Stabilization of a nucleotide-binding domain of the cystic fibrosis transmembrane conductance regulator yields insight into disease-causing mutations

Abstract

Characterization of the second nucleotide-binding domain (NBD2) of the cystic fibrosis transmembrane conductance regulator (CFTR) has lagged behind research into the NBD1 domain, in part because NBD1 contains the F508del mutation, which is the dominant cause of cystic fibrosis. Research on NBD2 has also been hampered by the overall instability of the domain and the difficulty of producing reagents. Nonetheless, multiple disease-causing mutations reside in NBD2, and the domain is critical for CFTR function, because channel gating involves NBD1/NBD2 dimerization, and NBD2 contains the catalytically active ATPase site in CFTR. Recognizing the paucity of structural and biophysical data on NBD2, here we have defined a bioinformatics-based method for manually identifying stabilizing substitutions in NBD2, and we used an iterative process of screening single substitutions against thermal melting points to both produce minimally mutated stable constructs and individually characterize mutations. We present a range of stable constructs with minimal mutations to help inform further research on NBD2. We have used this stabilized background to study the effects of NBD2 mutations identified in cystic fibrosis (CF) patients, demonstrating that mutants such as N1303K and G1349D are characterized by lower stability, as shown previously for some NBD1 mutations, suggesting a potential role for NBD2 instability in the pathology of CF.

Keywords: ABC transporter; cystic fibrosis transmembrane conductance regulator (CFTR); protein engineering; protein misfolding; protein stability.

Figure 1.

ABC(C/B) sequence conservation profiles. Sequence profiles in weblogo (65, 66) format are shown for the 36 highly conserved ABC(C/B) positions in A and for a selection of additional positions discussed in this study in B. Each column of the sequence data shows amino acid frequencies from a different subset of the data, from left to right, ABC(B) half-transporters, NBD1 of ABC(B) full transporters, NBD2 of ABC(B) full transporters, NBD1 of ABC(C) transporters, NBD2 of ABC(C) transporters, NBD1 of CFTR, and NBD2 of CFTR. Amino acid frequencies are shown by the relative size of the letter, with the consensus identity at the top. Rows are labeled with functional or motif annotations and with the CFTR mutations occurring at each position; with CF-causing mutants from the CFTR2 data set shown in red, CFTR2 mutations that do not cause CF shown in black, and stabilizing mutations found either in the literature or in this study shown in blue. C, Asymmetric Conservation Bias in NBD1 and NBD2. Structural representation of CFTR NBD2 (from PDB code 3GD7 at 2.6 Å resolution). The helical subdomain is pink with helix 5 in brown; the α-β subdomain is blue, and motifs have been highlighted with the signature sequence in brown; the Walker A motif in red; the Walker B motif in green, and the Q loop in yellow. Positions where highly conserved residues can be lost in one NBD and that have a tendency to be lost in the same NBD when comparing paralogous sequences from subfamily C and/or subfamily B are identified. Positions biased toward being lost in NBD1 are noted by red spheres, clustering primarily around the ATP-binding site, and those biased toward being lost in NBD2 are noted in blue, clustering primarily around the helix-5 signature sequence.

Figure 2.

Deviations from wild-type versus structure. A, positions in CFTR NBD2 of relevance to this study have been noted with spheres, with solubilizing mutations from the SGX 5sol background construct in black (five positions), our selection of new solubilizing mutations in blue (four positions), and positions of CF-causing mutations in red (four positions). Notably, N1303K is the sole CF-causing NBD2 substitution that is not in or adjacent to one of the named sequence motifs. B, expanded view of the polar contacts (yellow lines) and water molecules (small red spheres if found in PDB and red star if predicted by structure) that connect Asn-1303, helix 5, and the Q-loop. In this structure, Ser-1359 contacts the Gln-1291 directly, pulling it away from the ATP-binding pocket. C, homologous view for the NBD1 of Multidrug Resistance Protein 1 (PDB code 2CBZ at 1.7 Å resolution). The Ser-1359 analog in this structure, Ala-781, fits the ABC(C/B) consensus sequence. With alanine at this position, we see Gln-713, the Gln-1291 equivalent, extending out away from helix 5 and into the ATP-binding pocket. This change appears to open up space for a much larger network of water-mediated polar contacts underlying the Q-loop, and it coincides with a structural rearrangement enabling direct contact between helix 5 (through Gln-774) to a backbone oxygen adjacent to the Walker B motif. (Structural representations are colored as in Fig. 1.)

