A heteromeric complex of the two nucleotide binding domains of cystic fibrosis transmembrane conductance regulator (CFTR) mediates ATPase activity.
The cystic fibrosis transmembrane conductance regulator (CFTR) protein is a member of the ABC superfamily of transporter proteins. Recently, crystal structures of intact, prokaryotic members of this family have been described. These structures suggested that ATP binding and hydrolysis occurs at two sites formed at the interface between their nucleotide binding domains (NBDs). In contrast to the prokaryotic family members, the NBDs of CFTR are asymmetric (both structurally and functionally), and previous to the present studies, it was not clear whether both NBDs are required for ATP hydrolysis. In order to assess the relative roles of the two NBDs of human CFTR, we purified and reconstituted NBD1 and NBD2, separately and together. We found that NBD1 and NBD2 by themselves exhibited relatively low ATPase activity. Co-assembly of NBD1 and NBD2 exhibited a 2-3-fold enhancement in catalytic activity relative to the isolated domains and this increase reflected enhanced ATP turnover (V(max)). These data provide the first direct evidence that heterodimerization of the NBD1 and NBD2 domains of CFTR is required to generate optimal catalytic activity.
Programme in Structural Biology and Biochemistry, Hospital for Sick Children, Physiology and Biochemistry Departments, Faculty of Medicine, University of Toronto, Toronto, Ontario M5G 1X8, Canada.
Jackie F
JF
Kidd
Mohabir
M
Ramjeesingh
Fiona
F
Stratford
Ling-Jun
LJ
Huan
Christine E
CE
Bear
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D002460:Cell Line; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000737:chemistry* / Q000378:metabolism*; D019281:Dimerization; D006801:Humans; D007700:Kinetics; D009711:Nucleotides / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D014162:Transfection
C505032:CFTR protein, human; D009711:Nucleotides; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases