title: Mechanism of the Rpn13-induced activation of Uch37.
abstract: Uch37 is a de-ubiquitinating enzyme that is activated by Rpn13 and involved in the proteasomal degradation of proteins. The full-length Uch37 was shown to exhibit low iso-peptidase activity and is thought to be auto-inhibited. Structural comparisons revealed that within a homo-dimer of Uch37, each of the catalytic domains was blocking the other's ubiquitin (Ub)-binding site. This blockage likely prevented Ub from entering the active site of Uch37 and might form the basis of auto-inhibition. To understand the mode of auto-inhibition clearly and shed light on the activation mechanism of Uch37 by Rpn13, we investigated the Uch37-Rpn13 complex using a combination of mutagenesis, biochemical, NMR, and small-angle X-ray scattering (SAXS) techniques. Our results also proved that Uch37 oligomerized in solution and had very low activity against the fluorogenic substrate ubiquitin-7-amino-4-methylcoumarin (Ub-AMC) of de-ubiquitinating enzymes. Uch37Δ(Hb,Hc,KEKE), a truncation removal of the C-terminal extension region (residues 256-329) converted oligomeric Uch37 into a monomeric form that exhibited iso-peptidase activity comparable to that of a truncation-containing the Uch37 catalytic domain only. We also demonstrated that Rpn13C (Rpn13 residues 270-407) could disrupt the oligomerization of Uch37 by sequestering Uch37 and forming a Uch37-Rpn13 complex. Uch37 was activated in such a complex, exhibiting 12-fold-higher activity than Uch37 alone. Time-resolved SAXS (TR-SAXS) and FRET experiments supported the proposed mode of auto-inhibition and the activation mechanism of Uch37 by Rpn13. Rpn13 activated Uch37 by forming a 1:1 stoichiometric complex in which the active site of Uch37 was accessible to Ub.
authors.affiliation: National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China.
authors.forename: Lianying
authors.initials: L
authors.lastname: Jiao
authors.forename: Songying
authors.initials: S
authors.lastname: Ouyang
authors.forename: Neil
authors.initials: N
authors.lastname: Shaw
authors.forename: Gaojie
authors.initials: G
authors.lastname: Song
authors.forename: Yingang
authors.initials: Y
authors.lastname: Feng
authors.forename: Fengfeng
authors.initials: F
authors.lastname: Niu
authors.forename: Weicheng
authors.initials: W
authors.lastname: Qiu
authors.forename: Hongtao
authors.initials: H
authors.lastname: Zhu
authors.forename: Li-Wei
authors.initials: LW
authors.lastname: Hung
authors.forename: Xiaobing
authors.initials: X
authors.lastname: Zuo
authors.forename: V
authors.initials: V
authors.lastname: Eleonora Shtykova
authors.forename: Ping
authors.initials: P
authors.lastname: Zhu
authors.forename: Yu-Hui
authors.initials: YH
authors.lastname: Dong
authors.forename: Ruxiang
authors.initials: R
authors.lastname: Xu
authors.forename: Zhi-Jie
authors.initials: ZJ
authors.lastname: Liu
mesh_terms: D001665:Binding Sites; D020134:Catalytic Domain; D002850:Chromatography, Gel; D018360:Crystallography, X-Ray; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding; D011487:Protein Conformation; D055503:Protein Multimerization; D053838:Scattering, Small Angle; D043222:Ubiquitin Thiolesterase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014461:Ultracentrifugation
chemical_list: C500781:ADRM1 protein, human; D047908:Intracellular Signaling Peptides and Proteins; D008562:Membrane Glycoproteins; C421245:UCHL5 protein, human; D043222:Ubiquitin Thiolesterase
references.citation: J Biomol NMR. 1994 Sep;4(5):603-14
