Structural basis and specificity of human otubain 1-mediated deubiquitination

15 Pages·2009·1.07 MB·English

Biochem.J.(2009)418,379–390(PrintedinGreatBritain) doi:10.1042/BJ20081318 379
Structural basis and specificity of human otubain 1-mediated
deubiquitination
MariolaJ.EDELMANN*1,AlexanderIPHO¨FER*1,MasatoAKUTSU†,MikaelALTUN*,KatalinDIGLERIA‡,HolgerB.KRAMER*,
EddaFIEBIGER§,SiranoDHE-PAGANON†2 andBenediktM.KESSLER*2
*HenryWellcomeBuildingforMolecularPhysiology,DepartmentofClinicalMedicine,UniversityofOxford,RooseveltDrive,OxfordOX37BN,U.K.,†StructuralGenomicsConsortium
andtheDepartmentofPhysiology,UniversityofToronto,Ontario,Canada,M5G1L5,‡WeatherallInstituteofMolecularMedicine,UniversityofOxford,OxfordOX37BN,U.K.,and
§DepartmentofGastroenterology/Nutrition,Children’sHospital,Boston,MA02115,U.S.A.
OTUB (otubain) 1 is a human deubiquitinating enzyme that narrowP1(cid:2)site,asobservedinOTUB1,correlateswithitsability
is implicated in mediating lymphocyte antigen responsiveness, to preferentially cleave Lys48-linked ubiquitin chains. Analysis
but whose molecular function is generally not well defined. A of cellular interaction partners of OTUB1 by co-immunopre-
structural analysis of OTUB1 shows differences in accessibility cipitationandMS/MS(tandemmassspectrometry)experiments
totheactivesiteandinsurfacepropertiesofthesubstrate-binding demonstrated that FUS [fusion involved in t(12;6) in malignant
regions when compared with its close homologue, OTUB2, liposarcoma;alsoknownasTLS(translocationinliposarcoma)or
suggesting variations in regulatory mechanisms and substrate CHOP(CCAAT/enhancer-bindingproteinhomologousprotein)]
specificity.BiochemicalanalysisrevealsthatOTUB1hasapre- and RACK1 [receptor for activated kinase 1; also known as
ference for cleaving Lys48-linked polyubiquitin chains over GNB2L1 (guanine-nucleotide-binding protein β polypeptide
Lys63-linked polyubiquitin chains, and it is capable of cleaving 2-like 1)] are part of OTUB1-containing complexes, pointing
NEDD8(neural-precursor-cell-expresseddevelopmentallydown- towards a molecular function of this deubiquitinating enzyme
regulated 8), but not SUMO (small ubiquitin-related modifier) inRNAprocessingandcelladhesion/morphology.
1/2/3 and ISG15 (interferon-stimulated gene 15) conjugates.
A functional comparison of OTUB1 and OTUB2 indicated a Key words: deubiquitinating enzyme (DUB), interferon-stimu-
differential reactivity towards ubiquitin-based active-site probes latedgene15(ISG15),neural-precursor-cell-expresseddevelop-
carryingavinylmethylester,a2-chloroethylora2-bromoethyl mentallydown-regulated8(NEDD8),otubain1(OTUB1),small
group at the C-terminus. Mutational analysis suggested that a ubiquitin-relatedmodifier(SUMO),ubiquitin.
INTRODUCTION consistsofanapprox.130-amino-acidpapainfoldwithacatalytic
triad that is typically found in cysteine proteases [4]. OTUB
Ubiquitin is central for many biological processes, including (otubain)1and2areclosehomologuesexpressedinmammals[5].
proteinturnover,proteintargeting,signaltransductionandregu- Althoughubiquitouslyexpressed,OTUB1isimplicatedinanergy
lationoftranscription,andhasbeenimplicatedintumorigenesis, induction in CD4+ T-lymphocytes, affecting the stability of the
neurodegenerationandmicrobialpathogenesis.Therelevanceof lymphocyte-specific E3 ligase GRAIL (gene related to anergy
ubiquitin in these biological processes is reflected by the fact inlymphocytes)[6].Itsimmune-relatedfunctionswerepartially
that several hundred genes have so far been linked to ubiquitin ascribedtospecificsplicevariants,includingARF-1(alternative
conjugationanddeconjugation.PrimaryexamplesaretheE3lig- reading frame 1), found only in these cells [7]. In addition,
asesandDUBs(deubiquitinatingenzymes),theformerconsisting OTUB1 binds the Yersinia pathogen-encoded virulence factor
of approx. 600 genes that are classified into the RING (really YpkA[8].ExemplifiedbyA20[9,10],OTUB1alsoprobablyacts
interestingnewgene)andHECT(homologouswithE6-associated uponadiscretesetofsubstratesincontrastwithCezanne,which
proteinC-terminus)domainfamilies[1],andthelatterareclas- was shown to be a more general DUB [11]. Structural insights
sifiedintosixmainfamilies,suchasUBPs(ubiquitin-processing obtainedforsomeoftheOTU-domain-containingproteins,such
proteases), UCHs (ubiquitin C-terminal hydrolases), ataxin- as A20 [12,13], OTUB2 [14] and yeast OTU1 in complex with
3/Josephin domains, OTUs (ovarian-tumour-domain-containing a ubiquitin-Br3 derivative (in which Gly76 was replaced with a
proteases),pathogen-encodedubiquitin-processingproteasesand bromopropylamine group) [15] confirm that the OTU domain
JAMM(JAB1/MPN/MOV34metalloenzyme)proteases[2].The encodes a rudimentary papain-like domain with considerable
OTUfamilyhasrecentlyattractedattentionowingtothepresence variationsthatmayreflectfunctionaldiversity.
