Fig. 1.
Codon usage in NBD1 affects CFTR aggregation propensity. (A) Left, Orthogonal stereo ribbon representation of phosphorylated, ATP-bound human CFTR with the E1371Q mutation [Protein Data Bank (PDB) ID: 6MSM]10, generated using UCSF Chimera65. The unstructured R region is denoted in parentheses. The position of the plasma membrane is shown with the intracellular (In) and extracellular (Out) sides identified. Right, Magnified view of NBD1 (domain enclosed by a red box in the CFTR structure), rotated 90° about both the x- and y-axes relative to the CFTR structure. Secondary structural elements of NBD1 are indicated above the magnified view, with α-helices shown as cylinders and β-strands as filled arrows. Subdomains of NBD1 are color-coded: N-terminal (green), α-helical (gray), and β-sheet core (magenta). Helices H3-H5 in the α-helical subdomain and β-strands S3 and S6-S8 in the β-sheet core subdomain are labeled. The locations of the regulatory insertion (RI) and regulatory extension (RE) are also denoted by labels in parentheses. (B) Representative immunoblot of WT CFTR and variants bearing High-efficiency synonymous codon substitutions showing immature (band B) and mature (band C) CFTR protein in radioimmunoprecipitation assay (RIPA)-soluble and insoluble fractions. β-actin and co-expressed GFP serve as loading and transfection controls, respectively. Original uncropped blots are presented in Fig. S1. (C) Summary data show fold changes in total (band B + band C) relative to WT (indicated by dotted line) for CFTR variants bearing High-efficiency synonymous codon substitutions. Symbols show individual values and columns are means ± SEM (n = 3–11 independent experiments per variant); * P < 0.05 and ** P < 0.01 vs. WT CFTR by two-tailed Student’s t-test. (D) Relative ribosome occupancy across NBD1 (residues 389–673), calculated with a 15-amino acid moving window average, for WT CFTR (black) and the CFTR construct with High-codons in segment-abc (blue). Secondary conformations of NBD1 are indicated above the graph, with α-helices shown as cylinders and β-strands as filled arrows. The light blue shaded region (residues 525–593) marks the segment changed in the CFTR construct with High-codons in segment-abc. Data are the means of five independent experiments per construct. Asterisks denote residues with statistically significant differences in ribosome occupancy between WT and the CFTR construct with High-codons in segment-abc (*P < 0.05; two-tailed Student’s t-test). Ribosome profiling data shown in (D) are also represented in Figs. 2, 3A, S2, and S3A,B.