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. 2005 Jan;12(1):10-6.
doi: 10.1038/nsmb881. Epub 2004 Dec 26.

Side chain and backbone contributions of Phe508 to CFTR folding

Patrick H Thibodeau  1 , Chad A Brautigam, Mischa Machius, Philip J Thomas
Affiliations

Side chain and backbone contributions of Phe508 to CFTR folding

Patrick H Thibodeau et al. Nat Struct Mol Biol. 2005 Jan.

Abstract

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), an integral membrane protein, cause cystic fibrosis (CF). The most common CF-causing mutant, deletion of Phe508, fails to properly fold. To elucidate the role Phe508 plays in the folding of CFTR, missense mutations at this position were generated. Only one missense mutation had a pronounced effect on the stability and folding of the isolated domain in vitro. In contrast, many substitutions, including those of charged and bulky residues, disrupted folding of full-length CFTR in cells. Structures of two mutant nucleotide-binding domains (NBDs) reveal only local alterations of the surface near position 508. These results suggest that the peptide backbone plays a role in the proper folding of the domain, whereas the side chain plays a role in defining a surface of NBD1 that potentially interacts with other domains during the maturation of intact CFTR.

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Conflict of interest statement

COMPETING INTERESTS STATEMENT

The authors declare that they have no competing financial interests.

Figures

Figure 1
Figure 1
NBD1 folding efficiency as a function of folding temperature. NBD1 proteins were refolded at multiple temperatures and assessed for their ability to adopt native, soluble structures by tryptophan fluorescence. (a) The wild-type and ΔF508 proteins refolded with different efficiencies as temperature increased >4 °C. The ΔF508 was unable to refold with high efficiency at intermediate and high temperatures, whereas the wild type maintained a higher folding efficiency at these elevated temperatures. Substitution mutations at position 508 had little effect on the folding efficiency of NBD1 in vitro. (b,c) The hydrophobic substitutions (b) and the charged and polar substitutions (c) are superimposed on the wild-type and ΔF508 protein folding efficiencies. (b) The F508W mutant, the only mutant that deviated markedly from the wild type, was rescued by the introduction of a second missense mutation, W496F.
Figure 2
Figure 2
Structure of NBD1 proteins. (a) A smoothed trace of the main chain of the three proteins. Wild type, green; F508S variant, orange; F508R variant, blue. The r.m.s. deviations between the wild-type and F508S or F508R structures are ~0.3 Å. The side chain of Phe508 from the wild-type structure is red. (b,c) Electron density maps for the region near the 508 locus. Carbon atoms, green; oxygen, red; nitrogen, blue; sulfur, yellow; phosphorus, pink. The positions of the carbon atoms of Met496 (b) and Phe508 (b,c) from the wild-type structure are shown in orange for reference. The 2Fo – Fc electron density map (contoured at 1 σ) calculated with the F508S data at a resolution of 2.7 Å superposed on the final F508S model. (c) The 2Fo – Fc map (contoured at 1 σ) calculated from the F508R data at a resolution of 3.1 Å superposed on the final F508R model. (d) Molecular surfaces of the NBD1 proteins. The position of the 508 residue is circled for reference. Red, regions of negative electrostatic potential; blue, positive regions (range from −10 to +10 kT).
Figure 3
Figure 3
Maturation of full-length CFTR mutants. Maturation of full-length CFTR was monitored as a function of the formation of the upper molecular mass band, band C, indicative of post-ER trafficking. Band B, a marker for core-glycosylated ER resident protein is indicative of protein expression, but not of proper folding, assembly and post-ER trafficking. Closed arrowheads, CF-causing mutations; open arrowheads, known non-CF-causing variants. (F508S has been associated with congenital bilateral absence of (the vas deferens, but not CF.)
Figure 4
Figure 4
ABC transporter structure and CFTR biogenesis. (a) Crystal structure of the BtuCD ABC-transport systems. The Escherichia coli vitamin D transporter system BtuCD structures (PDB entry 1L7V) are shown with the Phe508-analogous residue Leu96 shown in red spheres at the NBD-TMD interfaces. The BtuC transmembrane proteins are blue and the BtuD NBDs are yellow. Two views of the BtuCD complex are shown rotated about the vertical axis by ~90°. (b) Hierarchical folding of CFTR. Step 1, TMD1 is translated and inserted into the membrane. Pale blue indicates the reduced stability of TMD1 in the absence of NBD1. Step 2, NBD1 is translated and folds into a native or near-native state. The blurred image of the mNBD1 structure indicates the attainment of a native or near-native state, which is most likely stabilized by interactions with additional CFTR domains. Step 3, NBD1 docks against TMD1. This event probably leads to the stabilization of both NBD1 and TMD1, as shown by the change in blue color in the TMD and the sharpening of the NBD1 structure. This is followed by the translation, folding and assembly of the domains C-terminal to NBD1. Mutations that putatively affect each step are in parentheses. The NBDs are represented by the mNBD1 structure and are oriented relative to the NBD dimer and TMD–NBD complex seen in BtuCD with the assumption that CFTR is monomeric with a functional NBD1–NBD2 heterodimer.
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Comment in

  • Arrest of CFTRDeltaF508 folding.
    Cyr DM. Cyr DM. Nat Struct Mol Biol. 2005 Jan;12(1):2-3. doi: 10.1038/nsmb0105-2. Nat Struct Mol Biol. 2005. PMID: 15689966 No abstract available.

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