Figures and data
Lumacaftor and tezacaftor bind to CFTR competitively
(A) The chemical structures of representative type I, II and III correctors. The 1,3-Benzodioxol-5-yl-Cyclopropane Carboxamide (BCC) headgroup is highlighted in grey. (B) Saturation binding and nonlinear regression analysis of [3H]lumacaftor binding to wtCFTR in the absence of phosphorylation and ATP (Kd = 8.3 ± 2.2 nM). Also shown is a negative control using a related ABC transporter MRP1. (C) Competition binding assay. The binding of [H3]lumacaftor (10 nM) was plotted as a function of the competitor’s concentration. Data were fit to a single-site competitive binding model. The Ki values for lumacaftor and tezacaftor are 7.7 ± 2.0 nM and 0.12 ± 0.04 μM, respectively. No competition was observed for elexacaftor and Corr-4a. Each data point represents the mean and the standard error of the mean (SEM) of 3-9 of measurements.
Lumacaftor binds to CFTR in both conformational states
(A) The overall structure of lumacaftor bound to the unphosphorylated, ATP-free wtCFTR (left) and phosphorylated, ATP-bound CFTR(E1371Q) (right). TMD1 and NBD1 are shown in blue, TMD2 and NBD2 in green. Lumacaftor is represented in yellow sticks and highlighted by the circle in magenta. The grey lines indicate the membrane boundaries. (B) Lumacaftor binds at the protein/membrane interface. The surface of the ATP-bound CFTR is shown by electrostatics and scaled from −10kT/e (red) to +10kT/e (blue). For reference, the location of the ivacaftor-binding site is indicated by an arrow. (C) Experimental density of the lumacaftor-binding site. Protein density is shown in grey and lumacaftor density in green. (D) Molecular recognition of lumacaftor. Residues within 4.5 Å of lumacaftor are shown as grey sticks. The salt bridge between K68 and lumacaftor is indicated by a magenta dashed line. (E) The lumacaftor-binding site is formed by TM 1, 2, 3, and 6. Cyan highlights the interactions between residues 371-375 and the N-terminal region of TM1. (F) Electrostatic surface representation of the same region as in Panel (E).
Tezacaftor binds CFTR at the same site as lumacaftor
(A) The overall structure of the CFTR/tezacaftor complex, with a zoom-in view of the binding site. Tezacaftor is represented in orange sticks and the protein surface is colored by electrostatics, scaled from −10kT/e (red) to +10kT/e (blue). (B) Experimental density of the tezacaftor-binding site. Protein density is represented in grey and tezacaftor density in green. (C) Molecular interaction at the binding site. Restudies within 4.5 Å distance from tezacaftor are shown as grey sticks. The H-bond between R74 and tezacaftor is indicated as a blue dashed line. (D) (E) Two views to compare the structures of lumacaftor (yellow) and tezacaftor (orange). The side chains of K68 and R74 are shown to highlight their different roles in drug binding. (F) (G) Schematic drawing of the CFTR-corrector interactions. All the restudies within 4.5 Å distance of the corrector are depicted. Residues mutated in the maturation and binding assays are indicated with colored circles.
See also Figures S5, S6, S10, and Table S1.
Mutations at the binding site diminished the efficacy of lumacaftor and tezacaftor
(A) Maturation assay of ΔF508-CFTR and binding-site mutations introduced to the ΔF508 background. Upper panel: SDS-PAGE of cell lysates from a single experiment, both mature and immature CFTR forms were visualized via the C-terminal GFP tag. Lower panel: Quantification of 3-6 repeats. The standard error of the mean is indicated as bars. Corrector concentrations: lumacaftor 1 μM, tezacaftor 10 μM, elexacaftor 0.2 μM, Corr-4a 10 μM in 0.1% DMSO. (B) SPA assay to measure the effects of mutations on lumacaftor binding. The Kd values of the polar residue substitutions K68I, R74A, and N71A were 0.19 ± 0.05 μM, 64 ± 13 nM, and 5.9 ± 2.9 nM, respectively. Those of the pocket-lining mutations A198Y, L195W, and S364F were 0.48 ± 0.11 μM, 0.86 ± 0.43 μM, and 1.43 ± 0.74 μM, respectively. Each data point represents the SEM of 6-9 of measurements. (C) Competition binding assay. The Ki values of K68I, N71A, and R74A CFTR were determined to be 0.12 ± 0.05 μM, 0.13 ± 0.03 μM and 0.41 ± 0.10 μM, respectively. For reference, the curves of the wtCFTR presented in Figure 1 were also shown (black line). Each data point represents the SEM of 6-9 of measurements. The concentration of [H3]lumacaftor was kept around the Kd value of the corresponding CFTR construct.
