The Walker B motif of the second nucleotide-binding domain (NBD2) of CFTR plays a key role in ATPase activity by the NBD1-NBD2 heterodimer.
CFTR (cystic fibrosis transmembrane conductance regulator), a member of the ABC (ATP-binding cassette) superfamily of membrane proteins, possesses two NBDs (nucleotide-binding domains) in addition to two MSDs (membrane spanning domains) and the regulatory 'R' domain. The two NBDs of CFTR have been modelled as a heterodimer, stabilized by ATP binding at two sites in the NBD interface. It has been suggested that ATP hydrolysis occurs at only one of these sites as the putative catalytic base is only conserved in NBD2 of CFTR (Glu1371), but not in NBD1 where the corresponding residue is a serine, Ser573. Previously, we showed that fragments of CFTR corresponding to NBD1 and NBD2 can be purified and co-reconstituted to form a heterodimer capable of ATPase activity. In the present study, we show that the two NBD fragments form a complex in vivo, supporting the utility of this model system to evaluate the role of Glu1371 in ATP binding and hydrolysis. The present studies revealed that a mutant NBD2 (E1371Q) retains wild-type nucleotide binding affinity of NBD2. On the other hand, this substitution abolished the ATPase activity formed by the co-purified complex. Interestingly, introduction of a glutamate residue in place of the non-conserved Ser573 in NBD1 did not confer additional ATPase activity by the heterodimer, implicating a vital role for multiple residues in formation of the catalytic site. These findings provide the first biochemical evidence suggesting that the Walker B residue: Glu1371, plays a primary role in the ATPase activity conferred by the NBD1-NBD2 heterodimer.
Stratford
Fiona L L
FL
Programme in Molecular Structure and Function, Research Institute, Hospital for Sick Children, Toronto, ON, Canada M5G 1X8.
Ramjeesingh
Mohabir
M
Cheung
Joanne C
JC
Huan
Ling-Jun
LJ
Bear
Christine E
CE
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001665:Binding Sites; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D018698:Glutamic Acid / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation; D009711:Nucleotides / Q000378:metabolism*
C009587:2',3'-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate; D009711:Nucleotides; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D018698:Glutamic Acid; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases