Fig. 1.

Identification of a potential VX-809 binding site within MSD1. A Western blot analysis of WT and mutant CFTR. CFTR fully glycosylated (band C) and core glycosylated (band B) are indicated. Cells were treated with VX-809 (3 µM, 24 h) when indicated. Tubulin (Tub) was probed to assess equal loading amounts. B Quantification of the C/(B + C) maturation ratio of the indicated mutants. Dark grey represents control conditions and light grey VX-809 treatments. Measures are means ± SEM of n = 3–20, with * indicating p < 0.05, ** indicating p < 0.01 and ns indicating no statistical differences compared to untreated condition (one-way ANOVA followed by Fischer test for p evaluation). C Two main pockets were identified at the level of the membrane inner and outer leaflets on the 3D structure of human CFTR MSD1, extracted from the cryo-EM 3D structure of the full-length protein (pdb: 6MSM). The membrane inner leaflet site corresponds to the best docking scores obtained after blind docking using SwissDock, with the surface envelope of VX-809 (representative members of the six first clusters) shaded in pink. The second binding site was identified at the level of the membrane outer leaflet, with fewer clusters (surface envelope (representative members of three clusters) shaded in purple) and less favorable docking scores. The lasso is shown in purple, MSD1 TM1 to TM3 in blue, MDS1 TM4 to TM6 in green. F508 and L206 are shown with pink spheres, L53 and F87 with yellow spheres. D Focus on amino acids within the potential VX-809 binding sites at the level of the membrane inner (bottom) and outer (top) leaflets. The conformations of VX-809 corresponding to the best docking scores in the two sites are shown (inner site (cluster 0): SwissDock FullFitness score of −1938; outer site (cluster 7): SwissDock FullFitness score of −1924). Tested amino acids are labeled in magenta