The first nucleotide binding fold of the cystic fibrosis transmembrane conductance regulator can function as an active ATPase.
Cystic fibrosis is caused by mutations in the cell membrane protein called CFTR (cystic fibrosis transmembrane conductance regulator) which functions as a regulated Cl- channel. Although it is known that CFTR contains two nucleotide domains, both of which exhibit the capacity to bind ATP, it has not been demonstrated directly whether one or both domains can function as an active ATPase. To address this question, we have studied the first CFTR nucleotide binding fold (NBF1) in fusion with the maltose-binding protein (MBP), which both stabilizes NBF1 and enhances its solubility. Three different ATPase assays conducted on MBP-NBF1 clearly demonstrate its capacity to catalyze the hydrolysis of ATP. Significantly, the mutations K464H and K464L in the Walker A consensus motif of NBF1 markedly impair its catalytic capacity. MBP alone exhibits no ATPase activity and MBP-NBF1 fails to catalyze the release of phosphate from AMP or ADP. The Vmax of ATP hydrolysis (approximately 30 nmol/min/mg of protein) is significant and is markedly inhibited by azide and by the ATP analogs 2'-(3')-O-(2,4,6-trinitrophenyl)-adenosine-5'-triphosphate and adenosine 5'-(beta, gamma-imido)triphosphate. As inherited mutations within NBF1 account for most cases of cystic fibrosis, results reported here are fundamental to our understanding of the molecular basis of the disease.
Ko
Y H
YH
Department of Biological Chemistry, Johns Hopkins University, School of Medicine, Baltimore, Maryland 21205-2185, USA.
Pedersen
P L
PL
D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D003550:Cystic Fibrosis / Q000201:enzymology*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D017931:DNA Primers / Q000737:chemistry; D006801:Humans; D066298:In Vitro Techniques; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D017354:Point Mutation; D011993:Recombinant Fusion Proteins; D013329:Structure-Activity Relationship
C505032:CFTR protein, human; D017931:DNA Primers; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases