Ligand binding to a remote site thermodynamically corrects the F508del mutation in the human cystic fibrosis transmembrane conductance regulator

Abstract

Many disease-causing mutations impair protein stability. Here, we explore a thermodynamic strategy to correct the disease-causing F508del mutation in the human cystic fibrosis transmembrane conductance regulator (hCFTR). F508del destabilizes nucleotide-binding domain 1 (hNBD1) in hCFTR relative to an aggregation-prone intermediate. We developed a fluorescence self-quenching assay for compounds that prevent aggregation of hNBD1 by stabilizing its native conformation. Unexpectedly, we found that dTTP and nucleotide analogs with exocyclic methyl groups bind to hNBD1 more strongly than ATP and preserve electrophysiological function of full-length F508del-hCFTR channels at temperatures up to 37 °C. Furthermore, nucleotides that increase open-channel probability, which reflects stabilization of an interdomain interface to hNBD1, thermally protect full-length F508del-hCFTR even when they do not stabilize isolated hNBD1. Therefore, stabilization of hNBD1 itself or of one of its interdomain interfaces by a small molecule indirectly offsets the destabilizing effect of the F508del mutation on full-length hCFTR. These results indicate that high-affinity binding of a small molecule to a remote site can correct a disease-causing mutation. We propose that the strategies described here should be applicable to identifying small molecules to help manage other human diseases caused by mutations that destabilize native protein conformation.

Keywords: F508del mutation; calorimetry; cystic fibrosis; cystic fibrosis transmembrane conductance regulator (CFTR); deoxythymidine; electrophysiology; fluorescence resonance energy transfer (FRET); high-throughput screening (HTS); isothermal titration calorimetry (ITC); protein aggregation; thermodynamics.

Figure 1.

Thermodynamic strategies to correct the disease-causing stability defect in F508del-CFTR. The structure of hCFTR is schematized at upper right. Its two nucleotide-binding domains, NBD1 (16–18, 44) (blue, PDB code 2BBO) and NBD2 (green, PDB code 3GD7) bind two molecules of Mg-ATP (yellow and orange) at their mutual interface (4–11). Its two transmembrane domains, TMD1 (blue) and TMD2 (green), structurally interdigitate to form two composite binding sites (20, 21), one for NBD1 and the other for NBD2. Residue Phe-508 (red), which is deleted in F508del-hCFTR, is located on the surface of NBD1 that binds to the cognate binding site formed by TMD1/TMD2 (1, 17, 18, 20, 21). The regulatory or R region (magenta) is believed to be predominantly disordered but to have segments that reversibly bind to the other domains in hCFTR to modulate their behavior (16, 91–93). The folding pathway of human NBD1 (hNBD1) is schematized in the center and at the left, above a free-energy diagram. The native conformation of hNBD1, which is stabilized by binding to ATP and the TMDs of hCFTR, is in equilibrium with a low-energy “molten-globule” conformation (94–101), which retains native-like secondary structure. Formation of this molten globule species is inhibited by a wide range of second-site mutations (10, 11, 16) that also suppress the trafficking defect (32, 34, 41) in F508del-hCFTR that is responsible for causing cystic fibrosis (24). These observations support the hypothesis that the F508del mutation causes the disease by promoting formation of the highly aggregation-prone molten-globule intermediate. Thermodynamic theory suggests that chemical compounds that bind to F508del-hNBD1, either alone or at its interface with the TMDs of hCFTR, should pull the domain away from the molten-globule conformation (20, 27–30) and thereby offset the defect caused by the mutation (orange and green dotted lines in the free-energy diagram). This thermodynamic effect can be thought of as a form of “mass action.”

Figure 2.

