Functional and Pharmacological Characterization of the Rare CFTR Mutation W361R

Abstract

Understanding the functional consequence of rare cystic fibrosis (CF) mutations is mandatory for the adoption of precision therapeutic approaches for CF. Here we studied the effect of the very rare CF mutation, W361R, on CFTR processing and function. We applied western blot, patch clamp and pharmacological modulators of CFTR to study the maturation and ion transport properties of pEGFP-WT and mutant CFTR constructs, W361R, F508del and L69H-CFTR, expressed in HEK293 cells. Structural analyses were also performed to study the molecular environment of the W361 residue. Western blot showed that W361R-CFTR was not efficiently processed to a mature band C, similar to F508del CFTR, but unlike F508del CFTR, it did exhibit significant transport activity at the cell surface in response to cAMP agonists. Importantly, W361R-CFTR also responded well to CFTR modulators: its maturation defect was efficiently corrected by VX-809 treatment and its channel activity further potentiated by VX-770. Based on these results, we postulate that W361R is a novel class-2 CF mutation that causes abnormal protein maturation which can be corrected by VX-809, and additionally potentiated by VX-770, two FDA-approved small molecules. At the structural level, W361 is located within a class-2 CF mutation hotspot that includes other mutations that induce variable disease severity. Analysis of the 3D structure of CFTR within a lipid environment indicated that W361, together with other mutations located in this hotspot, is at the edge of a groove which stably accommodates lipid acyl chains. We suggest this lipid environment impacts CFTR folding, maturation and response to CFTR modulators.

CFTR corrector efficacy is associated with occupancy of distinct binding sites

“…The two pockets located in MSD1 at the level of the inner and outer membrane leaflets share a common hydrophobic character, whose disruption by mutations may be one of the mechanism responsible for a weakened stability and protein misfolding. Worth noting is that MD simulations have proposed that lipids can be stably accommodated in the putative VX-809 binding site studied here (18), thereby suggesting that VX-809 may mimic such molecules for stabilizing these fragile areas.…”

“…S11). Such modified interactions with lipids, as also likely for W361R (18), might account for a modified architecture of the pocket, that can be rescued by displacement of lipids by the corrector. E56 in the lasso Lh2 plays a role in the architecture/stability of the pocket through a salt bridge established with R75 in the elbow helix, forming the cytoplasmic side of the pocket.…”

“…Binding to MSD1 was suggested by the stabilization of truncated CFTR forms, encompassing amino acids 1-373, likely by favoring a folding intermediate (15). This stabilizing effect appears to be sufficient to achieve a strong rescue of class II mutants located in the N-terminal part of CFTR, such as E56K, P67L, L69H, L206W or W361R (11,15,(18)(19)(20), indicating that targeting initial MSD1 folding and assembly accounted for the bulk VX-809 rescue. The VX-809-binding site on MSD1 has remained heretofore elusive.…”

“…This site is embedded within a hot spot for class II mutations associated with either mild folding defects (in pink on Fig. S2 : P67L, E56K, V232D, I336K, Q359K, T360K, R347P, S341P and I336K, and W361R) or severe folding defects (in red : G85E, E92K, G91R, H199Y, P205S, L206W and L227R) (7,18), consistent with a fragility in the folding and/or packing of the transmembrane helices (TM) in this region.…”

“…Corrective conformational changes or stabilization of MSD1 by VX-809 appears then to be communicated to other domains, including NBD1, MSD2 and NDB2. VX-809 is otherwise highly effective at correcting MSD1-specific disease-causing mutations (16,17), some of them forming a hot spot around a groove predicted on the 3D structure of the protein to bind phospholipids in a stable way (18). These mutations include P67L and L206W (11,15,17,(19)(20)(21) with the notable exception of G85E which was shown to be resistant to correction (4,22).…”