Can two wrongs make a right? F508del-CFTR ion channel rescue by second-site mutations in its transmembrane domains.

Autor:	Prins S; Department of Neuroscience, Physiology and Pharmacology, University College London, London, UK., Corradi V; Department of Biological Sciences, Centre for Molecular Simulation, University of Calgary, Calgary, Alberta, Canada., Sheppard DN; School of Physiology, Pharmacology and Neuroscience, University of Bristol, Bristol, UK., Tieleman DP; Department of Biological Sciences, Centre for Molecular Simulation, University of Calgary, Calgary, Alberta, Canada., Vergani P; Department of Neuroscience, Physiology and Pharmacology, University College London, London, UK. Electronic address: p.vergani@ucl.ac.uk.
Jazyk:	angličtina
Zdroj:	The Journal of biological chemistry [J Biol Chem] 2022 Mar; Vol. 298 (3), pp. 101615. Date of Electronic Publication: 2022 Jan 21.
DOI:	10.1016/j.jbc.2022.101615
Abstrakt:	Deletion of phenylalanine 508 (F508del) in the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel is the most common cause of cystic fibrosis. 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 11 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 WT CFTR). F508del-CFTR displayed a distorted aromatic stack, with F1068 displaced toward 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. Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article. (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)
Databáze:	MEDLINE
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