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Specific Inhibition of p97/VCP ATPase and Kinetic Analysis Demonstrate Interaction between D1 and D2 ATPase domains

Chou, Tsui-Fen and Bulfer, Stacie L. and Weihl, Conrad C. and Li, Kelin and Lis, Lev G. and Walters, Michael A. and Schoenen, Frank J. and Lin, Henry J. and Deshaies, Raymond J. and Arkin, Michelle R. (2014) Specific Inhibition of p97/VCP ATPase and Kinetic Analysis Demonstrate Interaction between D1 and D2 ATPase domains. Journal of Molecular Biology, 426 (15). pp. 2886-2899. ISSN 0022-2836. PMCID PMC4102644. http://resolver.caltech.edu/CaltechAUTHORS:20140609-114312310

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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 (k_(cat)/K_m) of D1 ATP hydrolysis 280-fold, by increasing k_(cat) 7-fold and decreasing K_m 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.


Item Type:Article
Related URLs:
URLURL TypeDescription
http://www.sciencedirect.com/science/article/pii/S0022283614002721PublisherArticle
http://dx.doi.org/10.1016/j.jmb.2014.05.022DOIArticle
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4102644/PubMed CentralArticle
ORCID:
AuthorORCID
Chou, Tsui-Fen0000-0003-2410-2186
Deshaies, Raymond J.0000-0002-3671-9354
Additional Information:© 2014 Elsevier. Received date: 6 February 2014. Revised date: 21 May 2014. Accepted date: 22 May 2014. Available online 27 May 2014. 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 UCLA CTSI 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 U.S. government. RJD is an Investigator of the Howard Hughes Medical Institute, and TFC is a member of UCLA Jonsson Comprehensive Cancer Center.
Funders:
Funding AgencyGrant Number
NIHUL1TR000124
LA BioMed Seed Grant program20826-01
NIHHHSN261200800001E
NIHU54HG005031
Howard Hughes Medical Institute (HHMI)UNSPECIFIED
Subject Keywords:p97/VCP AAA ATPase;IBMPFD/ALS; steady-state kinetics; SPR; p97 inhibitor
PubMed Central ID:PMC4102644
Record Number:CaltechAUTHORS:20140609-114312310
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:20140609-114312310
Official Citation: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, Specific Inhibition of p97/VCP ATPase and Kinetic Analysis Demonstrate Interaction between D1 and D2 ATPase Domains, Journal of Molecular Biology, Volume 426, Issue 15, 29 July 2014, Pages 2886-2899, ISSN 0022-2836, http://dx.doi.org/10.1016/j.jmb.2014.05.022. (http://www.sciencedirect.com/science/article/pii/S0022283614002721)
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:46152
Collection:CaltechAUTHORS
Deposited By: Ruth Sustaita
Deposited On:09 Jun 2014 19:14
Last Modified:20 Jul 2017 22:24

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