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Estimating the true stability of the prehydrolytic outward-facing state in an ABC protein
CFTR, the anion channel mutated in cystic fibrosis patients, is a model ABC protein whose ATP-driven conformational cycle is observable at single-molecule level in patch-clamp recordings. Bursts of CFTR pore openings are coupled to tight dimerization of its two nucleotide-binding domains (NBDs) and...
Autores principales: | , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
eLife Sciences Publications, Ltd
2023
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10569789/ https://www.ncbi.nlm.nih.gov/pubmed/37782012 http://dx.doi.org/10.7554/eLife.90736 |
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author | Simon, Márton A Iordanov, Iordan Szollosi, Andras Csanády, László |
author_facet | Simon, Márton A Iordanov, Iordan Szollosi, Andras Csanády, László |
author_sort | Simon, Márton A |
collection | PubMed |
description | CFTR, the anion channel mutated in cystic fibrosis patients, is a model ABC protein whose ATP-driven conformational cycle is observable at single-molecule level in patch-clamp recordings. Bursts of CFTR pore openings are coupled to tight dimerization of its two nucleotide-binding domains (NBDs) and in wild-type (WT) channels are mostly terminated by ATP hydrolysis. The slow rate of non-hydrolytic closure – which determines how tightly bursts and ATP hydrolysis are coupled – is unknown, as burst durations of catalytic site mutants span a range of ~200-fold. Here, we show that Walker A mutation K1250A, Walker B mutation D1370N, and catalytic glutamate mutations E1371S and E1371Q all completely disrupt ATP hydrolysis. True non-hydrolytic closing rate of WT CFTR approximates that of K1250A and E1371S. That rate is slowed ~15-fold in E1371Q by a non-native inter-NBD H-bond, and accelerated ~15-fold in D1370N. These findings uncover unique features of the NBD interface in human CFTR. |
format | Online Article Text |
id | pubmed-10569789 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | eLife Sciences Publications, Ltd |
record_format | MEDLINE/PubMed |
spelling | pubmed-105697892023-10-13 Estimating the true stability of the prehydrolytic outward-facing state in an ABC protein Simon, Márton A Iordanov, Iordan Szollosi, Andras Csanády, László eLife Structural Biology and Molecular Biophysics CFTR, the anion channel mutated in cystic fibrosis patients, is a model ABC protein whose ATP-driven conformational cycle is observable at single-molecule level in patch-clamp recordings. Bursts of CFTR pore openings are coupled to tight dimerization of its two nucleotide-binding domains (NBDs) and in wild-type (WT) channels are mostly terminated by ATP hydrolysis. The slow rate of non-hydrolytic closure – which determines how tightly bursts and ATP hydrolysis are coupled – is unknown, as burst durations of catalytic site mutants span a range of ~200-fold. Here, we show that Walker A mutation K1250A, Walker B mutation D1370N, and catalytic glutamate mutations E1371S and E1371Q all completely disrupt ATP hydrolysis. True non-hydrolytic closing rate of WT CFTR approximates that of K1250A and E1371S. That rate is slowed ~15-fold in E1371Q by a non-native inter-NBD H-bond, and accelerated ~15-fold in D1370N. These findings uncover unique features of the NBD interface in human CFTR. eLife Sciences Publications, Ltd 2023-10-02 /pmc/articles/PMC10569789/ /pubmed/37782012 http://dx.doi.org/10.7554/eLife.90736 Text en © 2023, Simon et al https://creativecommons.org/licenses/by/4.0/This article is distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use and redistribution provided that the original author and source are credited. |
spellingShingle | Structural Biology and Molecular Biophysics Simon, Márton A Iordanov, Iordan Szollosi, Andras Csanády, László Estimating the true stability of the prehydrolytic outward-facing state in an ABC protein |
title | Estimating the true stability of the prehydrolytic outward-facing state in an ABC protein |
title_full | Estimating the true stability of the prehydrolytic outward-facing state in an ABC protein |
title_fullStr | Estimating the true stability of the prehydrolytic outward-facing state in an ABC protein |
title_full_unstemmed | Estimating the true stability of the prehydrolytic outward-facing state in an ABC protein |
title_short | Estimating the true stability of the prehydrolytic outward-facing state in an ABC protein |
title_sort | estimating the true stability of the prehydrolytic outward-facing state in an abc protein |
topic | Structural Biology and Molecular Biophysics |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10569789/ https://www.ncbi.nlm.nih.gov/pubmed/37782012 http://dx.doi.org/10.7554/eLife.90736 |
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