of conserved sequences found in viruses, bacteria, plants, yeast Inthepresentpaper,weprovidethecrystalstructureofhuman
and humans, and its role in immunity and viral infection [3]. It OTUB1 and show significant differences when compared with
Abbreviationsused:AMC,7-amino-4-methylcoumarin;Br2,2-bromoethyl;Cl2,2-chloroethyl;DUB,deubiquitinatingenzyme;FUS,fusioninvolvedin
t(12;16)inmalignantliposarcoma;GRAIL,generelatedtoanergyinlymphocytes;HA,haemagglutinin;HEK,humanembryonickidney;ISG15,interferon-
stimulatedgene15;LC,liquidchromatography;MS/MS,tandemMS;NEDD8,neural-precursor-cell-expresseddevelopmentallydown-regulated8;NP-40,
NonidetP40;OTU,ovarian-tumour-domain-containingprotease;OTUB,otubain;RACK1,receptorforactivatedkinase1;SBP,streptavidin-bindingpeptide;
SUMO,smallubiquitin-relatedmodifier;SENP2,SUMO1/sentrin/SMT3(suppressorofmiftwo3homologue1)-specificpeptidase2;TEV,tobaccoetch
virus;TLS,translocationinliposarcoma;Ubl,ubiquitin-likeprotein;UCH,ubiquitinC-terminalhydrolase;USP,ubiquitin-specificpeptidase;VME,vinyl
methylester.
1 Theseauthorscontributedequallytothiswork.
2 Correspondencemaybeaddressedtoeitheroftheseauthors([email protected]@ccmp.ox.ac.uk).
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380 M.J.Edelmannandothers
OTUB2.Inadditiontobeingselectivetowardsubiquitinand,to Table1 Datacollectionandrefinementstatistics
some extent, NEDD8 (neural-precursor-cell-expressed develop-
Valuesinparenthesesareforthehighestresolutionshell.PDBcode2ZFY.RMSD,rootmean
mentallydown-regulated8),butnotISG15(interferon-stimulated squaredeviation.
gene 15) and SUMO (small ubiquitin-related modifier) 1/2/3,
OTUB1 prefers cleavage of Lys48-linked ubiquitin over Lys63- Parameter Value
linkedubiquitinchains,whichisprobablyattributabletoanarrow
cavityattheP1(cid:2)site.Massspectrometricidentificationofcellular Datacollectionstatistics
interaction partners suggests that OTUB1 may be generally Crystal Humanotubain1(residues40–271)
involvedinRNAprocessingandcelladhesion/morphology. Spacegroup C2
Unit-cellparameters a=81.25A˚,b=50.89A˚,c=54.72A˚,β=101.96◦
Wavelength(A˚) 1.54178
Resolution(A˚) 29.15–1.69
MATERIALSANDMETHODS Totalreflections 23651
Uniquereflections 7629
Celllines,reagentsandantibodies Completeness(%) 97.2(80.8)
R (%) 7.3(39.0)
ChemicalswerepurchasedfromSigma–Aldrich,unlessindicated merge
I/σ(I) 18.7(1.9)
otherwise. The cDNA for human OTUB1 was obtained by
Refinementstatistics
carrying out PCR with a spleen cDNA library (Invitrogen) as a Resolutionrange(A˚) 53.53–1.69
template with the following primers: 5(cid:2)-CTACTAGCTAGCAT- Numberofreflections 22222
GGCGGCGGAGGAACC-5(cid:2) (forward) and 5(cid:2)-CGGCGG- Numberofnon-hydrogenatoms
CTCGAGCTATTTGTAGAGGATATCGTAG-3(cid:2) (reverse). The Protein 1882
primers included a 5(cid:2) NheI and a 3(cid:2) XhoI site for directional Water 124
R (%) 19.5
cloning into a pcDNA3.1 vector containing a C-terminal SBP work
R (%) 23.9
free
(streptavidin-binding peptide)–TEV (tobacco etch virus)–HA RMSD
(haemagglutinin) tag. The OTUB1–SBP–TEV–HA (hereafter Bondlength(A˚) 0.016
referred to as outabain 1–HA) C91S mutant was created using Bondangle(◦) 1.435
the QuikChange® II site-directed mutagenesis kit (Stratagene). AverageB-factors(A˚2)
Otubain1 29.4
Wild-typeandcysteinemutantOTUB1–HAweresubclonedinto
Water 37.5
the bicistronic pEF-IRES vector [16], kindly provided by Dr
MarekCebecauer(SectionofMolecularandCellularMedicine,
NationalLungandHeartInstitute,FacultyofMedicine,Imperial
College London, London, U.K.), using the 5(cid:2) NheI and 3(cid:2) andRAXISIV++detectorandprocessedwithHKL2000[17].
