title: RING E3 mechanism for ubiquitin ligation to a disordered substrate visualized for human anaphase-promoting complex.
abstract: For many E3 ligases, a mobile RING (Really Interesting New Gene) domain stimulates ubiquitin (Ub) transfer from a thioester-linked E2∼Ub intermediate to a lysine on a remotely bound disordered substrate. One such E3 is the gigantic, multisubunit 1.2-MDa anaphase-promoting complex/cyclosome (APC), which controls cell division by ubiquitinating cell cycle regulators to drive their timely degradation. Intrinsically disordered substrates are typically recruited via their KEN-box, D-box, and/or other motifs binding to APC and a coactivator such as CDH1. On the opposite side of the APC, the dynamic catalytic core contains the cullin-like subunit APC2 and its RING partner APC11, which collaborates with the E2 UBCH10 (UBE2C) to ubiquitinate substrates. However, how dynamic RING-E2∼Ub catalytic modules such as APC11-UBCH10∼Ub collide with distally tethered disordered substrates remains poorly understood. We report structural mechanisms of UBCH10 recruitment to APC(CDH1) and substrate ubiquitination. Unexpectedly, in addition to binding APC11's RING, UBCH10 is corecruited via interactions with APC2, which we visualized in a trapped complex representing an APC(CDH1)-UBCH10∼Ub-substrate intermediate by cryo-electron microscopy, and in isolation by X-ray crystallography. To our knowledge, this is the first structural view of APC, or any cullin-RING E3, with E2 and substrate juxtaposed, and it reveals how tripartite cullin-RING-E2 interactions establish APC's specificity for UBCH10 and harness a flexible catalytic module to drive ubiquitination of lysines within an accessible zone. We propose that multisite interactions reduce the degrees of freedom available to dynamic RING E3-E2∼Ub catalytic modules, condense the search radius for target lysines, increase the chance of active-site collision with conformationally fluctuating substrates, and enable regulation.
authors.affiliation: Department of Structural Biology.
authors.forename: Nicholas G
authors.initials: NG
authors.lastname: Brown
authors.affiliation: Department of Structural Biology, Howard Hughes Medical Institute.
authors.forename: Ryan
authors.initials: R
authors.lastname: VanderLinden
authors.affiliation: Department of Structural Biology, Department of Microbiology, Immunology, and Biochemistry, University of Tennessee Health Sciences Center, Memphis, TN 38105;
authors.forename: Edmond R
authors.initials: ER
authors.lastname: Watson
authors.affiliation: Research Institute of Molecular Pathology (IMP), Vienna Biocenter (VBC), 1030 Vienna, Austria;
authors.forename: Renping
authors.initials: R
authors.lastname: Qiao
authors.affiliation: Department of Structural Biology.
authors.forename: Christy R R
authors.initials: CR
authors.lastname: Grace
authors.affiliation: Department of Structural Biology.
authors.forename: Masaya
authors.initials: M
authors.lastname: Yamaguchi
authors.affiliation: Research Institute of Molecular Pathology (IMP), Vienna Biocenter (VBC), 1030 Vienna, Austria;
authors.forename: Florian
authors.initials: F
authors.lastname: Weissmann
authors.affiliation: Department of Structural Biology.
authors.forename: Jeremiah J
authors.initials: JJ
authors.lastname: Frye
authors.affiliation: Max Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany; and Department of 3D Electron Cryomicroscopy, Institute of Microbiology and Genetics, Georg-August Universität, 37077 Göttingen, Germany.
authors.forename: Prakash
authors.initials: P
authors.lastname: Dube
authors.affiliation: Department of Structural Biology.
authors.forename: Shein
authors.initials: S
authors.lastname: Ei Cho
authors.affiliation: Department of Chemical Biology and Therapeutics, and.
authors.forename: Marcelo L
authors.initials: ML
authors.lastname: Actis
authors.affiliation: Hartwell Center for Bioinformatics and Biotechnology, St. Jude Children's Research Hospital, Memphis, TN 38105;
authors.forename: Patrick
authors.initials: P
authors.lastname: Rodrigues
authors.affiliation: Department of Chemical Biology and Therapeutics, and.