Engineering hNBD1 for single-site fluorescent labeling. A, ribbon diagram of hNBD1ΔRI with Mg-ATP (yellow), residue Phe-508 (red), and all cysteine residues in the native domain shown in space-filling representation. The Cys residues are colored like the protein backbone in the subdomain in which they are located (orange for the F1-like ATP binding core, green for the ABCβ subdomain, and blue for the ABCα subdomain. PDB code 2PZE). B, melting temperature (T_m) from DSC assays plotted against the change in the free energy of folding predicted by the program Eris (45, 80, 102, 103) for 11 Cys-reduced hNBD1ΔRI variants. Assays were conducted at 0.5 mg/ml protein using 2 mm ATP in SSB, which contains 3 mm MgCl₂, 150 mm NaCl, 10% (v/v) glycerol, 10% (v/v) ethylene glycol, 1 mm TCEP, and 20 mm Na-HEPES, pH 7.5. C, table showing the average value and sample variance of the T_m from at least two replicate DSC assays conducted in the same ATP-containing buffer on protein constructs used to develop or implement the fluorescence self-quenching assay for hNBD1ΔRI stability (Fig. 3). The magenta prefix AF546- indicates protein covalently labeled with the fluorescent dye Alexa Fluor 546.

Figure 3.

Efficient tFSQ assay for hNBD1 stability. A, triple detection experiment monitoring far-UV CD at 230 nm (top), SLS at 230 nm (middle), and fluorescence emission intensity at 573 nm (bottom) during thermal denaturation at 3 °C/min of 0.05 mg/ml (2 μm) AF546-labeled F508–C592L/G646C–hNBD1ΔRI in the presence of 30 or 430 μm Mg-ATP. The temperature corresponding to the steepest slope of decline in emission intensity, as determined using nonlinear curve fitting to the first derivative of the van't Hoff equation (104), is defined as the fluorescence self-quenching temperature (T_SQ).These experiments were conducted as described previously (10, 11) in a 1.6-ml cuvette using a fluorescence excitation wavelength of 556 nm. B, fluorescence emission intensity at 586 nm (top) and the first derivative of that intensity (bottom) during thermal denaturation of 0.005 mg/ml (∼0.2 μm) AF546-labeled F508del-C592L/G646C–hNBD1ΔRI in a 10-μl volume in a 96-well microtiter plate. Fluorescence in all wells was monitored in parallel using a real-time PCR machine with 545 nm excitation. Results are shown from three replicate assays conducted at each of four different Mg-ATP concentrations indicated on the graph. The T_SQ values here differ from A because the experiments employed different protein constructs (F508 versus F508del) and ATP concentrations. C, T_SQ values measured in eight replicate assays on AF546-labeled F508del-C592L/G646C–hNBD1ΔRI conducted using the same methods in 3 or 2003 μm Mg-ATP in the absence (left) or presence (right) of a 0.1% (w/v) concentration of the nonionic detergent C12E8 (∼10× cmc). All assays in A–C were conducted in SSB.

Figure 4.

Stabilization of hNBD1ΔRI by nucleotide analogs. A and B, T_SQ values from five replicate tFSQ assays conducted on AF546-labeled F508–C592L/G646C–hNBD1ΔRI in the presence of a 2 mm concentration of the Mg²⁺ complex of the indicated nucleotide. The control assays contained 3 μm Mg-ATP, which comes from the protein storage buffer and is present in all samples in addition to any added nucleotide. Assays were conducted in SSB using a heating rate of 3 °C/min in a microtiter plate as in Fig. 3, B and C. Equivalent differences in T_SQ are observed in assays conducted with AF546-labeled F508del-C592L/G646C–hNBD1ΔRI (Fig. S3B) or full-length C592L–hNBD1 (with native Cys-647) retaining the RI and RE sequences (Fig. S3, C and D). Sample variance for these assays ranged from 0.1 to 0.3 °C, as reported in Fig. S5B. Data from four nucleotides are duplicated in panels in A and B to illustrate the consistent effect of adding an exocyclic methyl group to the bases. C, DSC assays conducted on 0.5 mg/ml protein in the presence of a 0.5 mm concentration of the Mg²⁺ complex of the indicated nucleotide in SSB. D, T_m from DSC assays plotted against the T_SQ from the fluorescence self-quenching assays in Fig. 4, which were conducted in the same buffer containing a higher 2 mm concentration of the Mg²⁺ complex of the indicated nucleotide. Linear regression gives a slope of 1.03 ± 0.02 (red line). E, results from ITC measurements of ATP or dTTP binding to hNBD1ΔRI in the same buffer at 10 °C. The fitted binding stoichiometry likely reflects some aggregated protein that does not bind nucleotide, which should not perturb inferred thermodynamic parameters, as explained under “Experimental procedures.”

