TY - JOUR
T1 - Cystic fibrosis transmembrane conductance regulator
T2 - 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
AU - Annereau, Jean Philippe
AU - Ko, Young Hee
AU - Pedersen, Peter L.
PY - 2003/4/15
Y1 - 2003/4/15
N2 - 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 (Km, 8.7 μM) 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 Co2+, followed by Mn2+ and Mg2+. In contrast, Ca2+ is ineffective and Cd2+ 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.
AB - 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 (Km, 8.7 μM) 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 Co2+, followed by Mn2+ and Mg2+. In contrast, Ca2+ is ineffective and Cd2+ 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.
KW - ATP-binding-cassette transporter (ABC transporter)
KW - ATPase
KW - Anion channel
KW - Cystic fibrosis transmembrane conductance regulator (CFTR)
KW - Nucleotide domain
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U2 - 10.1042/BJ20021318
DO - 10.1042/BJ20021318
M3 - Article
C2 - 12523935
AN - SCOPUS:0037847523
SN - 0264-6021
VL - 371
SP - 451
EP - 462
JO - Biochemical Journal
JF - Biochemical Journal
IS - 2
ER -