authors.forename: Naoaki
authors.initials: N
authors.lastname: Fujii
authors.affiliation: Research Institute of Molecular Pathology (IMP), Vienna Biocenter (VBC), 1030 Vienna, Austria; jan-michael.peters@imp.ac.at hstark1@gwdg.de brenda.schulman@stjude.org.
authors.forename: Jan-Michael
authors.initials: JM
authors.lastname: Peters
authors.affiliation: Max Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany; and Department of 3D Electron Cryomicroscopy, Institute of Microbiology and Genetics, Georg-August Universität, 37077 Göttingen, Germany jan-michael.peters@imp.ac.at hstark1@gwdg.de brenda.schulman@stjude.org.
authors.forename: Holger
authors.initials: H
authors.lastname: Stark
authors.affiliation: Department of Structural Biology, Howard Hughes Medical Institute, Department of Microbiology, Immunology, and Biochemistry, University of Tennessee Health Sciences Center, Memphis, TN 38105; jan-michael.peters@imp.ac.at hstark1@gwdg.de brenda.schulman@stjude.org.
authors.forename: Brenda A
authors.initials: BA
authors.lastname: Schulman
mesh_terms: D064173:Anaphase-Promoting Complex-Cyclosome / Q000737:chemistry* / Q000378:metabolism; D064189:Apc1 Subunit, Anaphase-Promoting Complex-Cyclosome / Q000737:chemistry* / Q000378:metabolism; D064198:Apc11 Subunit, Anaphase-Promoting Complex-Cyclosome / Q000737:chemistry* / Q000378:metabolism; D018360:Crystallography, X-Ray; D004265:DNA Helicases / Q000737:chemistry* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D025801:Ubiquitin / Q000737:chemistry* / Q000378:metabolism; D044763:Ubiquitin-Conjugating Enzymes / Q000737:chemistry* / Q000378:metabolism
chemical_list: C577335:ANAPC1 protein, human; D064189:Apc1 Subunit, Anaphase-Promoting Complex-Cyclosome; D004268:DNA-Binding Proteins; D025801:Ubiquitin; C105156:UBE2C protein, human; D044763:Ubiquitin-Conjugating Enzymes; C577351:ANAPC11 protein, human; D064173:Anaphase-Promoting Complex-Cyclosome; D064198:Apc11 Subunit, Anaphase-Promoting Complex-Cyclosome; D004265:DNA Helicases; C080359:CHD1 protein, human
keywords: E3; anaphase-promoting complex; cullin; electron microscopy; ubiquitin
references.citation: Nat Struct Mol Biol. 2008 Mar;15(3):280-7
references.pmid: 18264111
references.citation: Mol Cell. 2013 May 23;50(4):516-27
references.pmid: 23665230
references.citation: Dev Cell. 2015 Feb 9;32(3):358-72
references.pmid: 25669885
references.citation: Acta Crystallogr D Biol Crystallogr. 2010 Apr;66(Pt 4):486-501
references.pmid: 20383002
references.citation: Cell. 1995 Apr 21;81(2):279-88
references.pmid: 7736580
references.citation: Nature. 2011 Feb 10;470(7333):274-8
references.pmid: 21107322
references.citation: Proc Natl Acad Sci U S A. 2010 Jan 26;107(4):1355-60
references.pmid: 20080579
references.citation: Nature. 2002 Apr 18;416(6882):703-9
references.pmid: 11961546
references.citation: Proc Natl Acad Sci U S A. 1996 Apr 30;93(9):4294-9
references.pmid: 8633058
references.citation: FEBS Open Bio. 2014 Jul 05;4:689-703
references.pmid: 25161877
references.citation: Nat Struct Mol Biol. 2011 Jan;18(1):6-13
references.pmid: 21186364
references.citation: Mol Cell. 2005 Dec 22;20(6):867-79
references.pmid: 16364912
references.citation: Proc Natl Acad Sci U S A. 2009 Oct 27;106(43):18213-8
references.pmid: 19822757
references.citation: Mol Cell. 2005 May 27;18(5):543-53
references.pmid: 15916961
references.citation: Nat Struct Mol Biol. 2013 Jul;20(7):827-35
references.pmid: 23708605
references.citation: Nat Struct Mol Biol. 2006 Oct;13(10):915-20
references.pmid: 16980971
