title: 2.3 Å resolution cryo-EM structure of human p97 and mechanism of allosteric inhibition.
abstract: p97 is a hexameric AAA+ adenosine triphosphatase (ATPase) that is an attractive target for cancer drug development. We report cryo-electron microscopy (cryo-EM) structures for adenosine diphosphate (ADP)-bound, full-length, hexameric wild-type p97 in the presence and absence of an allosteric inhibitor at resolutions of 2.3 and 2.4 angstroms, respectively. We also report cryo-EM structures (at resolutions of ~3.3, 3.2, and 3.3 angstroms, respectively) for three distinct, coexisting functional states of p97 with occupancies of zero, one, or two molecules of adenosine 5'-O-(3-thiotriphosphate) (ATPγS) per protomer. A large corkscrew-like change in molecular architecture, coupled with upward displacement of the N-terminal domain, is observed only when ATPγS is bound to both the D1 and D2 domains of the protomer. These cryo-EM structures establish the sequence of nucleotide-driven structural changes in p97 at atomic resolution. They also enable elucidation of the binding mode of an allosteric small-molecule inhibitor to p97 and illustrate how inhibitor binding at the interface between the D1 and D2 domains prevents propagation of the conformational changes necessary for p97 function.
authors.affiliation: Laboratory of Cell Biology, National Cancer Institute, Bethesda, MD 20892, USA.
authors.forename: Soojay
authors.initials: S
authors.lastname: Banerjee
authors.affiliation: Laboratory of Cell Biology, National Cancer Institute, Bethesda, MD 20892, USA.
authors.forename: Alberto
authors.initials: A
authors.lastname: Bartesaghi
authors.affiliation: Laboratory of Cell Biology, National Cancer Institute, Bethesda, MD 20892, USA.
authors.forename: Alan
authors.initials: A
authors.lastname: Merk
authors.affiliation: Laboratory of Cell Biology, National Cancer Institute, Bethesda, MD 20892, USA.
authors.forename: Prashant
authors.initials: P
authors.lastname: Rao
authors.affiliation: Small Molecule Discovery Center, Pharmaceutical Chemistry, School of Pharmacy, University of California, San Francisco, CA 94143, USA.
authors.forename: Stacie L
authors.initials: SL
authors.lastname: Bulfer
authors.affiliation: University of Pittsburgh Chemical Diversity Center, University of Pittsburgh, Pittsburgh, PA 15260, USA.
authors.forename: Yongzhao
authors.initials: Y
authors.lastname: Yan
authors.affiliation: Leidos Biomedical Research Inc., Frederick, MD 21702, USA.
authors.forename: Neal
authors.initials: N
authors.lastname: Green
authors.affiliation: Division of Cancer Treatment and Diagnosis, National Cancer Institute, Bethesda, MD 20892, USA.
authors.forename: Barbara
authors.initials: B
authors.lastname: Mroczkowski
authors.affiliation: Small Molecule Discovery Center, Pharmaceutical Chemistry, School of Pharmacy, University of California, San Francisco, CA 94143, USA.
authors.forename: R Jeffrey
authors.initials: RJ
authors.lastname: Neitz
authors.affiliation: University of Pittsburgh Chemical Diversity Center, University of Pittsburgh, Pittsburgh, PA 15260, USA.
authors.forename: Peter
authors.initials: P
authors.lastname: Wipf
authors.affiliation: Laboratory of Cell Biology, National Cancer Institute, Bethesda, MD 20892, USA.
authors.forename: Veronica
authors.initials: V
authors.lastname: Falconieri
authors.affiliation: Division of Biology and Biological Engineering and Howard Hughes Medical Institute, California Institute of Technology, Pasadena, CA 91107, USA.
authors.forename: Raymond J
authors.initials: RJ
authors.lastname: Deshaies
authors.affiliation: Laboratory of Cell Biology, National Cancer Institute, Bethesda, MD 20892, USA.
authors.forename: Jacqueline L S
authors.initials: JL
authors.lastname: Milne
authors.affiliation: University of Pittsburgh Chemical Diversity Center, University of Pittsburgh, Pittsburgh, PA 15260, USA.
authors.forename: Donna
authors.initials: D
authors.lastname: Huryn
authors.affiliation: Small Molecule Discovery Center, Pharmaceutical Chemistry, School of Pharmacy, University of California, San Francisco, CA 94143, USA.
