Role of E2-RING Interactions in Governing RNF4-Mediated Substrate Ubiquitination.
Members of the really interesting new gene (RING) E3 ubiquitin ligase family bind to both substrate and ubiquitin-charged E2 enzyme, promoting the transfer of ubiquitin from the E2 to substrate. Either a single ubiquitin or one of the several types of polyubiquitin chains can be conjugated to substrate proteins, with different types of ubiquitin modifications signaling the distinct outcomes. E2 enzymes play a central role in governing the nature of the ubiquitin modification, although the essential features of the E2 that differentiate mono- versus polyubiquitinating E2 enzymes remain unclear. RNF4 is a compact RING E3 ligase that directs the ubiquitination of polySUMO chains in concert with several different E2 enzymes. RNF4 monoubiquitinates polySUMO substrates in concert with RAD6B and polyubiquitinates substrates together with UBCH5B, a promiscuous E2 that can function with a broad range of E3 ligases. We find that the divergent ubiquitination activities of RAD6B and UBCH5B are governed by differences at the RING-binding surface of the E2. By mutating the RAD6B RING-binding surface to resemble that of UBCH5B, RAD6B can be transformed into a highly active UBCH5B-like E2 that polyubiquitinates SUMO chains in concert with RNF4. The switch in RAD6B activity correlates with increased affinity of the E2 for RNF4. These results point to an important role of the affinity between an E3 and its cognate E2 in governing the multiplicity of substrate ubiquitination.
Department of Biophysics and Biophysical Chemistry, The Johns Hopkins School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205, USA.
Anthony
A
DiBello
Department of Biophysics and Biophysical Chemistry, The Johns Hopkins School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205, USA.
Ajit B
AB
Datta
Department of Biophysics and Biophysical Chemistry, The Johns Hopkins School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205, USA.
Xiangbin
X
Zhang
Department of Biophysics and Biophysical Chemistry, The Johns Hopkins School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205, USA. Electronic address: cwolberg@jhmi.edu.
Cynthia
C
Wolberger
D006801:Humans; D009687:Nuclear Proteins / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D025841:Small Ubiquitin-Related Modifier Proteins / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*; D044763:Ubiquitin-Conjugating Enzymes / Q000378:metabolism*; D054875:Ubiquitination
D009687:Nuclear Proteins; C114070:RNF4 protein, human; D025841:Small Ubiquitin-Related Modifier Proteins; D014157:Transcription Factors; C471546:UBE2B protein, human; C097309:UBE2D2 protein, human; D044763:Ubiquitin-Conjugating Enzymes
E2 ubiquitin-conjugating enzyme; E3 ubiquitin ligase; RAD6B; RING finger protein 4 (RNF4); ubiquitin
Nat Cell Biol. 2016 May 27;18(6):579-86
27230526
Biochem J. 2013 Jul 1;453(1):137-45
23560854
Annu Rev Biochem. 2012;81:323-57
22404627
