Correction: Disease-relevant mutations alter amino acid co-evolution networks in the second nucleotide binding domain of CFTR.
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ABSTRACT:
SUBMITTER: Ivey G
PROVIDER: S-EPMC7046266 | biostudies-literature | 2020
REPOSITORIES: biostudies-literature
Publications
Correction: Disease-relevant mutations alter amino acid co-evolution networks in the second nucleotide binding domain of CFTR.
Ivey Gabrianne G Youker Robert T RT
PloS one 20200227 2
[This corrects the article DOI: 10.1371/journal.pone.0227668.]. ...[more]
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Project description:Cystic Fibrosis (CF) is an inherited disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) ion channel. Mutations in CFTR cause impaired chloride ion transport in the epithelial tissues of patients leading to cardiopulmonary decline and pancreatic insufficiency in the most severely affected patients. CFTR is composed of twelve membrane-spanning domains, two nucleotide-binding domains (NBDs), and a regulatory domain. The most common mutation in CFTR is a deletion of phenylalanine at position 508 (ΔF508) in NBD1. Previous research has primarily concentrated on the structure and dynamics of the NBD1 domain; However numerous pathological mutations have also been found in the lesser-studied NBD2 domain. We have investigated the amino acid co-evolved network of interactions in NBD2, and the changes that occur in that network upon the introduction of CF and CF-related mutations (S1251N(T), S1235R, D1270N, N1303K(T)). Extensive coupling between the α- and β-subdomains were identified with residues in, or near Walker A, Walker B, H-loop and C-loop motifs. Alterations in the predicted residue network varied from moderate for the S1251T perturbation to more severe for N1303T. The S1235R and D1270N networks varied greatly compared to the wildtype, but these CF mutations only affect ion transport preference and do not severely disrupt CFTR function, suggesting dynamic flexibility in the network of interactions in NBD2. Our results also suggest that inappropriate interactions between the β-subdomain and Q-loop could be detrimental. We also identified mutations predicted to stabilize the NBD2 residue network upon introduction of the CF and CF-related mutations, and these predicted mutations are scored as benign by the MUTPRED2 algorithm. Our results suggest the level of disruption of the co-evolution predictions of the amino acid networks in NBD2 does not have a straightforward correlation with the severity of the CF phenotypes observed.
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Project description:The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is an ABC transporter containing two transmembrane domains forming a chloride ion channel, and two nucleotide binding domains (NBD1 and NBD2). CFTR has presented a formidable challenge to obtain monodisperse, biophysically stable protein. Here we report a comprehensive study comparing effects of single and multiple NBD1 mutations on stability of both the NBD1 domain alone and on purified full length human CFTR. Single mutations S492P, A534P, I539T acted additively, and when combined with M470V, S495P, and R555K cumulatively yielded an NBD1 with highly improved structural stability. Strategic combinations of these mutations strongly stabilized the domain to attain a calorimetric Tm > 70 °C. Replica exchange molecular dynamics simulations on the most stable 6SS-NBD1 variant implicated fluctuations, electrostatic interactions and side chain packing as potential contributors to improved stability. Progressive stabilization of NBD1 directly correlated with enhanced structural stability of full-length CFTR protein. Thermal unfolding of the stabilized CFTR mutants, monitored by changes in intrinsic fluorescence, demonstrated that Tm could be shifted as high as 67.4 °C in 6SS-CFTR, more than 20 °C higher than wild-type. H1402S, an NBD2 mutation, conferred CFTR with additional thermal stability, possibly by stabilizing an NBD-dimerized conformation. CFTR variants with NBD1-stabilizing mutations were expressed at the cell surface in mammalian cells, exhibited ATPase and channel activity, and retained these functions to higher temperatures. The capability to produce enzymatically active CFTR with improved structural stability amenable to biophysical and structural studies will advance mechanistic investigations and future cystic fibrosis drug development.
