Abstract
Misfolding accounts for the endoplasmic reticulum–associated degradation of mutant cystic fibrosis transmembrane conductance regulators (CFTRs), including deletion of Phe508 (ΔF508) in the nucleotide-binding domain 1 (NBD1). To study the role of Phe508, the de novo folding and stability of NBD1, NBD2 and CFTR were compared in conjunction with mutagenesis of Phe508. ΔF508 and amino acid replacements that prevented CFTR folding disrupted the NBD2 fold and its native interaction with NBD1. ΔF508 caused limited alteration in NBD1 conformation. Whereas nonpolar and some aliphatic residues were permissive, charged residues and glycine compromised the post-translational folding and stability of NBD2 and CFTR. The results suggest that hydrophobic side chain interactions of Phe508 are required for vectorial folding of NBD2 and the domain-domain assembly of CFTR, representing a combined co- and post-translational folding mechanism that may be used by other multidomain membrane proteins.
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Acknowledgements
We are indebted to N. Kartner and D. Bedwell for generously providing M3A7, L12B4 and antibody 4562 anti-CFTRs. We thank C. Daniels for careful reading the manuscript. M.S. was supported by a Canadian Institutes of Health Research (CIHR) doctoral studentship. This work was supported by grants to G.L. from the Canadian Cystic Fibrosis Foundation, the CIHR and the Premier's Research Excellence Award of the Ontario Ministry of Energy and Education.
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Supplementary information
Supplementary Fig. 1
NBD1 fragment recognized by two antibodies. (PDF 245 kb)
Supplementary Fig. 2
Conformational maturation of NBDs. (PDF 1350 kb)
Supplementary Fig. 3
Anion channel function of X508 CFTR mutants. (PDF 329 kb)
Supplementary Fig. 4
Proteinase K susceptibility of NBD2. (PDF 314 kb)
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Du, K., Sharma, M. & Lukacs, G. The ΔF508 cystic fibrosis mutation impairs domain-domain interactions and arrests post-translational folding of CFTR. Nat Struct Mol Biol 12, 17–25 (2005). https://doi.org/10.1038/nsmb882
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DOI: https://doi.org/10.1038/nsmb882
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