(689) Pharmacological chaperones improve intra-domain stability and inter-domain assembly via distinct binding sites to rescue misfolded CFTR

Background: CFTR misfolding and subsequent premature degradation is a hallmark of class II mutations such as F508del or L206W that alter intra- and/or inter-domain folding steps. These mutants can be rescued by clinically approved correctors such as VX-809 / VX-661 (class I correctors) and VX-445 (class III correctors), with enhanced efficacy when used in combination. However, their binding sites and mechanisms of action are still incompletely understood.

Methods: Blind docking was performed using the EADock DSS tool provided by the online SwissDock server. Molecular dynamics (MD) simulations were performed using the CHARMM36 force field for the protein, lipids and ions, and the TIP3P model for water. Minimization, equilibration and production steps were performed using Gromacs 2019.1. The corrector parameters were generated using the CgenFF module in CHARMM-GUI server. Considered coordinates were those of the cryo-EM 3D structure of human CFTR in the ATP-bound conformation (pdb 6MSM) and the crystal structure of human F508del CFTR NBD1 (pdb 4WZ6). Mutagenesis of key amino acids located within the identified sites was performed in the CFTR WT, F508del and L206W background. The maturation of single and double mutants under basal and upon corrector treatment was evaluated by Western blot.

Results: Blind docking onto the 3D structures of both the first membrane-spanning domain (MSD1) and the first nucleotide-binding domain (NBD1), followed by molecular dynamics simulations, revealed the presence of two potential VX-809/VX-661 corrector binding sites which, when mutated, abrogated rescue. While F508del rescue required both MSD1 and NBD1 intact binding sites, L206W rescue was affected only by mutations in the MSD1 site, which was indepently reported in a cryo-EM 3D stucture (Fiedorczuk and Chen, 2022). We moreover highlighted a network of charged amino acids in the lasso helix Lh2 and the intracellular loops ICL1 and ICL4, which allosterically couple MSD1 to NBD1. Corrector VX-445 also occupied two potential binding sites on MSD1 and NBD1, the latter being shared with VX-809//VX-661. Binding of both correctors on MSD1 enhanced the allostery between MSD1 and NBD1, hence the increased efficacy of the corrector combination.



Conclusions: Taken together, these correctors improve both intra-domain folding by stabilizing fragile protein-lipid interfaces and inter-domain assembly via distant allosteric couplings. These results provide novel mechanistic insights into the rescue of misfolded CFTR by small molecules (Baatallah, Elbahnsi et al., 2021).

Keywords: Binding site; Chemical chaperone; Cystic fibrosis; Molecular dynamics; Protein folding.

Acknowledgements: This work was performed using HPC resources from GENCI-[CINES] (Grants 2017-A0020707206, 2018-A0040707206, 2019-A0060707206 and 2020-A0080707206) and benefited from the financial support of the French Association Vaincre la Mucoviscidose and Association pour l’Aide à la Recherche contre la Mucoviscidose (AARM).

References: Fiedorczuk K, Chen J. Mechanism of CFTR correction by type I folding correctors. Cell. 2022 Jan 6;185(1):158-168.e11.

Baatallah N, Elbahnsi A, Mornon JP, Chevalier B, Pranke I, Servel N, Zelli R, Décout JL, Edelman A, Sermet-Gaudelus I, Callebaut I, Hinzpeter A. Pharmacological chaperones improve intra-domain stability and inter-domain assembly via distinct binding sites to rescue misfolded CFTR. Cell Mol Life Sci. 2021 Dec;78(23):7813-7829.