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...
| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
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eLife Sciences Publications Ltd
2023-10-01
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| Series: | eLife |
| Subjects: | |
| Online Access: | https://elifesciences.org/articles/90736 |
| _version_ | 1797245291655593984 |
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| author | Márton A Simon Iordan Iordanov Andras Szollosi László Csanády |
| author_facet | Márton A Simon Iordan Iordanov Andras Szollosi László Csanády |
| author_sort | Márton A Simon |
| collection | DOAJ |
| 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. |
| first_indexed | 2024-03-11T18:39:08Z |
| format | Article |
| id | doaj.art-c434f17ce55f4fe781a3e677b787808d |
| institution | Directory Open Access Journal |
| issn | 2050-084X |
| language | English |
| last_indexed | 2024-04-24T19:24:34Z |
| publishDate | 2023-10-01 |
| publisher | eLife Sciences Publications Ltd |
| record_format | Article |
| series | eLife |
| spelling | doaj.art-c434f17ce55f4fe781a3e677b787808d2024-03-25T16:16:23ZengeLife Sciences Publications LtdeLife2050-084X2023-10-011210.7554/eLife.90736Estimating the true stability of the prehydrolytic outward-facing state in an ABC proteinMárton A Simon0Iordan Iordanov1https://orcid.org/0000-0001-8251-5857Andras Szollosi2https://orcid.org/0000-0002-5570-4609László Csanády3https://orcid.org/0000-0002-6547-5889Department of Biochemistry, Semmelweis University, Budapest, Hungary; HCEMM-SE Molecular Channelopathies Research Group, Budapest, Hungary; HUN-REN-SE Ion Channel Research Group, Budapest, HungaryDepartment of Biochemistry, Semmelweis University, Budapest, Hungary; HCEMM-SE Molecular Channelopathies Research Group, Budapest, Hungary; HUN-REN-SE Ion Channel Research Group, Budapest, HungaryDepartment of Biochemistry, Semmelweis University, Budapest, Hungary; HCEMM-SE Molecular Channelopathies Research Group, Budapest, Hungary; HUN-REN-SE Ion Channel Research Group, Budapest, HungaryDepartment of Biochemistry, Semmelweis University, Budapest, Hungary; HCEMM-SE Molecular Channelopathies Research Group, Budapest, Hungary; HUN-REN-SE Ion Channel Research Group, Budapest, HungaryCFTR, 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.https://elifesciences.org/articles/90736composite ATP-binding siteD-loopzebrafishflickery closuremutant cycle |
| spellingShingle | Márton A Simon Iordan Iordanov Andras Szollosi László Csanády Estimating the true stability of the prehydrolytic outward-facing state in an ABC protein eLife composite ATP-binding site D-loop zebrafish flickery closure mutant cycle |
| 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 | composite ATP-binding site D-loop zebrafish flickery closure mutant cycle |
| url | https://elifesciences.org/articles/90736 |
| work_keys_str_mv | AT martonasimon estimatingthetruestabilityoftheprehydrolyticoutwardfacingstateinanabcprotein AT iordaniordanov estimatingthetruestabilityoftheprehydrolyticoutwardfacingstateinanabcprotein AT andrasszollosi estimatingthetruestabilityoftheprehydrolyticoutwardfacingstateinanabcprotein AT laszlocsanady estimatingthetruestabilityoftheprehydrolyticoutwardfacingstateinanabcprotein |