CFTR trafficking mutations disrupt cotranslational protein folding by targeting biosynthetic intermediates

Abstract

Protein misfolding causes a wide spectrum of human disease, and therapies that target misfolding are transforming the clinical care of cystic fibrosis. Despite this success, however, very little is known about how disease-causing mutations affect the de novo folding landscape. Here we show that inherited, disease-causing mutations located within the first nucleotide-binding domain (NBD1) of the cystic fibrosis transmembrane conductance regulator (CFTR) have distinct effects on nascent polypeptides. Two of these mutations (A455E and L558S) delay compaction of the nascent NBD1 during a critical window of synthesis. The observed folding defect is highly dependent on nascent chain length as well as its attachment to the ribosome. Moreover, restoration of the NBD1 cotranslational folding defect by second site suppressor mutations also partially restores folding of full-length CFTR. These findings demonstrate that nascent folding intermediates can play an important role in disease pathogenesis and thus provide potential targets for pharmacological correction.

Fig. 1. CF-causing mutations perturb NBD1 biosynthetic intermediates.

a Location of CFTR processing mutations shown in the NBD1 structure taken from the human CFTR Cryo-EM structure (PDB: 5UAK). b Cartoon conceptually depicting FRET assay using translationally incorporated fluorophores to detect structural transitions of ribosome-attached nascent polypeptides. c Schematic of ribosome-attached CFP-NBD1 fusion protein truncated at CFTR residue 654 showing locations of FRET acceptor dye at CFTR residues Arg487, or Asp567, which report folding of the α-helical subdomain, or β-sheet core, respectively^,. Helices and β-strands are drawn as cylinders and filled arrows, respectively. d, e FRET efficiencies obtained for wild-type and mutant nascent polypeptides in (ribosome-attached) CFP-NBD1 constructs truncated at residue 654 and containing acceptor dye at residue Arg487 d or Asp567 e. Each dot represents data from an independent experiment. Each bar shows mean ± SEM, n ≥ 3 independent experiments. Two-tailed student’s t test comparing wild-type and indicated mutant, asterisk p < 0.05, double asterisk p < 0.01, triple asterisk p < 0.001, n.s. > 0.05 otherwise indicated. Red line shows mean of FRET efficiency for wild-type. Source data of d and e are provided as a Source Data file.

Fig. 2. A455E, L558S transiently disrupt NBD1 cotranslational folding.

a Immunoblot of full-length CFTR (wild-type, A455E, or L558S) expressed in human embryonic kidney (HEK) 293 cells showing core glycosylated (band B) and mature CFTR (band C). Uncropped blots in Source Data. b Schematic of ribosome-attached CFP-NBD1 showing approximate location of acceptor dye incorporation sites (residue Arg487 or Asp567) and truncation sites used in c–f. c–f Length-dependent FRET efficiencies for wild-type (red line) and A455E or L558S (blue line) obtained from ribosome-attached CFP-NBD1 constructs with acceptor dye located at R487UAG c, d or D567UAG e, f for each truncation site indicated. Data are mean ± SEM, n ≥ 3 independent experiments. Two-tailed student’s t test comparing wild-type and A455E or L558S, asterisk p < 0.05, double asterisk p < 0.01, triple asterisk p < 0.001, n.s. > 0.05 otherwise indicated. Note that wild-type data for truncation sites 500, 568, 584, and 664, and A455E data for truncation sites 500, 550, 568, 664 shown in c, and wild-type data for truncation sites 500, 550, 568, 584, and 664, and L558S data for truncation site 664 shown in d have error bars smaller than the dots. Some data for truncation sites 568, 604, 654, 666, 704, and 744 shown in c, d are also taken from Figs. 1d, 3a, b, 4a, b, & 5b. Source data of a, c–f are provided as a Source Data file.

