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J. Biol. Chem., Vol. 280, Issue 49, 40515-40523, December 9, 2005

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Multifunctional Roles of the Conserved Arg Residues in the Second Region of Homology of p97/Valosin-containing Protein^*

Qing Wang1, Changcheng Song, Lauren Irizarry, Renming Dai, Xiaodong Zhang¶, and Chou-Chi H. Li2

From the Laboratory of Cancer Prevention and the Basic Research Program, Science Applications International Corporation-Frederick, Center for Cancer Research, National Cancer Institute at Frederick, National Institutes of Health, Frederick, Maryland 21702 and the ^¶Centre for Structural Biology, Department of Biological Sciences, Imperial College London, South Kensington Campus, London SW7 2AZ, United Kingdom

The 97-kDa molecular chaperone valosin-containing protein (VCP) belongs to a highly conserved AAA family and forms a hexameric structure that is essential for its biological functions. The AAA domain contains highly conserved motifs, the Walker A, Walker B, and the second region of homology (SRH). Although Walker A and B motifs mediate ATP binding and hydrolysis, respectively, the function of the SRH in VCP is not clear. We examined the significance of the SRH in VCP, especially the conserved Arg³⁵⁹ and Arg³⁶² in the first AAA domain, D1 and Arg⁶³⁵ and Arg⁶³⁸ in the second AAA domain, D2. We show that Arg³⁵⁹ and Arg³⁶² in D1 are critical for maintaining the hexameric structure and the ability to bind the polyubiquitin chains. Although the rest of the tested SRH mutants retain the hexameric structure, all of them exhibit severely reduced ATPase activity. Tryptophan fluorescence analysis showed that all of the tested mutants can bind to ATP or ADP. Thus, the reduced ATPase activity likely results from the hampered communications among protomers during hydrolysis. Moreover, when the ATPase-defective mutant R635A or R638A is mixed with the Walker A mutant of D2, the ATPase activity is partially restored, suggesting that Arg⁶³⁵ and Arg⁶³⁸ can stimulate the ATPase activity of the neighboring protomer. Interestingly, mutation of Arg³⁵⁹ and Arg³⁶² uncouples the inhibitory effect of p47, a VCP co-factor, on the ATPase activity of VCP. Therefore, the Arg residues allow D1 to take on a specific conformation that is required for substrate binding and co-factor communications. Taken together, these results demonstrate that the conserved Arg residues in the SRH of both D1 and D2 play critical roles in communicating the conformational changes required for ATP hydrolysis, and SRH in D1 also contributes to substrate binding and co-factor communications.

Received for publication, September 1, 2005

^* This work was supported in part by National Cancer Institute, National Institutes of Health Contract NO1-CO-12400. 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.

¹ To whom correspondence may addressed. Tel.: 301-846-1588; Fax: 301-846-7034; E-mail: qwang{at}ncifcrf.gov. ² To whom correspondence may addressed. Tel.: 301-846-1478; Fax: 301-846-7034; E-mail: licc{at}ncifcrf.gov.

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