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) 6.7min−1] and an apparent affinity for ATP (Km, 8.7μM) 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.

Abbreviations used: ABC transporter, ATP-binding-cassette transporter; Caps, 3-(cyclohexylamino)-1-propane sulphonic acid; CF, cystic fibrosis; CFTR, cystic fibrosis transmembrane conductance regulator; ht, histidine tag; IPTG, isopropyl β-d-thiogalactoside; NBF, nucleotide-binding fold; Ni-NTA, Ni2+-nitrilotriacetic acid; R, regulatory domain; TBS, Tris-buffered saline; TM, transmembrane domain.

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Author notes


Present address: Laboratory of Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, U.S.A.