The CFTR P67L variant reveals a key role for N-terminal lasso helices in channel folding, maturation, and pharmacologic rescue

Abstract

Patients with cystic fibrosis (CF) harboring the P67L variant in the cystic fibrosis transmembrane conductance regulator (CFTR) often exhibit a typical CF phenotype, including severe respiratory compromise. This rare mutation (reported in <300 patients worldwide) responds robustly to CFTR correctors, such as lumacaftor and tezacaftor, with rescue in model systems that far exceed what can be achieved for the archetypical CFTR mutant F508del. However, the specific molecular consequences of the P67L mutation are poorly characterized. In this study, we conducted biochemical measurements following low-temperature growth and/or intragenic suppression, which suggest a mechanism underlying P67L that (1) shares key pathogenic features with F508del, including off-pathway (non-native) folding intermediates, (2) is linked to folding stability of nucleotide-binding domains 1 and 2, and (3) demonstrates pharmacologic rescue that requires domains in the carboxyl half of the protein. We also investigated the "lasso" helices 1 and 2, which occur immediately upstream of P67. Based on limited proteolysis, pulse chase, and molecular dynamics analysis of full-length CFTR and a series of deletion constructs, we argue that P67L and other maturational processing (class 2) defects impair the integrity of the lasso motif and confer misfolding of downstream domains. Thus, amino-terminal missense variants elicit a conformational change throughout CFTR that abrogates maturation while providing a robust substrate for pharmacologic repair.

Keywords: cystic fibrosis transmembrane conductance regulator; drug action; lumacaftor; protein misfolding; protein structure; protein trafficking.

Figure 1

Conservation of the lasso motif and P67 at the N terminus.A, alignment of CFTR amino terminus segment (aa 1–109) across multiple species, emblematic of a larger, ConSurf-based analysis ((72), Experimental procedures section). Color legend indicates highly variable (light blue) to average (white) to highly conserved (magenta) residues; yellow indicates insufficient data. B, residues conserved throughout species listed are highlighted in magenta (human CFTR; Protein Data Bank: 5UAK). C, the P67L variant is situated between lasso helix 2 (Lh2 shown in light blue) and elbow helix 1 (Eh1 shown in green) and in a region proximate to numerous transmembrane helices (THs) within CFTR membrane-spanning domains. CFTR, cystic fibrosis transmembrane conductance regulator.

Figure 2

P67L exhibits a molecular folding phenotype different from F508del.A, F508del lumacaftor functional correction in comparison to 27 °C (low temperature) rescue. Note change in y-axis in upper versus lower left panels. Arrows indicate forskolin (CFTR activator via cAMP/protein kinase A, 20 μM), ivacaftor (CFTR potentiator, 5 μM), and inh172 (CFTR inhibitor, 10 μM) (N = 6–9 filters/condition). Low-temperature incubation has been shown previously to confer diminished levels of WT CFTR expression (13). B, P67L band C (mature, post ER glycoform) in cis with R555K or R1070W second-site suppression following lumacaftor (N = 3 replicates/condition). Both second-site suppressors have been shown previously to rescue F508del processing (9, 10). Relative quantification (% wt band C) of the mature (mutant) glycoform shown on left was normalized to CFTR mRNA level in each sample (right). C, in Ussing chamber analysis, R555K was tested for effect on P67L activity. Short-circuit current tracings provided in the left panel are quantified on right (N = 3 replicates/condition). Acute additions were as previously mentioned except forskolin was tested at 10 μM. Lumacaftor was administered for 48 h at 3 μM prior to arrays shown. These studies were performed in the Fischer rat thyroid model. p Values indicate level of change as measured for band C CFTR or forskolin response (Student's t test). Error bars show mean ± SD (panel A and B) or mean ± SEM (panel C). CFTR, cystic fibrosis transmembrane conductance regulator.

Figure 3

Lasso helix 2 is necessary for CFTR maturation.A, construct encoding the first 380 residues of CFTR was used to probe modulator response of TMD1 (6) following deletion of Lh1 (Lh1del, aa 19–29) or loss of Lh2 (aa 46–61) (n = 3) during transient expression in human embryonic kidney 293 cells. p Values by Student's t test. B, response to lumacaftor (3 μM, 24 h incubation) of CFTR-380 (TMD1) constructs in human embryonic kidney 293 cells was measured following introduction of specific N-terminal point mutations. (P67L, E56K [within Lh2], R75E [a mutation near Lh2 at the tail of elbow helix 1], or W57G [a point mutation within Lh2]). Key aspects of this experiment have been repeated 3 to 5 times with similar results. Detection of CFTR was with MM13-4 antibody. CFTR, cystic fibrosis transmembrane conductance regulator; TMD, transmembrane domain.

