title: Cystic fibrosis transmembrane conductance regulator: the NBF1+R (nucleotide-binding fold 1 and regulatory domain) segment acting alone catalyses a Co2+/Mn2+/Mg2+-ATPase activity markedly inhibited by both Cd2+ and the transition-state analogue orthovanadate.
abstract: Cystic fibrosis (CF) is caused by mutations in the gene encoding CFTR (cystic fibrosis transmembrane conductance regulator), a regulated anion channel and member of the ATP-binding-cassette transporter (ABC transporter) superfamily. Of CFTR's five domains, the first nucleotide-binding fold (NBF1) has been of greatest interest both because it is the major 'hotspot' for mutations that cause CF, and because it is connected to a unique regulatory domain (R). However, attempts have failed to obtain a catalytically active NBF1+R protein in the absence of a fusion partner. Here, we report that such a protein can be obtained following its overexpression in bacteria. The pure NBF1+R protein exhibits significant ATPase activity [catalytic-centre activity (turnover number) 6.7 min(-1)] and an apparent affinity for ATP ( K (m), 8.7 microM) higher than reported previously for CFTR or segments thereof. As predicted, the ATPase activity is inhibited by mutations in the Walker A motif. It is also inhibited by vanadate, a transition-state analogue. Surprisingly, however, the best divalent metal activator is Co(2+), followed by Mn(2+) and Mg(2+). In contrast, Ca(2+) is ineffective and Cd(2+) is a potent inhibitor. These novel studies, while demonstrating clearly that CFTR's NBF1+R segment can act independently as an active, vanadate-sensitive ATPase, also identify its unique cation activators and a new inhibitor, thus providing insight into the nature of its active site.
authors.affiliation: Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, MD 21205-2185, USA.
authors.forename: Jean Philippe
authors.initials: JP
authors.lastname: Annereau
authors.forename: Young Hee
authors.initials: YH
authors.lastname: Ko
authors.forename: Peter L
authors.initials: PL
authors.lastname: Pedersen
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001665:Binding Sites; D017301:Ca(2+) Mg(2+)-ATPase / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D002104:Cadmium / Q000494:pharmacology*; D002384:Catalysis; D003001:Cloning, Molecular; D003035:Cobalt / Q000378:metabolism*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008345:Manganese / Q000494:pharmacology*; D008969:Molecular Sequence Data; D051264:Nuclear Respiratory Factors; D009711:Nucleotides / Q000378:metabolism*; D017433:Protein Structure, Secondary; D015534:Trans-Activators / Q000378:metabolism*; D014638:Vanadates / Q000494:pharmacology*
chemical_list: C505032:CFTR protein, human; D004268:DNA-Binding Proteins; D051264:Nuclear Respiratory Factors; D009711:Nucleotides; D015534:Trans-Activators; D002104:Cadmium; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D003035:Cobalt; D014638:Vanadates; D008345:Manganese; D000255:Adenosine Triphosphate; D017301:Ca(2+) Mg(2+)-ATPase