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

Stella Prins, Valentina Corradi, David N Sheppard, D. Peter Tieleman, Paola Vergani*

*Corresponding author for this work

Research output: Contribution to journalArticle (Academic Journal)peer-review

5 Citations (Scopus)
82 Downloads (Pure)

Abstract

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.
Original languageEnglish
Article number101615
Pages (from-to)1-14
Number of pages14
JournalJournal of Biological Chemistry
Volume298
Issue number3
Early online date21 Jan 2022
DOIs
Publication statusPublished - 1 Mar 2022

Bibliographical note

Funding Information:
Funding and additional information—S. P., D. N. S., and P. V. gratefully acknowledge funding by the Cystic Fibrosis Trust [SRC 005]. Work in D. P. T.’s group is supported by the Natural Sciences and Engineering Research Council (Canada) and the Canadian Institutes of Health Research. Further support came from the Canada Research Chairs program. Calculations were carried out on Compute Canada facilities supported by the Canada Foundation for Innovation and partners.

Publisher Copyright:
© 2022 THE AUTHORS.

Keywords

  • cystic fibrosis transmembrane conductance regulator (CFTR)
  • cystic fibrosis
  • ATP-binding cassette (ABC) transporters
  • ion channel
  • R1070W
  • YOR1 protein
  • domain interface
  • molecular dynamics simulations
  • cluster analysis

Fingerprint

Dive into the research topics of 'Can two wrongs make a right? F508del-CFTR ion channel rescue by second-site mutations in its transmembrane domains'. Together they form a unique fingerprint.

Cite this