Fig. 3. Ribosome influences cotranslational folding defects.

a, c FRET efficiencies of ribosome-bound and released CFP-NBD1 ± A455E or ± L558S polypeptides (acceptor dye at Arg487) truncated at residue 568 or 604 a, and 664 or 704 c. Each dot represents data from an independent experiment. Each bar shows mean ± SEM, n = 3 or 4 independent experiments. Two-tailed student’s t test, asterisk p < 0.05, double asterisk p < 0.01, triple asterisk p < 0.001, n.s. > 0.05 otherwise indicated. Ribosome-released (RNase A) (truncation 568 or 664) and ribosome-bound (truncation 604 or 704) polypeptides contains equivalent cytosolically exposed residues, therefore differences in FRET are due to ribosome attachment. b, d Illustration depicting results of a, or c showing schematic of wild-type and mutant ribosome-bound and released polypeptides (acceptor dye at Arg487) truncated at indicated residue. b NBD1 polypeptides truncated at residue 568 remain unfolded on the ribosome and show an increase in FRET, consistent with α-helical subdomain folding, following ribosome release. Wild-type ribosome-bound and released polypeptides are in the compact sate (high FRET) at truncation 604, whereas mutant polypeptides remain less compact (low FRET) both before and after ribosome release. d Wild-type ribosome-bound and released polypeptides are shown in the compact sate at truncation 664, whereas mutant polypeptides remain less compact (low FRET) both before and after ribosome release. In contrast, both wild-type and mutant polypeptides truncated at residue 704 have achieved a compact state (high FRET). Source data of a and b are provided as a Source Data file.

Fig. 4. Thermal stability of NBD1-folding intermediates.

a-d Temperature-dependent FRET efficiencies of wild-type (red line) and A455E or L558S (blue lines) ribosome-bound CFP-NBD1 polypeptides. Acceptor dye was located at Arg487 and polypeptides were truncated at residue 654 (intermediate) a, b or 744 (full-length) c, d. Data are mean ± SEM, n = 3 or 7 independent experiments. Two-tailed student’s t test, asterisk p < 0.05, double asterisk p < 0.01, triple asterisk p < 0.001, n.s. > 0.05 otherwise indicated. e, f Graphs show the difference between temperature-dependent FRET efficiencies for wild-type and mutants calculated from a–d. One-tailed student’s t test, *p < 0.05, **p < 0.01, n.s. > 0.05 otherwise indicated. Note that wild-type data at 45 °C in b–d, A455E data at 25, 40, 45, and 50 °C in c, and L558S data at 4, 35, 40, 45, and 50 °C in d have error bars smaller than the dots. Source data of a–f are provided as a Source Data file.

Fig. 5. Genetic correction of cotranslational folding also restores CFTR trafficking.

a–c S492P & I539T (PT) suppressor mutations restore FRET efficiency of the folding intermediate (truncation at 654, acceptor dye at Arg487) for A455E b but not L558S c. Each dot represents data from an independent experiment. Each bar shows mean ± SEM, n = 3 or 4 independent experiments. Two-tailed student’s t test, asterisk p < 0.05, double asterisk p < 0.01, triple asterisk p < 0.001, n.s. > 0.05 otherwise indicated. d, e Temperature-dependent FRET efficiencies of wild-type (red line), A455E (blue line), and A455E + PT (black line). Ribosome-bound CFP-NBD1 polypeptides were truncated at residue 654 (acceptor dye at Arg487). The difference between wild-type and mutants in e were calculated from the data in d. Data are mean ± SEM, n = 4 independent experiments. Two-tailed student’s t test comparing wild-type and A455E (red asterisks), A455E and A455E + PT (black asterisks), or wild-type and A455E + PT (n.s.) in d and one-tailed student’s t test in e, asterisk p < 0.05, double asterisk p < 0.01, n.s. > 0.05 otherwise indicated. Note that some points have error bars smaller than the dot. f, g Immunoblot of CFTR A455E or L558S and A455E or L558S plus PT mutations expressed in human embryonic kidney (HEK) 293 cells showing correction of folding defect for full-length CFTR A455E but not L558S. Uncropped blots in Source Data. Each dot represents data from an independent experiment. Each bar shows mean ± SEM, n = 4–6 independent experiments. Two-tailed student’s t test, double asterisk p < 0.01, n.s. > 0.05 otherwise indicated. Source data of b–g are provided as a Source Data file.