Figure 4

Lumacaftor correction of P67L in the presence of the second half of CFTR.A, ribbon diagrams of human CFTR showing location of R555 and CFTR half molecules. Blue inset is expanded on right. In panel (B), protein stability is tested following lumacaftor treatment (3 μM, 24 h incubation) of the WT-837X construct alone or in the presence of M837 CFTR. P67L–837X was also evaluated following lumacaftor with M837 CFTR coexpressed. Studies were conducted in human embryonic kidney 293 cells, and CFTR was detected with UNC432 and UNC769 antibodies. This experiment has been repeated three times with similar results. CFTR, cystic fibrosis transmembrane conductance regulator.

Figure 5

P67L affects late domain folding. Human embryonic kidney 293 cells were radiolabeled for 10 min and chased for 0 (left panels) or 2 h (right panels) in the presence or the absence of 3 μM VX-809 (lumacaftor). Detergent cell lysates were treated (bottom four panels) or not (top panels) with 25 μg/ml proteinase K. CFTR and fragments were immunoprecipitated with domain-specific antibodies: TMD1C (TMD1), Mr. Pink (which detects NBD1 fragments and nonproteolyzed CFTR NBD1) (30, 31, 32), TMD2C (TMD2), and 596 (NBD2) to probe conformation of each domain. Fragments are identified by domain according to nomenclature reported previously (31, 32). C, control transfection with empty plasmid; CFTR, cystic fibrosis transmembrane conductance regulator; NBD, nucleotide-binding domain; TMD, transmembrane domain.

Figure 6

R555K rescues P67L NBD2 destabilization in the inward model. Analysis of averaged (over three 100 ns simulations) root-mean-square fluctuation difference profiles comparing fluctuations of WT versus P67L-CFTR (P67L—WT in blue) and of WT versus P67L/R555K-CFTR (P67L/R555K—WT in red) for inward-facing CFTR. Positive and negative values correspond to regions where the mutants are fluctuating more or less than the WT, respectively. P67L destabilizes NBD2 (∼1200–1400 region), whereas the R555K mutation appears to counteract this effect. Statistically significant differences were noted in the NBD2 region for WT versus P67L CFTR, as well as for P67L versus P67L/R555K CFTR (p = 0.05, t test with two samples assuming unequal variances). CFTR, cystic fibrosis transmembrane conductance regulator; NBD, nucleotide-binding domain.

Figure 7

Molecular dynamics simulations of P67L-CFTR exhibit increased molecular separation between NBDs. Averaged (over three 100 ns simulations) distances between NBD1–NBD2 centers of mass derived from molecular dynamics simulations of models of WT (black) and P67L-CFTR (red) in the phosphorylated, ATP-bound, outward-facing conformation. Larger distances are clearly apparent for the P67L mutant and were found to be statistically significant at the p = 0.05 level (Student's t test). CFTR, cystic fibrosis transmembrane conductance regulator; NBD, nucleotide-binding domain.

Figure 8

Potential binding sites for lumacaftor flank Lasso helix 2. A model of the inward-facing cystic fibrosis transmembrane conductance regulator and the lumacaftor chemical structure is depicted. Insets focus on predicted binding sites for lumacaftor; potential interacting residues are highlighted in magenta. A1 and A2 suggest lumacaftor localization in proximity of the ICL4–NBD1 interface, adjacent to the end of transmembrane helix 6 (TMH6) (∼residue Y380). B1 and B2 portray an alternative binding site within the lasso region and involving the n terminus upstream of amino acid residue 80, transmembrane helices 1 and 2.

Figure 9

Model depicting structural defects conferred by P67L.A, the ribbon diagram on left demonstrates lasso arrangement relative to the transmembrane domains (TMDs). Lasso helix 1 (Lh1), lasso helix 2 (Lh2), and elbow helix 1 (Eh1) are shown (detail) on right. The lasso motif serves to constrain adjacent TMD helices (e.g., TMD1–TH2, TH3, and TH6) and (TMD2–TH10 and TH11). In panel (B), mutation of P67 leads to changes of an alpha-helical “kink” near the proline, contributing to disruption of lasso position and misalignment of transmembrane helices, global conformational change from loss of TMD structure, and overall protein misfolding/early degradation by the proteasome. Clinically important CFTR mutations near P67L (E56K and W57G) would also be expected to impact folding of the lasso motif. CFTR, cystic fibrosis transmembrane conductance regulator.