authors.forename: Michelle
authors.initials: M
authors.lastname: Arkin
authors.affiliation: Laboratory of Cell Biology, National Cancer Institute, Bethesda, MD 20892, USA. ss1@nih.gov.
authors.forename: Sriram
authors.initials: S
authors.lastname: Subramaniam
mesh_terms: D000244:Adenosine Diphosphate / Q000737:chemistry; D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors* / Q000737:chemistry*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000737:chemistry; D000494:Allosteric Regulation; D001665:Binding Sites; D020285:Cryoelectron Microscopy; D004791:Enzyme Inhibitors; D006801:Humans; D008958:Models, Molecular; D009687:Nuclear Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry*; D017434:Protein Structure, Tertiary
chemical_list: D004791:Enzyme Inhibitors; D009687:Nuclear Proteins; C022571:adenosine 5'-O-(3-thiotriphosphate); D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; C404942:p97 ATPase
references.citation: Structure. 2011 Jun 8;19(6):833-43
references.pmid: 21645854
references.citation: Mol Cell. 2000 Dec;6(6):1473-84
references.pmid: 11163219
references.citation: Nat Methods. 2014 Jan;11(1):63-5
references.pmid: 24213166
references.citation: J Biol Chem. 2014 May 2;289(18):12264-74
references.pmid: 24610782
references.citation: Open Biol. 2014 Mar 05;4:130142
references.pmid: 24598262
references.citation: Trends Cell Biol. 2015 Oct;25(10):611-622
references.pmid: 26410407
references.citation: J Mol Biol. 2016 May 8;428(9 Pt B):1861-9
references.pmid: 26608813
references.citation: Biochim Biophys Acta. 2015 Sep;1852(9):1950-9
references.pmid: 26073430
references.citation: J Biol Chem. 2013 Dec 20;288(51):36624-35
references.pmid: 24196964
references.citation: BMC Biol. 2014 Nov 11;12:94
references.pmid: 25385277
references.citation: J Med Chem. 2013 Jan 24;56(2):437-50
references.pmid: 23245311
references.citation: Structure. 2008 May;16(5):715-26
references.pmid: 18462676
references.citation: EMBO J. 2004 Mar 10;23(5):1030-9
references.pmid: 14988733
references.citation: Nat Rev Mol Cell Biol. 2005 Jul;6(7):519-29
references.pmid: 16072036
references.citation: PLoS One. 2012;7(12):e50490
references.pmid: 23226521
references.citation: ACS Med Chem Lett. 2015 Oct 23;6(12):1225-30
references.pmid: 26713109
references.citation: EMBO J. 2010 Jul 7;29(13):2217-29
references.pmid: 20512113
references.citation: Proc Natl Acad Sci U S A. 2011 Mar 22;108(12):4834-9
references.pmid: 21383145
references.citation: Autophagy. 2011 Sep;7(9):1091-2
references.pmid: 21606684
references.citation: Mol Cancer Ther. 2012 Dec;11(12):2610-20
references.pmid: 23041544
references.citation: J Cell Sci. 2014 Sep 15;127(Pt 18):3877-83
references.pmid: 25146396
references.citation: Molecules. 2015 Feb 12;20(2):3027-49
references.pmid: 25685910
references.citation: Genes Cells. 2001 Jul;6(7):575-97
references.pmid: 11473577
references.citation: Structure. 2013 Nov 5;21(11):1992-2002
references.pmid: 24055316
references.citation: J Biol Chem. 2012 Mar 9;287(11):8561-70
references.pmid: 22270372
references.citation: ChemMedChem. 2013 Feb;8(2):297-312
references.pmid: 23316025
references.citation: Front Cell Neurosci. 2015 Feb 02;9:16
references.pmid: 25698929
references.citation: Science. 2015 Jun 5;348(6239):1147-51
references.pmid: 25953817
references.citation: J Biomol Screen. 2015 Jul;20(6):788-800
references.pmid: 25690569
references.citation: EMBO J. 1999 Apr 15;18(8):2074-83
references.pmid: 10205162
references.citation: Nat Chem Biol. 2013 Sep;9(9):548-56
references.pmid: 23892893
references.citation: ACS Chem Biol. 2015 Aug 21;10(8):1916-1924
references.pmid: 26006219
references.citation: ChemMedChem. 2015 Jan;10(1):52-6
references.pmid: 25377500
references.citation: PLoS One. 2011;6(12):e29073
references.pmid: 22216170
references.citation: EMBO J. 2006 May 3;25(9):1967-76
references.pmid: 16601695