Mechanism of dual pharmacological correction and potentiation of human CFTR

Abstract

Cystic fibrosis (CF) is caused by mutations in a chloride channel called the human Cystic Fibrosis Transmembrane Conductance Regulator (hCFTR). We used cryo-EM global conformational ensemble reconstruction to characterize the mechanism by which the breakthrough drug VX445 (Elexacaftor) simultaneously corrects both protein-folding and channel-gating defects caused by CF mutations. VX445 drives hCFTR molecules harboring the gating-defective G551D mutation towards the open-channel conformation by binding to a site in the first transmembrane domain. This binding interaction reverses the usual pathway of allosteric structural communication by which ATP binding activates channel conductance, which is blocked by the G551D mutation. Our ensemble reconstructions include a 3.4 angstrom non-native structure demonstrating that detachment of the first nucleotide-binding domain of hCFTR is directly coupled to local unfolding of the VX445 binding site. Reversal of this unfolding transition likely contributes to its corrector activity by cooperatively stabilizing NBD1 and the transmembrane domains of hCFTR during biogenesis.

CFTR function, pathology and pharmacology at single-molecule resolution

“…8e,f). In agreement with these data, the findings of a recent cryo-EM study showed that the G551D variant adopts conformations in between those of the fully NBD-separated and NBD-dimerized conformations 33 .…”

“…4b,g). This observation, together with the recent cryo-EM study of the CFTR(G551D) variant in the presence of ivacaftor 33 , demonstrates that neither ivacaftor nor GLPG1837 promotes NBD dimerization. Similarly, for the L927P variant, the relative stimulation of open probability greatly exceeded the relative stimulation of dimerization probability (Fig.…”

Folding correctors can restore CFTR posttranslational folding landscape by allosteric domain-domain coupling

“…1c and Supplementary Table 1) 8,47 . Combination of VX-809 with the VX-445 corrector, which targets the TMD2(TMH10-11) and the N-terminal Lasso segment 15,17 , restored WT-like folding efficiency (∼36%) of ΔF508-CFTR (Fig. 1c), consistent with the mutant steady-state cellular accumulation based on functional studies 14 .…”

“…The CFTR cooperative domain-folding model dictates that following the formation of secondary and some tertiary structural elements co-translationally, the native domain-swapped conformational ensembles develop posttranslationally 15 , which requires dynamic and/or energetic interdomain coupling and the expression of the full-length CFTR or its minimal folding unit (TMD1-NBD1-RD- TMD2 or CFTR-ΔNBD2) 8,27 . The cooperative folding model aligns with the observation that the correctors binding sites overlap with the WT-CFTR conformational poses in the cryo-EM structures, 15,17 suggesting that conformers can accumulate synchronously with the posttranslational development of permissive inter-domain interactions. The intrinsically unstructured regulatory (R) domain (Supplementary Fig.1a) was found to be dispensable for CFTR expression 28 .…”

“…5d-e) may be attributed to their orthosteric versus allosteric stabilization of TMD1 (Fig. 5a), based on their binding site localization in the TMD1 and TMD2, respectively, in CFTR-E1371Q 15 , temperature rescued ΔF508-CFTR-E1371Q 64 and G551D-CFTR-6SS 17 . In line, the combination of VX-445 with VX-661/VX-809 exert a prominent rescue effect on the ΔF508-CFTR 12 (Fig.1c) and F508G-CFTR (Fig.…”