Analysis of nucleotide binding to P97 reveals the properties of a tandem AAA hexameric ATPase

Louise C. Briggs, Geoff S. Baldwin, Non Miyata, Hisao Kondo, Xiaodong Zhang, Paul S. Freemont

Research output: Contribution to journalArticle

59 Citations (Scopus)

Abstract

p97, an essential chaperone in endoplasmic reticulum-associated degradation and organelle biogenesis, contains two AAA domains (D1 and D2) and assembles as a stable hexamer. We present a quantitative analysis of nucleotide binding to both D1 and D2 domains of p97, the first detailed study of nucleotide binding to both AAA domains for this type of AAA+ ATPase. We report that adenosine 5′-O-(thiotriphosphate) (ATPγS) binds with similar affinity to D1 and D2, but ADP binds with higher affinity to D1 than D2, offering an explanation for the higher ATPase activity in D2. Stoichiometric measurements suggest that although both ADP and ATPγS can saturate all 6 nucleotide binding sites in D1, only 3-4 of the 6 D2 sites can bind ATPγS simultaneously. ATPγS binding triggers a downstream cooperative conformational change of at least three monomers, which involves conserved arginine fingers and is necessary for ATP hydrolysis.

Original languageEnglish
Pages (from-to)13745-13752
Number of pages8
JournalJournal of Biological Chemistry
Volume283
Issue number20
DOIs
Publication statusPublished - May 16 2008

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Adenosine Triphosphatases
Nucleotides
Adenosine Diphosphate
Endoplasmic Reticulum-Associated Degradation
Organelle Biogenesis
Fingers
Arginine
Hydrolysis
Adenosine Triphosphate
Monomers
Binding Sites
Degradation
Chemical analysis
adenosine 5'-O-(3-thiotriphosphate)

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Analysis of nucleotide binding to P97 reveals the properties of a tandem AAA hexameric ATPase. / Briggs, Louise C.; Baldwin, Geoff S.; Miyata, Non; Kondo, Hisao; Zhang, Xiaodong; Freemont, Paul S.

In: Journal of Biological Chemistry, Vol. 283, No. 20, 16.05.2008, p. 13745-13752.

Research output: Contribution to journalArticle

Briggs, Louise C. ; Baldwin, Geoff S. ; Miyata, Non ; Kondo, Hisao ; Zhang, Xiaodong ; Freemont, Paul S. / Analysis of nucleotide binding to P97 reveals the properties of a tandem AAA hexameric ATPase. In: Journal of Biological Chemistry. 2008 ; Vol. 283, No. 20. pp. 13745-13752.
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N2 - p97, an essential chaperone in endoplasmic reticulum-associated degradation and organelle biogenesis, contains two AAA domains (D1 and D2) and assembles as a stable hexamer. We present a quantitative analysis of nucleotide binding to both D1 and D2 domains of p97, the first detailed study of nucleotide binding to both AAA domains for this type of AAA+ ATPase. We report that adenosine 5′-O-(thiotriphosphate) (ATPγS) binds with similar affinity to D1 and D2, but ADP binds with higher affinity to D1 than D2, offering an explanation for the higher ATPase activity in D2. Stoichiometric measurements suggest that although both ADP and ATPγS can saturate all 6 nucleotide binding sites in D1, only 3-4 of the 6 D2 sites can bind ATPγS simultaneously. ATPγS binding triggers a downstream cooperative conformational change of at least three monomers, which involves conserved arginine fingers and is necessary for ATP hydrolysis.

AB - p97, an essential chaperone in endoplasmic reticulum-associated degradation and organelle biogenesis, contains two AAA domains (D1 and D2) and assembles as a stable hexamer. We present a quantitative analysis of nucleotide binding to both D1 and D2 domains of p97, the first detailed study of nucleotide binding to both AAA domains for this type of AAA+ ATPase. We report that adenosine 5′-O-(thiotriphosphate) (ATPγS) binds with similar affinity to D1 and D2, but ADP binds with higher affinity to D1 than D2, offering an explanation for the higher ATPase activity in D2. Stoichiometric measurements suggest that although both ADP and ATPγS can saturate all 6 nucleotide binding sites in D1, only 3-4 of the 6 D2 sites can bind ATPγS simultaneously. ATPγS binding triggers a downstream cooperative conformational change of at least three monomers, which involves conserved arginine fingers and is necessary for ATP hydrolysis.

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