Specific inhibition of p97/VCP ATPase and kinetic analysis demonstrate interaction between D1 and D2 ATPase domains

Tsui Fen Chou, Stacie L. Bulfer, Conrad C. Weihl, Kelin Li, Lev G. Lis, Michael A. Walters, Frank J. Schoenen, Henry J. Lin, Raymond J. Deshaies, Michelle R. Arkin

Research output: Contribution to journalArticlepeer-review

90 Scopus citations

Abstract

The p97 AAA (ATPase associated with diverse cellular activities), also called VCP (valosin-containing protein), is an important therapeutic target for cancer and neurodegenerative diseases. p97 forms a hexamer composed of two AAA domains (D1 and D2) that form two stacked rings and an N-terminal domain that binds numerous cofactor proteins. The interplay between the three domains in p97 is complex, and a deeper biochemical understanding is needed in order to design selective p97 inhibitors as therapeutic agents. It is clear that the D2 ATPase domain hydrolyzes ATP in vitro, but whether D1 contributes to ATPase activity is controversial. Here, we use Walker A and B mutants to demonstrate that D1 is capable of hydrolyzing ATP and show for the first time that nucleotide binding in the D2 domain increases the catalytic efficiency (kcat/K m) of D1 ATP hydrolysis 280-fold, by increasing kcat 7-fold and decreasing Km about 40-fold. We further show that an ND1 construct lacking D2 but including the linker between D1 and D2 is catalytically active, resolving a conflict in the literature. Applying enzymatic observations to small-molecule inhibitors, we show that four p97 inhibitors (DBeQ, ML240, ML241, and NMS-873) have differential responses to Walker A and B mutations, to disease-causing IBMPFD mutations, and to the presence of the N domain binding cofactor protein p47. These differential effects provide the first evidence that p97 cofactors and disease mutations can alter p97 inhibitor potency and suggest the possibility of developing context-dependent inhibitors of p97.

Original languageEnglish (US)
Pages (from-to)2886-2899
Number of pages14
JournalJournal of Molecular Biology
Volume426
Issue number15
DOIs
StatePublished - Jul 29 2014

Bibliographical note

Funding Information:
We thank Michelina Iacovino for critical reading and suggestions on this manuscript. We thank David Myszka for assistance in fitting SPR data in CLAMP. The project was in part supported by the National Center for Advancing Translational Sciences through University of California, Los Angeles, Clinical and Translational Science Institute Grant UL1TR000124 and the LA BioMed Seed Grant program ( 20826-01 ). This project has been funded in part with federal funds from the National Cancer Institute, National Institutes of Health , under Chemical Biology Consortium Contract No. HHSN261200800001E , and by the National Human Genome Research Institute of the National Institutes of Health under Award Number U54HG005031 (Jeffrey Aubé, PI). 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. R.J.D. is an investigator of the Howard Hughes Medical Institute, and T.F.C. is a member of University of California, Los Angeles Jonsson Comprehensive Cancer Center.

Keywords

  • IBMPFD/ALS
  • SPR
  • p97 inhibitor
  • p97/VCP AAA ATPase
  • steady-state kinetics

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