references.pmid: 22911360
references.citation: EMBO J. 2006 Oct 4;25(19):4524-36
references.pmid: 16990800
references.citation: Biochem Biophys Res Commun. 2009 Dec 18;390(3):855-60
references.pmid: 19836345
references.citation: J Biomol NMR. 2009 Aug;44(4):213-23
references.pmid: 19548092
references.citation: Acta Crystallogr D Biol Crystallogr. 1998 Sep 1;54(Pt 5):905-21
references.pmid: 9757107
references.citation: Nature. 1997 Feb 20;385(6618):737-40
references.pmid: 9034192
references.citation: J Mol Graph. 1996 Feb;14(1):51-5, 29-32
references.pmid: 8744573
references.citation: Nat Cell Biol. 2006 Sep;8(9):994-1002
references.pmid: 16906146
references.citation: Nature. 2008 May 22;453(7194):548-52
references.pmid: 18497827
references.citation: Mol Cell. 2010 May 14;38(3):404-15
references.pmid: 20471946
references.citation: EMBO J. 1997 Jul 1;16(13):3787-96
references.pmid: 9233788
references.citation: EMBO J. 2006 Dec 13;25(24):5742-53
references.pmid: 17139257
references.citation: Biochem J. 2012 Jan 1;441(1):143-9
references.pmid: 21851340
references.citation: Proc Natl Acad Sci U S A. 2006 Mar 21;103(12):4675-80
references.pmid: 16537382
references.citation: Proc Natl Acad Sci U S A. 2013 May 28;110(22):9054-9
references.pmid: 23569220
references.citation: Biophys J. 2000 Mar;78(3):1606-19
references.pmid: 10692345
references.citation: Mol Cell. 2009 Dec 25;36(6):1018-33
references.pmid: 20064467
references.citation: Proteins. 2012 Feb;80(2):649-54
references.pmid: 21953935
references.citation: J Mol Biol. 1997 Oct 17;273(1):283-98
references.pmid: 9367762
references.citation: Oncogene. 2005 Dec 1;24(54):8080-4
references.pmid: 16027725
references.citation: J Biol Chem. 2005 Jan 14;280(2):1512-20
references.pmid: 15531586
references.citation: Annu Rev Biochem. 2009;78:363-97
references.pmid: 19489724
references.citation: Biochem Biophys Res Commun. 2011 Dec 9;416(1-2):76-9
references.pmid: 22086173
references.citation: J Mol Biol. 2007 Sep 21;372(3):774-97
references.pmid: 17681537
references.citation: Mol Biol Cell. 2008 Mar;19(3):1072-82
references.pmid: 18162577
references.citation: Proteins. 2005 Jun 1;59(4):662-72
references.pmid: 15822098
references.citation: Mol Cell. 2008 Sep 26;31(6):909-17
references.pmid: 18922472
references.citation: J Biomol NMR. 2001 Apr;19(4):321-9
references.pmid: 11370778
references.citation: J Mol Biol. 2009 Sep 11;392(1):228-41
references.pmid: 19591838
references.citation: Biochem J. 2008 Sep 1;414(2):161-75
references.pmid: 18687060
references.citation: EMBO J. 1999 Jul 15;18(14):3877-87
references.pmid: 10406793
references.citation: Mol Cell. 2006 Oct 6;24(1):39-50
references.pmid: 17018291
references.citation: PLoS One. 2010 Feb 04;5(2):e9046
references.pmid: 20140230
references.citation: Annu Rev Biochem. 1999;68:1015-68
references.pmid: 10872471
references.citation: Bioinformatics. 2011 Jul 15;27(14):1908-14
references.pmid: 21576179
references.citation: J Biomol NMR. 1996 Dec;8(4):477-86
references.pmid: 9008363
references.citation: Biophys J. 2001 Jun;80(6):2946-53
references.pmid: 11371467
references.citation: Nature. 2008 May 22;453(7194):481-8
references.pmid: 18497817
references.citation: J Biol Chem. 2009 Feb 6;284(6):3593-602
references.pmid: 19047059
references.citation: FEBS J. 2011 Dec;278(24):4917-26
references.pmid: 21995438
references.citation: J Biol Chem. 1998 Mar 6;273(10):5461-7
references.pmid: 9488668
references.citation: Exp Cell Res. 2005 Jul 15;307(2):436-51
references.pmid: 15950624