Structural Comparative Modeling of Multi-Domain F508del CFTR

Abstract

Cystic fibrosis (CF) is a rare genetic disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), an epithelial anion channel expressed in several vital organs. Absence of functional CFTR results in imbalanced osmotic equilibrium and subsequent mucus build up in the lungs-which increases the risk of infection and eventually causes death. CFTR is an ATP-binding cassette (ABC) transporter family protein composed of two transmembrane domains (TMDs), two nucleotide binding domains (NBDs), and an unstructured regulatory domain. The most prevalent patient mutation is the deletion of F508 (F508del), making F508del CFTR the primary target for current FDA approved CF therapies. However, no experimental multi-domain F508del CFTR structure has been determined and few studies have modeled F508del using multi-domain WT CFTR structures. Here, we used cryo-EM density data and Rosetta comparative modeling (RosettaCM) to compare a F508del model with published experimental data on CFTR NBD1 thermodynamics. We then apply this modeling method to generate multi-domain WT and F508del CFTR structural models. These models demonstrate the destabilizing effects of F508del on NBD1 and the NBD1/TMD interface in both the inactive and active conformation of CFTR. Furthermore, we modeled F508del/R1070W and F508del bound to the CFTR corrector VX-809. Our models reveal the stabilizing effects of VX-809 on multi-domain models of F508del CFTR and pave the way for rational design of additional drugs that target F508del CFTR for treatment of CF.

Keywords: VX-809; comparative modeling; computational protein modeling; cystic fibrosis; pharmacological chaperones; protein folding disease; structure-based drug discovery.

Figure 1

Comparative modeling captures multi-domain CFTR thermodynamics. (A) The complex topology of CFTR involves interdomain contacts formed during the folding process that include intercellular loops (ICLs) interfacing with the cytosolic NDBs. The inactive PBD ID 5UAK [2] (left) and active PBD ID 6MSM [6] (right). (B) Our workflow for generating ensembles of F508del models in this study (see Methods).

Figure 2

Refinement into the cryo-EM density generates a diverse ensemble of structures. (A) Average Cα RMSD of the best scoring (lowest 10% by potential energy function) 5UAK cryo-EM refinement models mapped onto 5UAK. (B) Average Cα RMSD of the best scoring (lowest 10% by potential energy function 6MSM cryo-EM refinement models mapped onto 6MSM. (C) The average NBD1 Cα RMSD of the best scoring 100 5UAK refinement models. The blue shading represents a 95% confidence interval, and the large Cα RMSD demonstrates high structural diversity in the SDR (residues 526–547). (D) The average NBD1 RMSD of the best scoring 100 6MSM refinement models. The blue shading represents a 95% confidence interval.

Figure 3

Comparative modeling of F508del NBD1 using five templates correlates well with experimental data. (A) An overlay of WT and F508del CFTR NBD1 structures at the H3/H4 loop. WT is depicted in blue and F508del is depicted in red with just the α-helical subdomain shown for clarity. Deletion of F508 leaves surrounding residues I506, I507 and V510 relatively unaltered. (B) The model for testing included NBD1 bound to ATP. Residues mutated in second site suppressor mutations are shown in blue including F494N, V510, I539, G550, R553, R555, and Q637. (C) Testing correlation between Rosetta score given in REU and ΔΔG values from the literature (Table S1, Supplementary Materials). Error bars represent standard error of the mean. Error in experimental data likely lies lower than ±1–2 kcal/mol. R squared represents Pearson correlation coefficient. (D) Testing correlation between Rosetta score given in REU and ΔTM values from the literature (Table S2, Supplementary Materials). Error bars represent standard error of the mean. Error in experimental data likely ranges with ±1–2 C. R squared represents Pearson correlation coefficient.

