {CLC CI-/H+ Transporters Constrained by Covalent Cross-Linking},
author = {Nguitragool,W. and Miller, C.},
abstractNote = {CLC Cl-/H+ exchangers are homodimers with Cl--binding and H+-coupling residues contained within each subunit. It is not known whether the transport mechanism requires conformational rearrangement between subunits or whether each subunit operates as a separate exchanger. We designed various cysteine substitution mutants on a cysteine-less background of CLC-ec1, a bacterial CLC exchanger of known structure, with the aim of covalently linking the subunits. The constructs were cross-linked in air or with exogenous oxidant, and the cross-linked proteins were reconstituted to assess their function. In addition to conventional disulfides, a cysteine-lysine cross-bridge was formed with I2 as an oxidant. The constructs, all of which contained one, two, or four cross-bridges, were functionally active and kinetically competent with respect to Cl- turnover rate, Cl-/H+ exchange stoichiometry, and H+ pumping driven by a Cl- gradient. These results imply that large quaternary rearrangements, such as those known to occur for 'common gating' in CLC channels, are not necessary for the ion transport cycle and that it is therefore likely that the transport mechanism is carried out by the subunits working individually, as with 'fast gating' of the CLC channels.},
doi = {10.1073/pnas.0708639104},
journal = {Proceedings of the National Academy of Sciences of the USA},
number = 52,
volume = 104,
place = {United States},
year = {Mon Jan 01 00:00:00 EST 2007},
month = {Mon Jan 01 00:00:00 EST 2007}
}