title: Impact of the deltaF508 mutation in first nucleotide-binding domain of human cystic fibrosis transmembrane conductance regulator on domain folding and structure.
abstract: Cystic fibrosis is caused by defects in the cystic fibrosis transmembrane conductance regulator (CFTR), commonly the deletion of residue Phe-508 (DeltaF508) in the first nucleotide-binding domain (NBD1), which results in a severe reduction in the population of functional channels at the epithelial cell surface. Previous studies employing incomplete NBD1 domains have attributed this to aberrant folding of DeltaF508 NBD1. We report structural and biophysical studies on complete human NBD1 domains, which fail to demonstrate significant changes of in vitro stability or folding kinetics in the presence or absence of the DeltaF508 mutation. Crystal structures show minimal changes in protein conformation but substantial changes in local surface topography at the site of the mutation, which is located in the region of NBD1 believed to interact with the first membrane spanning domain of CFTR. These results raise the possibility that the primary effect of DeltaF508 is a disruption of proper interdomain interactions at this site in CFTR rather than interference with the folding of NBD1. Interestingly, increases in the stability of NBD1 constructs are observed upon introduction of second-site mutations that suppress the trafficking defect caused by the DeltaF508 mutation, suggesting that these suppressors might function indirectly by improving the folding efficiency of NBD1 in the context of the full-length protein. The human NBD1 structures also solidify the understanding of CFTR regulation by showing that its two protein segments that can be phosphorylated both adopt multiple conformations that modulate access to the ATPase active site and functional interdomain interfaces.
authors.affiliation: Structural GenomiX, Inc., San Diego, California 92121, USA. hal_lewis@stromix.com
authors.forename: Hal A
authors.initials: HA
authors.lastname: Lewis
authors.forename: Xun
authors.initials: X
authors.lastname: Zhao
authors.forename: Chi
authors.initials: C
authors.lastname: Wang
authors.forename: J Michael
authors.initials: JM
authors.lastname: Sauder
authors.forename: Isabelle
authors.initials: I
authors.lastname: Rooney
authors.forename: Brian W
authors.initials: BW
authors.lastname: Noland
authors.forename: Don
authors.initials: D
authors.lastname: Lorimer
authors.forename: Margaret C
authors.initials: MC
authors.lastname: Kearins
authors.forename: Kris
authors.initials: K
authors.lastname: Conners
authors.forename: Brad
authors.initials: B
authors.lastname: Condon
authors.forename: Peter C
authors.initials: PC
authors.lastname: Maloney
authors.forename: William B
authors.initials: WB
authors.lastname: Guggino
authors.forename: John F
authors.initials: JF
authors.lastname: Hunt
authors.forename: Spencer
authors.initials: S
authors.lastname: Emtage
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D003550:Cystic Fibrosis / Q000235:genetics*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009711:Nucleotides / Q000378:metabolism*; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D020673:Protein Renaturation; D017434:Protein Structure, Tertiary; D017384:Sequence Deletion / Q000235:genetics*; D012995:Solubility
chemical_list: C505032:CFTR protein, human; D009711:Nucleotides; D019005:Cystic Fibrosis Transmembrane Conductance Regulator