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J. Biol. Chem., Vol. 283, Issue 20, 13745-13752, May 16, 2008

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Analysis of Nucleotide Binding to P97 Reveals the Properties of a Tandem AAA Hexameric ATPase^*

Louise C. Briggs¹, Geoff S. Baldwin, Non Miyata, Hisao Kondo, Xiaodong Zhang, and Paul S. Freemont²

From the Division of Molecular Biosciences, Imperial College London, South Kensington, London SW7 2AZ, United Kingdom and the Department of Molecular Biology, Graduate School of Medical Science, Kyushi University, Fukuoka 812-8582, Japan

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) (ATPS) 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 ATPS can saturate all 6 nucleotide binding sites in D1, only 3–4 of the 6 D2 sites can bind ATPS simultaneously. ATPS binding triggers a downstream cooperative conformational change of at least three monomers, which involves conserved arginine fingers and is necessary for ATP hydrolysis.

Received for publication, November 27, 2007 , and in revised form, February 15, 2008.

^* This work was supported by a Welcome Trust grant (to P. S. F. and X. Z.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains four supplemental figures, three supplemental tables, and supplemental methods.

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¹ The recipient of a Biotechnology and Biological Science Research Council studentship.

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² To whom correspondence should be addressed: Rm. 502, Biochemistry, Imperial College London, South Kensington, London SW7 2AZ, UK. Fax: 44-20-75943057; E-mail: p.freemont{at}imperial.ac.uk.