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Molecular pathology of the R117H cystic fibrosis mutation is explained by loss of a hydrogen bond

The phosphorylation-activated anion channel cystic fibrosis transmembrane conductance regulator (CFTR) is gated by an ATP hydrolysis cycle at its two cytosolic nucleotide-binding domains, and is essential for epithelial salt-water transport. A large number of CFTR mutations cause cystic fibrosis. Si...

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Autores principales: Simon, Márton A, Csanády, László
Formato: Online Artículo Texto
Lenguaje:English
Publicado: eLife Sciences Publications, Ltd 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8673840/
https://www.ncbi.nlm.nih.gov/pubmed/34870594
http://dx.doi.org/10.7554/eLife.74693
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author Simon, Márton A
Csanády, László
author_facet Simon, Márton A
Csanády, László
author_sort Simon, Márton A
collection PubMed
description The phosphorylation-activated anion channel cystic fibrosis transmembrane conductance regulator (CFTR) is gated by an ATP hydrolysis cycle at its two cytosolic nucleotide-binding domains, and is essential for epithelial salt-water transport. A large number of CFTR mutations cause cystic fibrosis. Since recent breakthrough in targeted pharmacotherapy, CFTR mutants with impaired gating are candidates for stimulation by potentiator drugs. Thus, understanding the molecular pathology of individual mutations has become important. The relatively common R117H mutation affects an extracellular loop, but nevertheless causes a strong gating defect. Here, we identify a hydrogen bond between the side chain of arginine 117 and the backbone carbonyl group of glutamate 1124 in the cryo-electronmicroscopic structure of phosphorylated, ATP-bound CFTR. We address the functional relevance of that interaction for CFTR gating using macroscopic and microscopic inside-out patch-clamp recordings. Employing thermodynamic double-mutant cycles, we systematically track gating-state-dependent changes in the strength of the R117-E1124 interaction. We find that the H-bond is formed only in the open state, but neither in the short-lived ‘flickery’ nor in the long-lived ‘interburst’ closed state. Loss of this H-bond explains the strong gating phenotype of the R117H mutant, including robustly shortened burst durations and strongly reduced intraburst open probability. The findings may help targeted potentiator design.
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spelling pubmed-86738402021-12-17 Molecular pathology of the R117H cystic fibrosis mutation is explained by loss of a hydrogen bond Simon, Márton A Csanády, László eLife Structural Biology and Molecular Biophysics The phosphorylation-activated anion channel cystic fibrosis transmembrane conductance regulator (CFTR) is gated by an ATP hydrolysis cycle at its two cytosolic nucleotide-binding domains, and is essential for epithelial salt-water transport. A large number of CFTR mutations cause cystic fibrosis. Since recent breakthrough in targeted pharmacotherapy, CFTR mutants with impaired gating are candidates for stimulation by potentiator drugs. Thus, understanding the molecular pathology of individual mutations has become important. The relatively common R117H mutation affects an extracellular loop, but nevertheless causes a strong gating defect. Here, we identify a hydrogen bond between the side chain of arginine 117 and the backbone carbonyl group of glutamate 1124 in the cryo-electronmicroscopic structure of phosphorylated, ATP-bound CFTR. We address the functional relevance of that interaction for CFTR gating using macroscopic and microscopic inside-out patch-clamp recordings. Employing thermodynamic double-mutant cycles, we systematically track gating-state-dependent changes in the strength of the R117-E1124 interaction. We find that the H-bond is formed only in the open state, but neither in the short-lived ‘flickery’ nor in the long-lived ‘interburst’ closed state. Loss of this H-bond explains the strong gating phenotype of the R117H mutant, including robustly shortened burst durations and strongly reduced intraburst open probability. The findings may help targeted potentiator design. eLife Sciences Publications, Ltd 2021-12-06 /pmc/articles/PMC8673840/ /pubmed/34870594 http://dx.doi.org/10.7554/eLife.74693 Text en © 2021, Simon and Csanády 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
Csanády, László
Molecular pathology of the R117H cystic fibrosis mutation is explained by loss of a hydrogen bond
title Molecular pathology of the R117H cystic fibrosis mutation is explained by loss of a hydrogen bond
title_full Molecular pathology of the R117H cystic fibrosis mutation is explained by loss of a hydrogen bond
title_fullStr Molecular pathology of the R117H cystic fibrosis mutation is explained by loss of a hydrogen bond
title_full_unstemmed Molecular pathology of the R117H cystic fibrosis mutation is explained by loss of a hydrogen bond
title_short Molecular pathology of the R117H cystic fibrosis mutation is explained by loss of a hydrogen bond
title_sort molecular pathology of the r117h cystic fibrosis mutation is explained by loss of a hydrogen bond
topic Structural Biology and Molecular Biophysics
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8673840/
https://www.ncbi.nlm.nih.gov/pubmed/34870594
http://dx.doi.org/10.7554/eLife.74693
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