Figure 4

Comparative modeling of multi-domain F508del CFTR shows thermodynamic instability and lose of interaction energy at key domain-domain interfaces. (A) Cα RMSD vs. score plot of the lowest scoring 100 inactive conformation models from ensembles of WT (blue) and F508del (red) CFTR. RMSD is calculated relative to the lowest scoring WT model. Score is shown in REU. Statistical significance was calculated with a Mann–Whitney U test and all p-values are depicted by * < 0.05, *** < 0.001, and **** < 0.0001. (B) Cα RMSD vs. score plot of the lowest scoring 100 active conformation models from ensembles of WT (blue) and F508del (red) CFTR. (C) Average residue Cα RMSD of the lowest scoring 100 inactive state WT models subtracted from the Cα RMSD of the lowest scoring 100 inactive state F508del models mapped on 5UAK. Here, red represents region where the RMSD was higher in F508del than WT, and blue represents regions where the RMSD was lower. (D) Average residue Cα RMSD of the lowest scoring 100 inactive state WT models subtracted from the Cα RMSD of the lowest scoring 100 inactive state F508del models mapped on 6MSM. (E) Quantification of the residue–residue interactions at the NBD1/NBD2 interface across the lowest scoring 100 models. Only the active state is considered as the inactive state lack the NBD dimer and hence there are no residue interactions to measure. (F) Quantification of the residue-residue interactions at the NBD1/TMD2 interface across the lowest scoring 100 models. The box limits represent the upper and lower quartile with a line at the median, the whiskers represent 1.5 times the interquartile range, statistical significance was calculated with a Mann–Whitney U test.

Figure 5

R1070W stabilizes the NBD1/TMD2 interface. (A) F508 in 5UAK CFTR (gold spheres) contacts an aromatic pocket in ICL4 formed by F1068, Y1073, and F1074 (yellow spheres). This aromatic pocket is filled with R1070 (blue) is mutated to a tryptophan (left). (B) Average residue Cα RMSD of the lowest scoring 100 inactive state F508del /R1070W models subtracted from the Cα RMSD of the lowest scoring 100 inactive state F508del models mapped on 5UAK. Here, red represents region where the RMSD was higher in F508del/R1070W than F508del alone, and blue represents regions where the RMSD was lower and hence stabilized by R1070W. (C) Average residue Cα RMSD of the lowest scoring 100 inactive state F508del /R1070W models subtracted from the Cα RMSD of the lowest scoring 100 inactive state F508del models mapped on 6MSM. (D) Quantification of the residue-residue interactions at the NBD1/TMD2 interface across the lowest scoring 100 models. The box limits represent the upper and lower quartile with a line at the median, the whiskers represent 1.5 times the interquartile range, statistical significance was calculated with a Mann–Whitney U test and p-values are depicted by * < 0.05, and **** < 0.0001. (E) Quantification of the residue-residue interactions at the TMD1/TMD2 interface across the lowest scoring 100 models. R1070W likely stabilize TMD2 enough to reduce the interaction energy between the TMDs in the active conformation.

Figure 6

Comparative modeling of VX-809 bound to TMD1 F508del reveals local stability changes including the TMD1 domain-domain interfaces. (A) VX-809 chemical structure. (B) VX-809 docked to 6MSM CFTR structure in a putative binding site recently published by two parallel studies [16,17]. Interactions with important residues are shown in blue, VX-809 is shown in green with colored hetero-atoms. (C) Average residue Cα RMSD of the lowest scoring 100 inactive state F508del+VX-809 models subtracted from the Cα RMSD of the lowest scoring 100 inactive state F508del models mapped on 5UAK. Here, red represents region where the RMSD was higher in F508del +VX-809 than F508del alone, and blue represents regions where the RMSD was lower hence the structure was stabilized by VX-809. The inset shows the RMSD of the region surrounding VX-809 demonstrates reduced RMSD. (D) Average residue Cα RMSD of the lowest scoring 100 inactive state F508del+VX-809 models subtracted from the Cα RMSD of the lowest scoring 100 inactive state F508del models mapped on 6MSM. (E) Quantification of the residue-residue interactions at the TMD1/NBD2 interface across the lowest scoring 100 models. The box limits represent the upper and lower quartile with a line at the median, the whiskers represent 1.5 times the interquartile range, statistical significance was calculated with a Mann–Whitney U test and p-values are depicted by * < 0.05, ** < 0.01, and **** < 0.0001. (F) Quantification of the residue-residue interactions at the TMD1/TMD2 interface across the lowest scoring 100 models.