The first nucleotide binding fold of the cystic fibrosis transmembrane conductance regulator can function as an active ATPase

Young Hee Ko, Peter L. Pedersen

Research output: Contribution to journalArticlepeer-review

Abstract

Cystic fibrosis is caused by mutations in the cell membrane protein called CFTR (cystic fibrosis transmembrane conductance regulator) which functions as a regulated Cl- channel. Although it is known that CFTR contains two nucleotide domains, both of which exhibit the capacity to bind ATP, it has not been demonstrated directly whether one or both domains can function as an active ATPase. To address this question, we have studied the first CFTR nucleotide binding fold (NBF1) in fusion with the maltose-binding protein (MBP), which both stabilizes NBF1 and enhances its solubility. Three different ATPase assays conducted on MBP-NBF1 clearly demonstrate its capacity to catalyze the hydrolysis of ATP. Significantly, the mutations K464H and K464L in the Walker A consensus motif of NBF1 markedly impair its catalytic capacity. MBP alone exhibits no ATPase activity and MBP-NBF1 fails to catalyze the release of phosphate from AMP or ADP. The V(max) of ATP hydrolysis (~30 nmol/min/mg of protein) is significant and is markedly inhibited by azide and by the ATP analogs 2'-(3')-O-(2,4,6-trinitrophenyl)- adenosine-5'-triphosphate and adenosine 5'-(β,γ-imido)triphosphate. As inherited mutations within NBF1 account for most cases of cystic fibrosis, results reported here are fundamental to our understanding of the molecular basis of the disease.

Original languageEnglish (US)
Pages (from-to)22093-22096
Number of pages4
JournalJournal of Biological Chemistry
Volume270
Issue number38
DOIs
StatePublished - Sep 22 1995

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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