A minimal helical-hairpin motif provides molecular-level insights into misfolding and pharmacological rescue of CFTR

Abstract

Our meagre understanding of CFTR misfolding and its reversal by small-molecule correctors hampers the development of mechanism-based therapies of cystic fibrosis. Here we exploit a helical-hairpin construct—the simplest proxy of membrane-protein tertiary contacts—containing CFTR’s transmembrane helices 3 and 4 and its corresponding disease phenotypic mutant V232D to gain molecular-level insights into CFTR misfolding and drug rescue by the corrector Lumacaftor. Using a single-molecule FRET approach to study hairpin conformations in lipid bilayers, we find that the wild-type hairpin is well folded, whereas the V232D mutant assumes an open conformation in bilayer thicknesses mimicking the endoplasmic reticulum. Addition of Lumacaftor reverses the aberrant opening of the mutant hairpin to restore a compact state as in the wild type. The observed membrane escape of the V232D hairpin and its reversal by Lumacaftor complement cell-based analyses of the full-length protein, thereby providing in vivo and in vitro correlates of CFTR misfolding and drug-action mechanisms.

CFTR corrector efficacy is associated with occupancy of distinct binding sites

“…In the second situation (G85E, facing the potential VX-809 binding site), alterations in TM1 span integration in the membrane was shown to be defective (41), possibly leading to the production of a nonrescuable CFTR. This was not the case of V232D which is corrected by VX-809 (42) and was recently associated with mis-insertion of the TM3/4 hairpin within the membrane (43). Interestingly, the hydrophobic pocket at the vicinity of which V232D is located, well discussed by Loo and Clarke (42), corresponds to the VX-445 binding site in MSD1 highlighted here at the level of the membrane outer leaflet (Fig.…”

SDS-induced multi-stage unfolding of a small globular protein through different denatured states revealed by single-molecule fluorescence

“…Calculation of uorescence lifetimes (s D,A ) and anisotropies (r D,A ) as well as steady-state rotational correlation times (r D,A ) were performed as described previously; 26 also plots of relative donor uorescence lifetime (s D(A) /s D(0) ) versus E were created as described. 43 Analysis of single-molecule bursts for millisecond dynamics was done by FRET-2CDE analysis. 44 Quantication of millisecond interconversion dynamics from FRET efficiency histograms was performed by dPDA as described in the ESI † and detailed elsewhere.…”

Mechanism of CFTR correction by type I folding correctors

“…In one study it was suggested that lumacaftor (formally VX-809) acts through perturbation of membranes as it appeared to be homogeneously distributed throughout the lipid bilayer (Baroni et al, 2014). In multiple other studies, a direct action on the channel was proposed, but the location of the binding site remains in dispute (Eckford et al, 2014; Farinha and Canato, 2017; He et al, 2013; Hudson et al, 2017; Krainer et al, 2020; Krainer et al, 2018; Laselva et al, 2018; Loo et al, 2013; Loo and Clarke, 2017; Okiyoneda et al, 2013; Ren et al, 2013; Sinha et al, 2015). In this study, we determined cryo-EM structures of CFTR in complex with either lumacaftor or tezacaftor (formally VX-661).…”