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

Jean Philippe Annereau, Young Hee Ko, Peter L. Pedersen

Research output: Contribution to journalArticle

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 (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.

Original languageEnglish (US)
Pages (from-to)451-462
Number of pages12
JournalBiochemical Journal
Volume371
Issue number2
DOIs
StatePublished - Apr 15 2003

Keywords

  • ATP-binding-cassette transporter (ABC transporter)
  • ATPase
  • Anion channel
  • Cystic fibrosis transmembrane conductance regulator (CFTR)
  • Nucleotide domain

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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