EMBO J. 2009 Dec 2;28(23 ):3657-66
19851286
Mol Immunol. 2009 Apr;46(7):1319-25
19118899
Methods. 2011 May;54(1):145-56
21167941
J Biol Chem. 2003 Mar 14;278(11):9448-57
12524449
J Biol Chem. 2015 Feb 27;290(9):5298-310
25548288
J Biol Chem. 2010 Dec 31;285(53):41300-9
21041297
Crit Rev Biochem Mol Biol. 2012 Jan-Feb;47(1):17-28
21981143
Mol Cell. 2014 Oct 23;56(2):246-60
25306923
Biochemistry. 2014 Jun 24;53(24):4004-14
24901938
Biochim Biophys Acta. 2004 Nov 29;1695(1-3):55-72
15571809
Protein Expr Purif. 2005 May;41(1):207-34
15915565
Dev Cell. 2004 Nov;7(5):663-76
15525528
Mol Cell. 2015 Apr 16;58(2):297-310
25801170
Nat Cell Biol. 2009 Feb;11(2):123-32
19136968
Annu Rev Cell Dev Biol. 2006;22:159-80
16753028
J Biol Chem. 1991 Mar 15;266(8):5104-12
1848239
Nat Cell Biol. 2010 Dec;12 (12 ):1177-85
21076411
Mol Cell Biol. 1998 Sep;18(9):5128-39
9710597
Cell. 2016 Jun 2;165(6):1440-53
27259151
Nat Cell Biol. 2008 May;10 (5):538-46
18408734
Cell. 2005 Dec 16;123(6):1107-20
16360039
Nat Struct Mol Biol. 2011 Aug 21;18(9):1052-9
21857666
Nat Struct Mol Biol. 2014 Apr;21(4):308-16
24699079
Mol Cell. 2003 Jan;11(1):267-74
12535539
Mol Cell. 2006 Mar 17;21(6):873-80
16543155
J Biol Chem. 2011 Jun 17;286(24):21277-86
21518764
Nature. 2002 Sep 12;419(6903):135-41
12226657
J Biol Chem. 2009 Jul 31;284(31):20582-92
19531475
Nucleic Acids Res. 2015 Oct 15;43(18):9039-50
26286193
EMBO J. 2006 Oct 18;25(20):4877-87
17006537
Structure. 2004 Aug;12(8):1519-31
15296745
Biochem J. 2011 Jan 1;433(1):31-42
21158740
Anal Biochem. 2004 Mar 15;326(2):234-56
15003564
J Biol Chem. 1985 Feb 10;260(3):1573-81
2981864
Cell. 2014 Jun 19;157(7):1671-84
24949976
Nat Struct Mol Biol. 2007 Oct;14 (10 ):941-8
17873885
J Biol Chem. 2012 Jan 2;287(1):311-21
22069333
Biochim Biophys Acta. 2014 Jan;1843(1):75-85
24018209
Cell. 2000 Aug 18;102(4):533-9
10966114
Mol Cell. 2014 Oct 23;56(2):232-45
25306918
J Biol Chem. 2013 Jun 28;288(26):18784-8
23696636
Nature. 2012 Sep 6;489(7414):115-20
22842904
Nature. 2004 Oct 14;431(7010):873-8
15386022
Nat Chem Biol. 2015 Jan;11(1):83-9
25436519
Proc Natl Acad Sci U S A. 2011 Apr 5;108(14 ):5590-5
21422291
EMBO J. 2006 Jun 7;25(11):2465-74
16710298
Nature. 2014 Oct 30;514(7524):591-6
25355358
EMBO J. 2007 Sep 19;26(18):4089-101
17762865
Nat Chem Biol. 2013 Mar;9(3):154-6
23292652
Cell Res. 2016 Apr;26(4):399-422
27012465
Exp Cell Res. 2005 Mar 10;304(1):224-33
15707587
Nat Methods. 2008 Oct;5(10):853-4
18825126
J Mol Biol. 2015 Jul 3;427(13):2290-304
25960396
Nat Struct Mol Biol. 2012 Sep;19(9):876-83
22902369
Annu Rev Biochem. 2012;81:203-29
22524316
Mol Cell. 2014 Mar 20;53(6):880-92
24656128
Mol Cell. 2012 Sep 28;47(6):933-42
22885007
EMBO J. 2011 Jul 19;30(16):3285-97
21772249
Annu Rev Biochem. 2009;78:399-434
19489725
Methods Enzymol. 2005;399:21-36
16338346
Nat Cell Biol. 2006 Jul;8(7):700-10
16799550
Mol Cell. 2003 Jan;11(1):261-6
12535538
Proc Natl Acad Sci U S A. 2013 Apr 2;110(14 ):E1263-72
23509263
Nat Struct Mol Biol. 2014 Apr;21(4):301-7
24699078