Functional roles of the nucleotide-binding folds in the activation of the cystic fibrosis transmembrane conductance regulator.
The cystic fibrosis transmembrane conductance regulator (CFTR), a member of the traffic ATPase superfamily, possesses two putative nucleotide-binding folds (NBFs). The NBFs are sufficiently similar that sequence alignment of highly conserved regions can be used to identify analogous residues in the two domains. To determine whether this structural homology is paralleled in function, we compared the activation of chloride conductance by forskolin and 3-isobutyl-1-methylxanthine in Xenopus oocytes expressing CFTRs bearing mutations in NBF1 or NBF2. Mutation of a conserved glycine in the putative linker domain in either NBF produced virtually identical changes in the sensitivity of chloride conductance to activating conditions, and mutation of this site in both NBFs produced additive effects, suggesting that in the two NBFs this region plays a similar and critical role in the activation process. In contrast, amino acid substitutions in the Walker A and B motifs, thought to form an integral part of the nucleotide-binding pockets, produced strikingly different effects in NBF1 and NBF2. Substitutions for the conserved lysine (Walker A) or aspartate (Walker B) in NBF1 resulted in a marked decrease in sensitivity to activation, whereas the same changes in NBF2 produced an increase in sensitivity. These results are consistent with a model for the activation of CFTR in which both NBF1 and NBF2 are required for normal function but in which either the nature or the exact consequences of nucleotide binding differ for the two domains.
Smit
L S
LS
Department of Human Genetics, University of Michigan, Ann Arbor 48109.
Wilkinson
D J
DJ
Mansoura
M K
MK
Collins
F S
FS
Dawson
D C
DC
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D002712:Chlorides / Q000378:metabolism; D005576:Colforsin / Q000494:pharmacology; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D005260:Female; D006801:Humans; D066298:In Vitro Techniques; D007473:Ion Channels / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008564:Membrane Potentials; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008954:Models, Biological; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000187:drug effects / Q000502:physiology*; D017510:Protein Folding; D017386:Sequence Homology, Amino Acid; D014982:Xenopus laevis
C505032:CFTR protein, human; D002712:Chlorides; D007473:Ion Channels; D008565:Membrane Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D005576:Colforsin; D015056:1-Methyl-3-isobutylxanthine