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Restoration of domain folding and interdomain assembly by second‐site suppressors of the ΔF508 mutation in CFTR
Author(s) -
He Lihua,
Aleksandrov Luba A.,
Cui Liying,
Jensen Timothy J.,
Nesbitt Kenneth L.,
Riordan John R.
Publication year - 2010
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fj.09-141788
Subject(s) - cyclic nucleotide binding domain , δf508 , mutant , suppressor , protein folding , context (archaeology) , protein structure , chemistry , mutation , biology , microbiology and biotechnology , nucleotide , biophysics , biochemistry , gene , paleontology
Deletion of PHE508 (ΔF508) from the first nucleotide‐binding domain (NBD1) of CFTR, which causes most cystic fibrosis, disrupts the folding and assembly of the protein. Although the folding pathways and yield of isolated NBD1 are altered, its global structure is not, and details of the changes in the rest of the protein remain unclear. To gain further insight into how the whole mutant protein is altered, we have determined the influence of known second‐site suppressor mutations in NBD1 on the conformation of this domain and key interfaces between domains. We found that the suppressors restored maturation of only those processing mutations located in NBD1, but not in other domains, including those in the C‐terminal cytoplasmic loop of the second membrane‐spanning domain, which forms an interface with the NBD1 surface. Nevertheless, the suppressors promoted the formation of this interface and others in the absence of F508. The suppressors restored maturation in a ΔF508 construct from which NBD2 was absent but to a lesser extent than in the full‐length, indicating that ΔF508 disrupts interactions involving NBD2, as well as other domains. Rescue of ΔF508‐CFTR by suppressors required the biosynthesis of the entire full‐length protein in continuity, as it did not occur when N‐ and C‐terminal “halves” were coexpressed. Simultaneous with these interdomain perturbations, ΔF508 resulted in suppressor reversed alterations in accessibility of residues both in the F508‐containing NBD1 surface loop and in the Q loop within the domain core. Thus, in the context of the full‐length protein, ΔF508 mutation causes detectable changes in NBD1 conformation, as well as interdomain interactions.—He, L., Aleksandrov, L. A., Cui, L., Jensen, T. J., Nesbitt, K. L., Riordan, J. R. Restoration of domain folding and interdomain assembly by second‐site suppressors of the ΔF508 mutation in CFTR. FASEB J . 24, 3103–3112 (2010). www.fasebj.org