Figure 3.

NMR spectra. NMR HSQC spectra were obtained for the SGX 5sol NBD2 background construct (A) and 9sol NBD2, our highest stability final construct (B). There is a 20-fold protein concentration difference between A and B, because the SGX 5sol construct was not soluble enough to produce an NMR sample with the concentration typically required. Thus, these spectra represent the best spectra we could produce using each construct, although they are not directly comparable. The SGX 5sol background has intense resolved peaks primarily in the disordered region of the protein, consistent with an aggregation-prone sample. In contrast, the 9sol sample has a larger number of dispersed peaks with a greater degree of uniformity in peak intensity, indicating predominantly monodisperse, folded protein, with peaks corresponding to the vast majority of resonances expected.

Figure 4.

Expression of CFTR with NBD2-stabilizing mutations. Western blottings of WT and F508del CFTR, with and without the S1359A NBD2-stabilizing mutation and other stabilizing mutations, are shown at the top of the figure. Following expression for 48–60 h, the lysates were analyzed by Western blotting using the mAb 660 α-CFTR antibody. The upper band in the WT samples corresponds to band C, and the lower band corresponds to band B. Tubulin (Tub) bands are shown as a loading control.

Figure 5.

Effect of S1359A on full-length CFTR. A, detection of S1359A and wild-type CFTR proteins in microsomal membranes of transfected HEK293 cells. 10 μg of total membrane protein were resolved by SDS-PAGE (7.5% acrylamide) and electroblotted onto a nitrocellulose membrane that was probed with primary mAb 596. B, single-channel recording after fusion of S1359A membrane vesicles with a planar lipid bilayer. The unitary conductance and open probability are indicated above the recording, and the amplitude histogram is shown to the left. Dwell time histograms below the recording were used to estimate mean closed and open times.

Figure 6.

Effect of ATP concentration and protein concentration on NBD2 stability. Thermal melts were performed by DSC on the purified 9sol NBD2 in a buffer containing 20 mm NaPO₄, 150 mm NaCl, 10% glycerol, 5 mm BME, 4 mm MgCl₂, and ATP. A, effect of varying ATP concentrations on NBD2 stability, measured at 20 μm protein. B, effect of varying NBD2 concentrations on stability, measured at 10 mm ATP.

Figure 7.

Stability of disease-causing mutants. Thermal melts were performed on the purified 9sol NBD2 as background and three disease-causing mutant variants in buffer containing 20 mm NaPO₄, 150 mm NaCl, 10% glycerol, 5 mm BME, 4 mm MgCl₂ and ATP, G1244E, S1251N, and N1303K. A, thermal melt assays using DSC at two different ATP concentrations, with 10 mm in black and 4 mm in red. B, normalized thermal melts of the same set of proteins measured by CD at a wavelength of 230 nm in the same buffer as used for the DSC experiments. Protein concentration was 0.5 mg/ml (18 μm), and ATP concentration was 10 mm. Circles represent data points, and the red line represents the fit obtained by linear regression as described previously (63).

Figure 8.

Return-to-consensus score post-validation. Back-to-consensus score value ranges for different categories of substitution mutations in both NBD1 (red) and NBD2 (blue), shown as standard box plots (points show statistical outliers, whiskers show lower and upper extremes, and box shows lower quartile, median, and upper quartile) with values on the x axis, and outliers labeled by their identity. A, comparison of mutations whose expression and stability were tested in isolated NBD2 during this study, with potential processing mutants defined as either < −1 °C ΔT_m or a ≥70% drop in expression, neutral mutations having ΔT_m values in the range of −1 to 1, and stabilizing mutations having either >1 °C ΔT_m or, in the case of Gln-1411, cannot be removed from the construct without a ≥70% drop in expression. Overall, the score matches effects on stability, with the one outlier, H1402A, being a special case known to stabilize by loss of a conserved function in ATP hydrolysis. B, comparison of score ranges by disease associations, splitting substitutions into four categories based on the CFTR1, CFTR2, and SNP databases. Mutations known to cause CF consistently have the lowest scores; those associated with the less severe disease CBAVD have intermediate but still negative scores, and the three conclusive non-disease-causing mutations in the CFTR2 database have neutral or slightly positive scores, showing that the general progression of disease association correlates with deviation from the general ABC(C/B) consensus. The SNP database covers the full range of potential scores with a median at 0, being predominantly neutral with regard to the consensus.