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Mutant cycles at CFTR’s non-canonical ATP-binding site support little interface separation during gating

Cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel belonging to the adenosine triphosphate (ATP)-binding cassette (ABC) superfamily. ABC proteins share a common molecular mechanism that couples ATP binding and hydrolysis at two nucleotide-binding domains (NBDs) to diver...

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Autores principales: Szollosi, Andras, Muallem, Daniella R., Csanády, László, Vergani, Paola
Formato: Texto
Lenguaje:English
Publicado: The Rockefeller University Press 2011
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3105517/
https://www.ncbi.nlm.nih.gov/pubmed/21576373
http://dx.doi.org/10.1085/jgp.201110608
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author Szollosi, Andras
Muallem, Daniella R.
Csanády, László
Vergani, Paola
author_facet Szollosi, Andras
Muallem, Daniella R.
Csanády, László
Vergani, Paola
author_sort Szollosi, Andras
collection PubMed
description Cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel belonging to the adenosine triphosphate (ATP)-binding cassette (ABC) superfamily. ABC proteins share a common molecular mechanism that couples ATP binding and hydrolysis at two nucleotide-binding domains (NBDs) to diverse functions. This involves formation of NBD dimers, with ATP bound at two composite interfacial sites. In CFTR, intramolecular NBD dimerization is coupled to channel opening. Channel closing is triggered by hydrolysis of the ATP molecule bound at composite site 2. Site 1, which is non-canonical, binds nucleotide tightly but is not hydrolytic. Recently, based on kinetic arguments, it was suggested that this site remains closed for several gating cycles. To investigate movements at site 1 by an independent technique, we studied changes in thermodynamic coupling between pairs of residues on opposite sides of this site. The chosen targets are likely to interact based on both phylogenetic analysis and closeness on structural models. First, we mutated T460 in NBD1 and L1353 in NBD2 (the corresponding site-2 residues become energetically coupled as channels open). Mutation T460S accelerated closure in hydrolytic conditions and in the nonhydrolytic K1250R background; mutation L1353M did not affect these rates. Analysis of the double mutant showed additive effects of mutations, suggesting that energetic coupling between the two residues remains unchanged during the gating cycle. We next investigated pairs 460–1348 and 460–1375. Although both mutations H1348A and H1375A produced dramatic changes in hydrolytic and nonhydrolytic channel closing rates, in the corresponding double mutants these changes proved mostly additive with those caused by mutation T460S, suggesting little change in energetic coupling between either positions 460–1348 or positions 460–1375 during gating. These results provide independent support for a gating model in which ATP-bound composite site 1 remains closed throughout the gating cycle.
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spelling pubmed-31055172011-12-01 Mutant cycles at CFTR’s non-canonical ATP-binding site support little interface separation during gating Szollosi, Andras Muallem, Daniella R. Csanády, László Vergani, Paola J Gen Physiol Article Cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel belonging to the adenosine triphosphate (ATP)-binding cassette (ABC) superfamily. ABC proteins share a common molecular mechanism that couples ATP binding and hydrolysis at two nucleotide-binding domains (NBDs) to diverse functions. This involves formation of NBD dimers, with ATP bound at two composite interfacial sites. In CFTR, intramolecular NBD dimerization is coupled to channel opening. Channel closing is triggered by hydrolysis of the ATP molecule bound at composite site 2. Site 1, which is non-canonical, binds nucleotide tightly but is not hydrolytic. Recently, based on kinetic arguments, it was suggested that this site remains closed for several gating cycles. To investigate movements at site 1 by an independent technique, we studied changes in thermodynamic coupling between pairs of residues on opposite sides of this site. The chosen targets are likely to interact based on both phylogenetic analysis and closeness on structural models. First, we mutated T460 in NBD1 and L1353 in NBD2 (the corresponding site-2 residues become energetically coupled as channels open). Mutation T460S accelerated closure in hydrolytic conditions and in the nonhydrolytic K1250R background; mutation L1353M did not affect these rates. Analysis of the double mutant showed additive effects of mutations, suggesting that energetic coupling between the two residues remains unchanged during the gating cycle. We next investigated pairs 460–1348 and 460–1375. Although both mutations H1348A and H1375A produced dramatic changes in hydrolytic and nonhydrolytic channel closing rates, in the corresponding double mutants these changes proved mostly additive with those caused by mutation T460S, suggesting little change in energetic coupling between either positions 460–1348 or positions 460–1375 during gating. These results provide independent support for a gating model in which ATP-bound composite site 1 remains closed throughout the gating cycle. The Rockefeller University Press 2011-06 /pmc/articles/PMC3105517/ /pubmed/21576373 http://dx.doi.org/10.1085/jgp.201110608 Text en © 2011 Szollosi et al. This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).
spellingShingle Article
Szollosi, Andras
Muallem, Daniella R.
Csanády, László
Vergani, Paola
Mutant cycles at CFTR’s non-canonical ATP-binding site support little interface separation during gating
title Mutant cycles at CFTR’s non-canonical ATP-binding site support little interface separation during gating
title_full Mutant cycles at CFTR’s non-canonical ATP-binding site support little interface separation during gating
title_fullStr Mutant cycles at CFTR’s non-canonical ATP-binding site support little interface separation during gating
title_full_unstemmed Mutant cycles at CFTR’s non-canonical ATP-binding site support little interface separation during gating
title_short Mutant cycles at CFTR’s non-canonical ATP-binding site support little interface separation during gating
title_sort mutant cycles at cftr’s non-canonical atp-binding site support little interface separation during gating
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3105517/
https://www.ncbi.nlm.nih.gov/pubmed/21576373
http://dx.doi.org/10.1085/jgp.201110608
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