Prins, S;
Corradi, V;
Sheppard, DN;
Tieleman, DP;
Vergani, P;
(2022)
Can two wrongs make a right? F508del-CFTR ion channel rescue by second-site mutations in its transmembrane domains.
Journal of Biological Chemistry
, Article 101615. 10.1016/j.jbc.2022.101615.
(In press).
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Vergani_Can two wrongs make a right_ F508del-CFTR ion channel rescue by second-site mutations in its transmembrane domains_AOP.pdf Download (2MB) | Preview |
Abstract
Deletion of phenylalanine 508 (F508del) in the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel is the most common cause of cystic fibrosis (CF). The F508 residue is located on nucleotide-binding domain 1 (NBD1) in contact with the cytosolic extensions of the transmembrane helices, in particular intracellular loop 4 (ICL4). To investigate how absence of F508 at this interface impacts the CFTR protein, we carried out a mutagenesis scan of ICL4 by introducing second-site mutations at eleven positions in cis with F508del. Using an image-based fluorescence assay, we measured how each mutation affected membrane proximity and ion-channel function. The scan strongly validated the effectiveness of R1070W at rescuing F508del defects. Molecular dynamics simulations highlighted two features characterizing the ICL4/NBD1 interface of F508del/R1070W-CFTR: flexibility, with frequent transient formation of interdomain hydrogen bonds, and loosely stacked aromatic sidechains (F1068, R1070W, and F1074, mimicking F1068, F508 and F1074 in wild-type CFTR). F508del-CFTR displayed a distorted aromatic stack, with F1068 displaced towards the space vacated by F508, while, in F508del/R1070F-CFTR, which largely retained F508del defects, R1070F could not form hydrogen bonds and the interface was less flexible. Other ICL4 second-site mutations which partially rescued F508del-CFTR included F1068M and F1074M. Methionine side chains allow hydrophobic interactions without the steric rigidity of aromatic rings, possibly conferring flexibility to accommodate the absence of F508 and retain a dynamic interface. These studies highlight how both hydrophobic interactions and conformational flexibility might be important at the ICL4/NBD1 interface, suggesting possible structural underpinnings of F508del-induced dysfunction.
| Type: | Article |
|---|---|
| Title: | Can two wrongs make a right? F508del-CFTR ion channel rescue by second-site mutations in its transmembrane domains |
| Open access status: | An open access version is available from UCL Discovery |
| DOI: | 10.1016/j.jbc.2022.101615 |
| Publisher version: | https://doi.org/10.1016/j.jbc.2022.101615 |
| Language: | English |
| Additional information: | © 2022 THE AUTHORS. Published by Elsevier Inc on behalf of American Society for Biochemistry and Molecular Biology under a Creative Commons license (https://creativecommons.org/licenses/by/4.0/). |
| Keywords: | Cystic fibrosis transmembrane conductance regulator (CFTR), cystic fibrosis, ATP-binding cassette (ABC) transporters, ion channel, R1070W, YOR1 protein, domain interface, methionine, molecular dynamics simulations, cluster analysis |
| UCL classification: | UCL UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences > Neuro, Physiology and Pharmacology |
| URI: | https://discovery.ucl.ac.uk/id/eprint/10142440 |
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