Hexamerization of p97-VCP is promoted by ATP binding to the D1 domain and required for ATPase and biological activities

doi:10.1016/S0006-291X(02)02840-1

Biochemical and Biophysical Research Communications

Volume 300, Issue 2, 10 January 2003, Pages 253-260

https://doi.org/10.1016/S0006-291X(02)02840-1 Get rights and content

Abstract

The 97-kDa valosin-containing protein (p97-VCP or VCP), a hexameric AAA ATPase, plays an important role in diverse cell activities, including ubiquitin–proteasome mediated protein degradation. In this report, we studied dissociation–reassembly kinetics to analyze the structure–function relationship in VCP. Urea-dissociated VCP can reassemble by itself, but addition of ATP, ADP, or ATP-γS accelerates the reassembly. Mutation in the ATP-binding site of D1, but not D2, domain abolishes the ATP acceleration effect and further delays the reassembly. Using hybrid hexamers of the wild type and ATP-binding site mutant, we show that hexameric structure and proper communication among the subunits are required for the ATPase activity and ubiquitin–proteasome mediated degradation. Thus, ATP-binding site in D1 plays a major role in VCP hexamerization, of which proper inter-subunit interaction is essential for the activities.

Section snippets

Materials and methods

Site-directed mutagenesis and bacterial strains. Site-specific mutants were generated using Quikchange Site-Directed Mutagenesis kit (Stratagene) with the wild-type pVCP-His [11] as a template. In A1 or A2 mutant, a lysine to threonine mutation was introduced in residues 251 in D1 or 524 in D2, respectively. For A1A2 mutant with double mutations, A1 was used as a template in the site-directed mutagenesis system. In B1B2 mutant, a glutamic acid was changed to glutamine in residues 305 in D1 and

Purification of site-specific mutants

Wild type and mutant VCP-His fusion proteins were expressed in E. coli and affinity-purified to near homogeneity (Fig. 1). The A1 (K251T) and A2 (K524T) mutants harbor defective ATP-binding sites in the Walker A motif of D1 and D2, respectively. A1A2 mutant contains the same K→T mutations in D1 and D2 (Fig. 4A). B1B2 harbors E→Q mutations in the ATP-hydrolysis sites (Walker B motif) in D1 and D2 (Fig. 4A).

Self-assembly of urea-dissociated VCP and its acceleration by nucleotide binding

To explore the stability of VCP hexamers and how nucleotide affects VCP hexamerization, we

Discussion

Our studies show that VCP has a high propensity to form hexamer, which is essential for supporting the diverse functions of VCP. Harsh treatment with acid (pH 3), alkaline (pH 10), ethanol (30%), heat (65 °C), apyrase (an enzyme that destroys ATP and ADP), numerous detergents, and low protein concentration (0.01 mg/ml) has no impact on the hexameric structure (data not shown). These stress-tolerant properties of VCP underscore the nature of an important molecular chaperone. It is expected that

Acknowledgements

We thank R.M. Dai for technical assistance and Drs. S. Gottesman, M. Maurizi, and S. Wickner for helpful discussion. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services nor does mention of trade names, commercial products, or organizations imply endorsement by the US Government. The publisher or recipient acknowledges the right of the US Government to retain a nonexclusive, royalty-free license in and to any copyright

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These prior findings prompted the question that we tackle in this study: might we reduce the toxicity of pathogenic ataxin-3 by disrupting its interaction with VCP through a ‘decoy’ approach targeting the VBM? We utilized a truncated protein that consists of the VCP N-terminus (amino acids 1–199), denoted at ‘N-VCP’, that binds ataxin-3 (Boeddrich et al., 2006), but cannot hexamerize (DeLaBarre et al., 2006; Kobayashi et al., 2002; Mori-Konya et al., 2009; Wang et al., 2003) (Fig. 1A). As VCP is a critical protein, whereas ataxin-3 is dispensable in mice (Costa and Paulson, 2012; Matos et al., 2011; Schmitt et al., 2007; Switonski et al., 2011; Zeng et al., 2013), we reasoned that it would be more practical to target ataxin-3 rather than VCP so as not to impede VCP functions while still hindering the interaction of interest.
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The AAA⁺ ATPase p97 regulates ubiquitin-dependent protein homeostasis and has been pursued as a cancer drug target. The ATP-competitive inhibitor CB-5083 and allosteric inhibitor NMS-873 are the most advanced p97 inhibitors described to date. Previous studies have reported that their cytotoxicity can be readily overcome and involves single p97 mutations in the linker between the D1 and D2 ATPase domains and within D2. We report here that the proline 472 to leucine (P472L) mutation, in the D1–D2 linker and identified in CB-5083–resistant cells, desensitizes p97 to both inhibitor classes. This mutation does not disrupt the distinct D2-binding sites of the inhibitors. Instead, P472L changes ATPase domain communication within the p97 hexamer. P472L enhances cooperative D2 ATP binding and hydrolysis. This mechanism alters the function of the D1–D2 linker in the control of D2 activity involving the ATP-bound state of D1. Although increased D2 activity is sufficient to desensitize the P472L mutant to NMS-873, the mutant’s desensitization to CB-5083 also requires D1 ATPase domain function. Our study highlights the remarkable adaptability of p97 ATPase domain communication that enables escape from mechanistically distinct classes of cytotoxic p97 inhibitors.

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^☆: Abbreviations: VCP, valosin-containing protein; AAA, ATPases associated with a variety of activities; PAGE, polyacrylamide gel electrophoresis.

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Hexamerization of p97-VCP is promoted by ATP binding to the D1 domain and required for ATPase and biological activities☆

Abstract

Section snippets

Materials and methods

Purification of site-specific mutants

Self-assembly of urea-dissociated VCP and its acceleration by nucleotide binding

Discussion

Acknowledgements

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