Estimating the true stability of the prehydrolytic outward-facing state in an ABC protein
Abstract
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.
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All data generated or analysed during this study are included in the manuscript and the figures.
Article and author information
Author details
Funding
EU Horizon 2020 Research and Innovation Program (739593)
- László Csanády
Cystic Fibrosis Foundation (CSANAD21G0)
- László Csanády
National Research, Development and Innovation Office (KKP 144199)
- László Csanády
Ministry for Innovation and Technology of Hungary (ÚNKP-22-3-II-SE-12)
- Márton A Simon
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Ethics
Animal experimentation: This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocols were approved by the Institutional Animal Care and Use Committee of Semmelweis University (Assurance number:SEMMAWB/2023-001).
Copyright
© 2023, Simon et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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