references.citation: Mol Cell. 2014 Oct 23;56(2):246-60
references.pmid: 25306923
references.citation: Mol Biol Cell. 2001 Dec;12(12):3839-51
references.pmid: 11739784
references.citation: J Biol Chem. 2002 Jun 14;277(24):21913-21
references.pmid: 11927573
references.citation: Mol Cell. 2009 Jun 26;34(6):674-85
references.pmid: 19560420
references.citation: Mol Biol Cell. 1995 Feb;6(2):185-97
references.pmid: 7787245
references.citation: J Comput Chem. 2004 Oct;25(13):1605-12
references.pmid: 15264254
references.citation: Curr Biol. 1996 Apr 1;6(4):455-66
references.pmid: 8723350
references.citation: Nat Cell Biol. 2009 Nov;11(11):1363-9
references.pmid: 19820702
references.citation: Mol Cell. 2013 Jun 6;50(5):649-60
references.pmid: 23707760
references.citation: Nat Cell Biol. 2013 Jul;15(7):797-806
references.pmid: 23708001
references.citation: Elife. 2013;2:e00828
references.pmid: 23936628
references.citation: Nature. 2004 Dec 2;432(7017):588-95
references.pmid: 15558010
references.citation: Biochemistry. 2013 Apr 30;52(17):2991-9
references.pmid: 23550736
references.citation: Curr Opin Struct Biol. 2011 Apr;21(2):257-64
references.pmid: 21288713
references.citation: Curr Biol. 2014 Jul 7;24(13):1556-62
references.pmid: 24930963
references.citation: Mol Cell. 2008 Aug 22;31(4):544-56
references.pmid: 18722180
references.citation: Science. 1998 Feb 20;279(5354):1216-9
references.pmid: 9469814
references.citation: Genes Dev. 2006 Sep 1;20(17):2410-20
references.pmid: 16921029
references.citation: Mol Cell. 2006 Mar 17;21(6):873-80
references.pmid: 16543155
references.citation: Nature. 2005 Jun 2;435(7042):687-92
references.pmid: 15931224
references.citation: Proc Natl Acad Sci U S A. 2012 Nov 6;109(45):18419-24
references.pmid: 23091007
references.citation: Cell. 2006 Jan 13;124(1):89-103
references.pmid: 16413484
references.citation: Cell. 2009 Nov 25;139(5):957-68
references.pmid: 19945379
references.citation: Nat Cell Biol. 2012 Feb;14(2):168-76
references.pmid: 22286100
references.citation: Cell. 2014 Jun 19;157(7):1671-84
references.pmid: 24949976
references.citation: Mol Cell. 2014 Oct 23;56(2):232-45
references.pmid: 25306918
references.citation: Genes Dev. 2001 Sep 15;15(18):2381-95
references.pmid: 11562348
references.citation: Cell. 2008 Sep 19;134(6):995-1006
references.pmid: 18805092
references.citation: Nature. 2012 Sep 6;489(7414):115-20
references.pmid: 22842904
references.citation: Curr Opin Struct Biol. 2010 Dec;20(6):714-21
references.pmid: 20880695
references.citation: Science. 1998 Feb 20;279(5354):1219-22
references.pmid: 9469815
references.citation: Nature. 2014 Sep 18;513(7518):388-93
references.pmid: 25043029
references.citation: Mol Cell. 2011 Jun 24;42(6):744-57
references.pmid: 21700221
references.citation: Nature. 2014 Oct 30;514(7524):591-6
references.pmid: 25355358
references.citation: Nat Struct Mol Biol. 2012 Sep;19(9):876-83
references.pmid: 22902369
references.citation: J Mol Biol. 2015 Apr 24;427(8):1748-64
references.pmid: 25490258
references.citation: Mol Cell. 2012 Sep 28;47(6):933-42
references.pmid: 22885007
references.citation: Annu Rev Biochem. 2009;78:399-434
references.pmid: 19489725
references.citation: Cell. 2007 Jul 13;130(1):127-39
references.pmid: 17632060
references.citation: Mol Cell. 2008 Nov 21;32(4):576-83
references.pmid: 19026787
references.citation: Nat Struct Mol Biol. 2012 Nov;19(11):1116-23
references.pmid: 23007861
references.citation: Proc Natl Acad Sci U S A. 2011 Apr 5;108(14):5590-5
references.pmid: 21422291