FUNCTIONAL ROLES OF THE NUCLEOTIDE-BINDING FOLDS IN THE ACTIVATION OFTHE CYSTIC-FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR

The cystic fibrosis transmembrane conductance regulator (CFTR), a memb er of the traffic ATPase superfamily, possesses two putative nucleotid e-binding folds (NBFs). The NBFs are sufficiently similar that sequenc e alignment of highly conserved regions can be used to identify analog ous residues in the two domains. To determine whether this structural homology is paralleled in function, we compared the activation of chlo ride conductance by forskolin and 3-isobutyl-1-methylxanthine in Xenop us oocytes expressing CFTRs bearing mutations in NBF1 or NBF2. Mutatio n of a conserved glycine in the putative linker domain in either NBF p roduced virtually identical changes in the sensitivity of chloride con ductance to activating conditions, and mutation of this site in both N BFs produced additive effects, suggesting that in the two NBFs this re gion plays a similar and critical role in the activation process. In c ontrast, amino acid substitutions in the Walker A and B motifs, though t 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 i n a marked decrease in sensitivity to activation, whereas the same cha nges in NBF2 produced an increase in sensitivity. These results are co nsistent with a model for the activation of CFTR in which both NBF1 an d NBF2 are required for normal function but in which either the nature or the exact consequences of nucleotide binding differ for the two do mains.