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Project description:Most CF (cystic fibrosis) results from deletion of a phenylalanine (F508) in the CFTR {CF transmembrane-conductance regulator; ABCC7 [ABC (ATP-binding cassette) sub-family C member 7]} which causes ER (endoplasmic reticulum) degradation of the mutant. Using stably CFTR-expressing BHK (baby-hamster kidney) cell lines we demonstrated that wild-type CTFR and the F508delCFTR mutant are cleaved into differently sized N- and C-terminal-bearing fragments, with each hemi-CFTR carrying its nearest NBD (nucleotide-binding domain), reflecting differential cleavage through the central CFTR R-domain. Similar NBD1-bearing fragments are present in the natively expressing HBE (human bronchial epithelial) cell line. We also observe multiple smaller fragments of different sizes in BHK cells, particularly after F508del mutation (ladder pattern). Trapping wild-type CFTR in the ER did not generate a F508del fragmentation fingerprint. Fragments change their size/pattern again post-mutation at sites involved in CFTR's in vitro interaction with the pleiotropic protein kinase CK2 (S511A in NBD1). The F508del and S511A mutations generate different fragmentation fingerprints that are each unlike the wild-type; yet, both mutants generate new N-terminal-bearing CFTR fragments that are not observed with other CK2-related mutations (S511D, S422A/D and T1471A/D). We conclude that the F508delCFTR mutant is not degraded completely and there exists a relationship between CFTR's fragmentation fingerprint and the CFTR sequence through putative CK2-interactive sites that lie near F508.
| S-EPMC3542821 | biostudies-other
Project description:The human ATP-binding cassette (ABC) protein CFTR (cystic fibrosis transmembrane conductance regulator) is a chloride channel, whose dysfunction causes cystic fibrosis. To gain structural insight into the dynamic interaction between CFTR's nucleotide-binding domains (NBDs) proposed to underlie channel gating, we introduced target cysteines into the NBDs, expressed the channels in Xenopus oocytes, and used in vivo sulfhydryl-specific crosslinking to directly examine the cysteines' proximity. We tested five cysteine pairs, each comprising one introduced cysteine in the NH(2)-terminal NBD1 and another in the COOH-terminal NBD2. Identification of crosslinked product was facilitated by co-expression of NH(2)-terminal and COOH-terminal CFTR half channels each containing one NBD. The COOH-terminal half channel lacked all native cysteines. None of CFTR's 18 native cysteines was found essential for wild type-like, phosphorylation- and ATP-dependent, channel gating. The observed crosslinks demonstrate that NBD1 and NBD2 interact in a head-to-tail configuration analogous to that in homodimeric crystal structures of nucleotide-bound prokaryotic NBDs. CFTR phosphorylation by PKA strongly promoted both crosslinking and opening of the split channels, firmly linking head-to-tail NBD1-NBD2 association to channel opening.
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Project description:The deletion of Phe508 (?F508) in the first nucleotide binding domain (NBD1) of CFTR is the most common mutation associated with cystic fibrosis. The ?F508-CFTR mutant is recognized as improperly folded and targeted for proteasomal degradation. Based on molecular dynamics simulation results, we hypothesized that interaction between ?F508-NBD1 and housekeeping proteins prevents ?F508-CFTR delivery to the plasma membrane. Based on this assumption we applied structure-based virtual screening to identify new low-molecular-weight compounds that should bind to ?F508-NBD1 and act as protein-protein interaction inhibitors. Using different functional assays for CFTR activity, we demonstrated that in silico-selected compounds induced functional expression of ?F508-CFTR in transfected HeLa cells, human bronchial CF cells in primary culture, and in the nasal epithelium of homozygous ?F508-CFTR mice. The proposed compounds disrupt keratin8-?F508-CFTR interaction in ?F508-CFTR HeLa cells. Structural analysis of ?F508-NBD1 in the presence of these compounds suggests their binding to NBD1. We conclude that our strategy leads to the discovery of new compounds that are among the most potent correctors of ?F508-CFTR trafficking defect known to date.
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Project description:Mutations in the CFTR gene that lead to premature stop codons or splicing defects cause cystic fibrosis (CF) and are not amenable to treatment by small-molecule modulators. Here, we investigate the use of adenine base editor (ABE) ribonucleoproteins (RNPs) that convert A•T to G•C base pairs as a therapeutic strategy for three CF-causing mutations. Using ABE RNPs, we corrected in human airway epithelial cells premature stop codon mutations (R553X and W1282X) and a splice-site mutation (3849 + 10 kb C > T). Following ABE delivery, DNA sequencing revealed correction of these pathogenic mutations at efficiencies that reached 38-82% with minimal bystander edits or indels. This range of editing was sufficient to attain functional correction of CFTR-dependent anion channel activity in primary epithelial cells from CF patients and in a CF patient-derived cell line. These results demonstrate the utility of base editor RNPs to repair CFTR mutations that are not currently treatable with approved therapeutics.
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