title: Specific inhibition of p97/VCP ATPase and kinetic analysis demonstrate interaction between D1 and D2 ATPase domains.
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/Km) 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.
authors.affiliation: Division of Medical Genetics, Department of Pediatrics, Harbor-UCLA Medical Center and Los Angeles Biomedical Research Institute, Torrance, CA 90502, USA. Electronic address: tsuifenchou@ucla.edu.
authors.forename: Tsui-Fen
authors.initials: TF
authors.lastname: Chou
authors.affiliation: Department of Pharmaceutical Chemistry, Small Molecule Discovery Center, University of California, San Francisco, CA 94158, USA.
authors.forename: Stacie L
authors.initials: SL
authors.lastname: Bulfer
authors.affiliation: Department of Neurology, Washington University School of Medicine, St. Louis, MO 63110, USA.
authors.forename: Conrad C
authors.initials: CC
authors.lastname: Weihl
authors.affiliation: Specialized Chemistry Center, University of Kansas, Lawrence, KS 66047, USA.
authors.forename: Kelin
authors.initials: K
authors.lastname: Li
authors.affiliation: Department of Medicinal Chemistry, Institute for Therapeutics Discovery and Development, College of Pharmacy, University of Minnesota, Minneapolis, MN 55414, USA.
authors.forename: Lev G
authors.initials: LG
authors.lastname: Lis
authors.affiliation: Department of Medicinal Chemistry, Institute for Therapeutics Discovery and Development, College of Pharmacy, University of Minnesota, Minneapolis, MN 55414, USA.
authors.forename: Michael A
authors.initials: MA
authors.lastname: Walters
authors.affiliation: Specialized Chemistry Center, University of Kansas, Lawrence, KS 66047, USA.
authors.forename: Frank J
authors.initials: FJ
authors.lastname: Schoenen
authors.affiliation: Division of Medical Genetics, Department of Pediatrics, Harbor-UCLA Medical Center and Los Angeles Biomedical Research Institute, Torrance, CA 90502, USA.
authors.forename: Henry J
authors.initials: HJ
authors.lastname: Lin
authors.affiliation: Division of Biology, California Institute of Technology, Pasadena, CA 91125, USA; Howard Hughes Medical Institute, Chevy Chase, MD 20815-6789, USA.
authors.forename: Raymond J
authors.initials: RJ
authors.lastname: Deshaies
authors.affiliation: Department of Pharmaceutical Chemistry, Small Molecule Discovery Center, University of California, San Francisco, CA 94158, USA.
authors.forename: Michelle R
authors.initials: MR
authors.lastname: Arkin
mesh_terms: D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D001667:Binding, Competitive; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D057180:Frontotemporal Dementia / Q000235:genetics; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D009154:Mutation / Q000235:genetics; D009687:Nuclear Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D020349:Surface Plasmon Resonance
chemical_list: D004791:Enzyme Inhibitors; D009687:Nuclear Proteins; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; C404942:p97 ATPase
keywords: IBMPFD/ALS; SPR; p97 inhibitor; p97/VCP AAA ATPase; steady-state kinetics