Figure 5.

X-ray crystal structures of nucleotides bound to hNBD1ΔRI. A–D, nucleotide-bound hNBD1ΔRI structures are in a tetragonal lattice in which the nucleotides do not participate in crystal packing. The protein backbone and carbon atoms in two residues from the dTTP structure are shown in gray. The Mg²⁺ cofactor bound to dTTP is shown as a yellow sphere. Four nucleotides from different structures are shown in ball-and-stick representation with carbon atoms colored according to nucleotide identity: magenta for dTTP (PDB code 5TF8, R_free = 18.7% at 1.86 Å); green for ATP (PDB code 5TF7, R_free = 20.0% at 1.93 Å); cyan for 7Me-GTP (PDB code 5TFB, R_free = 19.9% at 1.87 Å); pink for dUTP (PDB code 5TFA, R_free = 19.6% at 1 .87 Å), and ruby for GTP (PDB code 5TFC, R_free = 20.2% at 1.92 Å). Oxygen, nitrogen, and phosphorous atoms are colored red, blue, and orange, respectively. Dotted lines connect atoms for which internuclear distances are given.

Figure 6.

Temperature-dependent single-channel electrophysiology analyses of full-length hCFTR. A–C, single-channel electrophysiology measurements conducted at the indicated temperatures on wildtype (WT) hCFTR or VX809-rescued (105) F508del-hCFTR (rF508del-hCFTR) in the presence of the indicated NTPs at 2 mm concentration. Measurements were performed at 25 °C (A), 30 °C (B), or 37 °C (C) on proteins reconstituted into black lipid membranes (39, 106). Electrical current is calibrated in picoamps (pA), and time is indicated in seconds (sec). The plots on the left in B and C show all-points histograms with the observed current on the ordinate, which were used to calculate channel P_o. The lower expanded traces in A show 60-s segments from the upper traces, which were recorded for 10 min, and the traces in B represent the final 2 min of a 20-min recording under each condition. Three different experiments of 10 min duration were used to calculate the single-channel parameters for both WT and rF058del-hCFTR at 25 °C in the presence of 2 mm dTTP: γ_WT = γ_rF508del = 10.6 ± 0.1 pS; P_o-WT = 0.15 ± 0.02; and P_o-rF08del = 0.10 ± 0.02. D, Eadie-Hofstee–like plots showing P_o versus (P_o/[NTP]) values derived from electrophysiology experiments like those in A–C conducted at 30 °C on WT-hCFTR at varying concentrations of the indicated NTPs (raw data not shown). The slopes of the solid lines are proportional to the effective nucleotide dissociation constant or affinity in the open state of the channel, as described by the parameter K_eff in the Eadie-Hofstee equation presented in the text. The dotted lines connect points from experiments conducted at the same concentration of different nucleotides. At least five different experiments of 2 min duration were used to calculate the mean P_o value of for each NTP type and concentration. The values of the standard error of the mean are less than the size of the symbols and therefore not shown. E, Gibbs free energy change for opening the WT-hCFTR channel at saturating nucleotide concentration (ΔG⁰_{hNBD1–hNBD2}) plotted against the fluorescence T_SQ of AF546-labeled F508–C592L/G646C–hNBD1ΔRI at a 2 mm concentration of the same nucleotide (from Fig. 4, A and B). The value of ΔG⁰_{hNBD1–hNBD2} is calculated as −RT·ln(P_o-max/(1 − P_o-max)). Decreasing ΔG⁰_{hNBD1–hNBD2} reflects greater stabilization of the functional hNBD1–hNBD2 interface, which is required for stable channel opening, whereas increasing T_SQ reflects greater stabilization of hNBD1 itself. Closed symbols are used for nucleotides that maintain normal activity of rF508del-hCFTR channels at 30 °C, and open symbols are used for those that do not. The ellipses highlight the correlation predicted by the thermodynamic scheme in Fig. 1 between the ability of nucleotides to rescue the thermal defect caused by the F508del mutation (B and C) and their efficacy in stabilizing either hNBD1 itself (orange dotted lines here and in Fig. 1) or the hNBD1–hNBD2 interface (green dotted lines here and in Fig. 1).