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

Biochem J. 2003 Apr 15;371(Pt 2):451-62. doi: 10.1042/BJ20021318.


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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Adenosine Triphosphate / metabolism
  • Amino Acid Sequence
  • Binding Sites
  • Ca(2+) Mg(2+)-ATPase / antagonists & inhibitors*
  • Ca(2+) Mg(2+)-ATPase / metabolism*
  • Cadmium / pharmacology*
  • Catalysis
  • Cloning, Molecular
  • Cobalt / metabolism*
  • Cystic Fibrosis Transmembrane Conductance Regulator / chemistry
  • Cystic Fibrosis Transmembrane Conductance Regulator / metabolism*
  • DNA-Binding Proteins / metabolism*
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Humans
  • Kinetics
  • Manganese / pharmacology*
  • Molecular Sequence Data
  • Nuclear Respiratory Factors
  • Nucleotides / metabolism*
  • Protein Structure, Secondary
  • Trans-Activators / metabolism*
  • Vanadates / pharmacology*


  • CFTR protein, human
  • DNA-Binding Proteins
  • Nuclear Respiratory Factors
  • Nucleotides
  • Trans-Activators
  • Cadmium
  • Cystic Fibrosis Transmembrane Conductance Regulator
  • Cobalt
  • Vanadates
  • Manganese
  • Adenosine Triphosphate
  • Ca(2+) Mg(2+)-ATPase