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

Briggs LC, Baldwin GS, Miyata N, Kondo H, Zhang X, Freemont PS - J. Biol. Chem. (2008)

Bottom Line: 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.Stoichiometric measurements suggest that although both ADP and ATPgammaS can saturate all 6 nucleotide binding sites in D1, only 3-4 of the 6 D2 sites can bind ATPgammaS simultaneously.ATPgammaS binding triggers a downstream cooperative conformational change of at least three monomers, which involves conserved arginine fingers and is necessary for ATP hydrolysis.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Biosciences, Imperial College London, South Kensington, London SW7 2AZ, United Kingdom.

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) (ATPgammaS) 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 ATPgammaS can saturate all 6 nucleotide binding sites in D1, only 3-4 of the 6 D2 sites can bind ATPgammaS simultaneously. ATPgammaS 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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Models of ATP binding to p97 D2 domain. The complete D2 ring is shown uppermost with the D1 ring and N domains below (partially obscured). Four of the possible arrangements of ATP binding consistent with our measurements of stoichiometry and cooperativity of ATPγS binding are shown. 3 or 4 molecules of ATP could bind symmetrically as a trimer of dimers (A) or a dimer of trimers (B), or alternatively, in an asymmetric organization (C and D). Additionally, ATP could bind to three or four neighboring D2 domains (not shown).
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fig5: Models of ATP binding to p97 D2 domain. The complete D2 ring is shown uppermost with the D1 ring and N domains below (partially obscured). Four of the possible arrangements of ATP binding consistent with our measurements of stoichiometry and cooperativity of ATPγS binding are shown. 3 or 4 molecules of ATP could bind symmetrically as a trimer of dimers (A) or a dimer of trimers (B), or alternatively, in an asymmetric organization (C and D). Additionally, ATP could bind to three or four neighboring D2 domains (not shown).

Mentions: Although the affinity of ATPγS for D1 and D2 is similar, our results show that the stoichiometry of binding is distinct; ATPγS binding is limited to a subset of D2 domains (3–4 out of 6 possible sites), whereas the D1 ring binds ATPγS to full occupancy. The stoichiometry of ATPγS binding to D2 predicts that ATPγS binding to the first 3–4 D2 sites leads to changes in the final 2–3 D2 sites to prevent ATPγS binding. Fig. 5 summarizes possible symmetrical and random arrangements of ATPγS binding to D2 consistent with our results. The partial occupancy of ATPγS in D2 is in agreement with earlier studies. i) Only 2.23 molecules of photoactivated ATP derivative were cross-linked to endogenous p97 (21), and ii) only one D2 domain of the three monomers in the asymmetric unit of the p97-ADP-AlF3 crystal structure had full occupancy of AlF3 (14). In the AAA+ ATPase superfamily, there is a precedent for partial occupancy of ATP in homohexameric AAA proteins with single rings such as ClpX, RuvB, and Hs1U (29–31), but this is the first report for a tandem AAA protein and suggests a potential conservation of mechanism despite the wide variety of AAA+ ATPase functions.


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

Briggs LC, Baldwin GS, Miyata N, Kondo H, Zhang X, Freemont PS - J. Biol. Chem. (2008)

Models of ATP binding to p97 D2 domain. The complete D2 ring is shown uppermost with the D1 ring and N domains below (partially obscured). Four of the possible arrangements of ATP binding consistent with our measurements of stoichiometry and cooperativity of ATPγS binding are shown. 3 or 4 molecules of ATP could bind symmetrically as a trimer of dimers (A) or a dimer of trimers (B), or alternatively, in an asymmetric organization (C and D). Additionally, ATP could bind to three or four neighboring D2 domains (not shown).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2376215&req=5

fig5: Models of ATP binding to p97 D2 domain. The complete D2 ring is shown uppermost with the D1 ring and N domains below (partially obscured). Four of the possible arrangements of ATP binding consistent with our measurements of stoichiometry and cooperativity of ATPγS binding are shown. 3 or 4 molecules of ATP could bind symmetrically as a trimer of dimers (A) or a dimer of trimers (B), or alternatively, in an asymmetric organization (C and D). Additionally, ATP could bind to three or four neighboring D2 domains (not shown).
Mentions: Although the affinity of ATPγS for D1 and D2 is similar, our results show that the stoichiometry of binding is distinct; ATPγS binding is limited to a subset of D2 domains (3–4 out of 6 possible sites), whereas the D1 ring binds ATPγS to full occupancy. The stoichiometry of ATPγS binding to D2 predicts that ATPγS binding to the first 3–4 D2 sites leads to changes in the final 2–3 D2 sites to prevent ATPγS binding. Fig. 5 summarizes possible symmetrical and random arrangements of ATPγS binding to D2 consistent with our results. The partial occupancy of ATPγS in D2 is in agreement with earlier studies. i) Only 2.23 molecules of photoactivated ATP derivative were cross-linked to endogenous p97 (21), and ii) only one D2 domain of the three monomers in the asymmetric unit of the p97-ADP-AlF3 crystal structure had full occupancy of AlF3 (14). In the AAA+ ATPase superfamily, there is a precedent for partial occupancy of ATP in homohexameric AAA proteins with single rings such as ClpX, RuvB, and Hs1U (29–31), but this is the first report for a tandem AAA protein and suggests a potential conservation of mechanism despite the wide variety of AAA+ ATPase functions.

Bottom Line: 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.Stoichiometric measurements suggest that although both ADP and ATPgammaS can saturate all 6 nucleotide binding sites in D1, only 3-4 of the 6 D2 sites can bind ATPgammaS simultaneously.ATPgammaS binding triggers a downstream cooperative conformational change of at least three monomers, which involves conserved arginine fingers and is necessary for ATP hydrolysis.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Biosciences, Imperial College London, South Kensington, London SW7 2AZ, United Kingdom.

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) (ATPgammaS) 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 ATPgammaS can saturate all 6 nucleotide binding sites in D1, only 3-4 of the 6 D2 sites can bind ATPgammaS simultaneously. ATPgammaS 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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