See also Figures S8, S9.
The proposed mechanism of type I correctors.
CFTR folds co-translationally as individual domains are synthesized, followed by assembly of the mature tertiary structure. The N-terminal TMD1, synthesized in the early phase, is thermodynamically unstable. The binding of the corrector (yellow sticks) stabilizes TMD1 in the ER membrane, makes it less susceptible to degradation. Increasing the lifetime of TMD1 can partially rescue folding defects in other parts of CFTR, such as ΔF508 in NBD1 (indicated in red). For simplicity, the chaperones that assist CFTR folding are not shown.
See also Table S2.
Cryo-EM analysis of the CFTR/lumacaftor complex.
(A) Image processing procedure and representative example of a micrograph. (B) Fourier shell correlation curves of the final map. (C) Local resolution estimation of the final map. (D) Particles orientation distribution histograms. Related to Figure 2.
Quality of the CFTR/lumacaftor reconstruction.
(A) Model-to-map fit for the full map (black), half-map 1 (blue), half-map 2 (red). (B) EM density of NBD2. (C) EM density of the four helices forming the lumacaftor-binding site. Related to Figure 2.
Cryo-EM analysis of the CFTR/lumacaftor+ATP complex.
(A) Image processing procedure and representative example of a micrograph. (B) Fourier shell correlation curves of the final map. (C) Local resolution estimation of the final map. (D) Particles orientation distribution histograms. Related to Figure 2.
Quality of the CFTR/lumacaftor+ATP complex.
(A) Model-to-map fit for the full map (black), halfmap 1 (blue), half-map 2 (red). (B) EM density at the degenerate ATP binding site. (C) EM density of the four helices forming the lumacaftor-binding site. Related to Figure 2.
Cryo-EM analysis of the CFTR/tezacaftor+ATP complex.
(A) Image processing procedure and representative example of a micrograph. (B) Particles orientation distribution histograms. (C) Fourier shell correlation curves of the final map. (D) Local resolution estimation of the final map. Related to Figure 3.
Quality of the CFTR/tezacaftor+ATP complex.
(A) Model-to-map fit for the full map (black), halfmap 1 (blue), half-map 2 (red). (B) EM density at the tezacaftor-binding site. The lipid acyl chain is represented as magenta sticks. (C) EM density of the four helices forming the tezacaftor-binding site. Related to Figure 3.
Comparison of the EM density at the corrector-binding site
(A) The lumacaftor-bound, NBD-separated structure (this study). (B) The apo structure (PDB:5UAK and EMD-8516). (C) The lumacaftor-bound, NBD-dimerized structure (this study). (D) The ivacaftor-bound structure (PDB:6O2P and EMD-0611). The black arrow indicates binding site of the potentiator ivacaftor (magenta sticks). All maps were contoured to show similar density for the CFTR main chain and side chains.
Related to Figure 2.
The mature, glycosylated form of CFTR is sensitive to PNGase F treatment.
Related to Figure 4.
Folding and stability assessment of CFTR mutants used in this study.
(A) Size exclusion chromatography profiles of the wt and mutant CFTR. The position of monomeric CFTR is indicated by an arrow. (B) Quantitative measurement of CFTR-ivacaftor interactions. The Kd values of the wt, K68I, R74A, N71A, L195W, A198Y and S364F CFTR were calculated to be 11.4 ± 2.5 nM, 6.1 ± 2.0 nM, 8.2 ± 2.0 nM, 12.1 ± 3.9 nM, 7.3 ± 2.6 nM, 21.9 ± 8.6 nM and 9.0 ± 2.6 nM respectively. Related to Figure 4.
Comparison of the corrector-bound and drug-free CFTR structures.
(A) Superposition of the CFTR/lumacaftor (blue) and drug-free (red) (PDB:6MSM) structures. Inset: local rearrangements of sidechains at the lumacaftor-binding site. Lumacaftor is represented in yellow sticks. (B) Superposition of the CFTR/tezacaftor (blue) and drug-free (red)(PDB:6MSM) structures. Inset: local rearrangements of sidechains the tezacaftor-binding site. Tezacaftor is represented in orange sticks. Related to Figure 2 and 3.
Cryo-EM data collection, refinement and validation statistics
