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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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Stoichiometry and cooperativity associated with ATPγS binding p97. A, ITC profile of wild-type p97 binding to ATPγS. The data are fitted to a competition model calculating that p97 is saturated at 1.6 ATPγS per monomer. B, modified heat denaturation experiments in which ATPγS was added to p97 (with ADP prebound or removed by apyrase digest). Results show that ATPγS binding to p97 is saturated at 1.5–1.6 molecules of nucleotide per monomer (9–10 per hexamer) and is independent of ADP prebound to D1. C, tryptophan fluorescence changes of wild type and Walker A mutants in response to ATPγS binding show that the fluorescence change results from ATPγS binding to D2 and is not altered by nucleotide bound to D1. Fits to a cooperative binding equation calculate greater than 2.8 monomers positively cooperate. D, tryptophan fluorescence of SRH mutant (R359A,R635A) shows no positive cooperativity. Fitting to a single site model shows that ATPγS is bound with similar affinity to ITC measurements of wild-type p97. E, ITC data of R359A,R635A binding to ATPγS indicate that the stoichiometry of ATPγS binding is 1.66 molecules of ATPγS per monomer (10 per hexamer), similar to wild-type p97.
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fig4: Stoichiometry and cooperativity associated with ATPγS binding p97. A, ITC profile of wild-type p97 binding to ATPγS. The data are fitted to a competition model calculating that p97 is saturated at 1.6 ATPγS per monomer. B, modified heat denaturation experiments in which ATPγS was added to p97 (with ADP prebound or removed by apyrase digest). Results show that ATPγS binding to p97 is saturated at 1.5–1.6 molecules of nucleotide per monomer (9–10 per hexamer) and is independent of ADP prebound to D1. C, tryptophan fluorescence changes of wild type and Walker A mutants in response to ATPγS binding show that the fluorescence change results from ATPγS binding to D2 and is not altered by nucleotide bound to D1. Fits to a cooperative binding equation calculate greater than 2.8 monomers positively cooperate. D, tryptophan fluorescence of SRH mutant (R359A,R635A) shows no positive cooperativity. Fitting to a single site model shows that ATPγS is bound with similar affinity to ITC measurements of wild-type p97. E, ITC data of R359A,R635A binding to ATPγS indicate that the stoichiometry of ATPγS binding is 1.66 molecules of ATPγS per monomer (10 per hexamer), similar to wild-type p97.

Mentions: Quantification of Bound Nucleotide by Heat Denaturation— Bound nucleotide was released by incubating 75 μl of 100 μm p97 at 100 °C. The precipitated protein was removed by centrifugation, the supernatant was decanted, and the volume was measured. The concentration of adenine nucleotide was calculated from absorbance at 260 nm (ε = 15 400 m-1 cm-1), and the amount of nucleotide released was expressed as the ratio of nucleotide to p97 monomer. Error bars in the legends for Figs. 2 and 4 represent the standard deviation of greater than six measurements.


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)

Stoichiometry and cooperativity associated with ATPγS binding p97. A, ITC profile of wild-type p97 binding to ATPγS. The data are fitted to a competition model calculating that p97 is saturated at 1.6 ATPγS per monomer. B, modified heat denaturation experiments in which ATPγS was added to p97 (with ADP prebound or removed by apyrase digest). Results show that ATPγS binding to p97 is saturated at 1.5–1.6 molecules of nucleotide per monomer (9–10 per hexamer) and is independent of ADP prebound to D1. C, tryptophan fluorescence changes of wild type and Walker A mutants in response to ATPγS binding show that the fluorescence change results from ATPγS binding to D2 and is not altered by nucleotide bound to D1. Fits to a cooperative binding equation calculate greater than 2.8 monomers positively cooperate. D, tryptophan fluorescence of SRH mutant (R359A,R635A) shows no positive cooperativity. Fitting to a single site model shows that ATPγS is bound with similar affinity to ITC measurements of wild-type p97. E, ITC data of R359A,R635A binding to ATPγS indicate that the stoichiometry of ATPγS binding is 1.66 molecules of ATPγS per monomer (10 per hexamer), similar to wild-type p97.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig4: Stoichiometry and cooperativity associated with ATPγS binding p97. A, ITC profile of wild-type p97 binding to ATPγS. The data are fitted to a competition model calculating that p97 is saturated at 1.6 ATPγS per monomer. B, modified heat denaturation experiments in which ATPγS was added to p97 (with ADP prebound or removed by apyrase digest). Results show that ATPγS binding to p97 is saturated at 1.5–1.6 molecules of nucleotide per monomer (9–10 per hexamer) and is independent of ADP prebound to D1. C, tryptophan fluorescence changes of wild type and Walker A mutants in response to ATPγS binding show that the fluorescence change results from ATPγS binding to D2 and is not altered by nucleotide bound to D1. Fits to a cooperative binding equation calculate greater than 2.8 monomers positively cooperate. D, tryptophan fluorescence of SRH mutant (R359A,R635A) shows no positive cooperativity. Fitting to a single site model shows that ATPγS is bound with similar affinity to ITC measurements of wild-type p97. E, ITC data of R359A,R635A binding to ATPγS indicate that the stoichiometry of ATPγS binding is 1.66 molecules of ATPγS per monomer (10 per hexamer), similar to wild-type p97.
Mentions: Quantification of Bound Nucleotide by Heat Denaturation— Bound nucleotide was released by incubating 75 μl of 100 μm p97 at 100 °C. The precipitated protein was removed by centrifugation, the supernatant was decanted, and the volume was measured. The concentration of adenine nucleotide was calculated from absorbance at 260 nm (ε = 15 400 m-1 cm-1), and the amount of nucleotide released was expressed as the ratio of nucleotide to p97 monomer. Error bars in the legends for Figs. 2 and 4 represent the standard deviation of greater than six measurements.

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.

Show MeSH
Related in: MedlinePlus