XbaI restriction sites. Residues 40–271 were subcloned into A molecular replacement solution using OTUB2 (PDB code
pET28a-LIC for structural studies. The cDNA for human 1TFF)wasfoundwiththeprogramMolRep[18].Therefinement
OTUB2 was obtained by carrying out PCR with a spleen procedureswerecarriedoutwithREFMAC5[19].Modelfitting
cDNA library as a template with the following primers: 5(cid:2)- to electron-density maps was performed manually using Coot
CTACTAGCTAGCATGAGTGAAACATCTTTCAACC-3(cid:2) (for- [20].FinalrefinementstatisticsaresummarizedinTable1.The
ward) and 5(cid:2)-CGGCGGCTCGAGTCAATGTTTATCGGCTGC- generation of recombinant wild-type and P87G mutant OTUB1
ATAAAGG-3(cid:2) (reverse). The primers contained a 5(cid:2) NheI and G47P mutant OTUB2 protein is described in detail in the
and a 3(cid:2) XhoI site for directional cloning into a pcDNA3.1 SupplementaryOnlineData.
vector containing a C-terminal SBP–TEV–HA tag as well as a
pET28a-LIC vector for bacterial expression. The proteasome PreparationofubiquitinandUbl(ubiquitin-likeprotein)substrates
inhibitor ZL3VS was kindly provided by Professor Hermen andisopeptidaseassays
Overkleeft (Department of Bio-organic Synthesis, Leiden
ThesynthesisandpreparationofubiquitinandUblsubstratebased
InstituteofChemistry,GorlaeusLaboratories,LeidenUniversity,
ontheubiquitin/UblorthogonalpeptidescaffoldcontaininganN-
Leiden, The Netherlands). The antibodies used in the present
terminalbiotinmoietywasperformedessentiallyasdescribedin
study are described in the Supplementary Online Data at
[21]. Further details can be found in the Supplementary Online
http://www.BiochemJ.org/bj/418/bj4180379add.htm.
Data.
Proteinpurification,crystallizationandstructuredetermination
Preparationofubiquitin-specificactive-siteprobesand
OTUB1-(40–271)wasexpressedinEscherichiacoliBL21(DE3) activity-basedprofilingassays
cells and grown in Terrific broth in the presence of 50μg/ml
The synthesis and preparation of HA-tagged ubiquitin active-
kanamycinat37◦CtoaD of0.5.Cellswereinducedovernight
600 siteprobescontainingaC-terminalBr2(2-bromoethyl),Cl2(2-
at15◦Cwith0.05mMIPTG(isopropylβ-D-thiogalactoside).The chloroethyl)andVME(vinylmethylester)groupwasperformed
expressedproteinwaspurifiedfromcellpelletsusingaTALON®
essentially as reported previously [22]. Further details are
metal-affinity resin column (BD Biosciences) at 4◦C, cleaved
describedintheSupplementaryOnlineData.
with thrombin (Sigma T9681), and purified by gel filtration
on a HighLoad 16/60 Superdex 200 column (GE Healthcare).
Lineardi-ubiquitin,Lys48-/Lys63-linkedtetra-ubiquitin,di-SUMO
Purifiedprotein(68mg/ml)wascrystallizedatroomtemperature
(25◦C) using the hanging-drop vapour-diffusion method when andubiquitin-AMC(7-amino-4-methylcoumarin)cleavageassays
mixedwithanequalvolumeofthereservoirsolution{30%PEG Linear di-ubiquitin, Lys48-, Lys63-linked tetra-ubiquitin, di-
[poly(ethylene glycol)] 8000, 0.2M sodium acetate and 0.1M SUMO2, di-SUMO3, ubiquitin-AMC, UCH-L3 and SENP2
sodium cacodylate (pH6.5)}. Crystals were cryoprotected in a [SUMO1/sentrin/SMT3(suppressorofmiftwo3homologue1)-
50:50mixtureofParatone-Nandmineraloilandfrozeninliquid specific peptidase 2] were purchased from Boston Biochem.
nitrogen.DiffractiondatawereobtainedusinganFR-Egenerator A 20μl solution containing reaction buffer (50mM Tris/HCl,
(cid:3)c TheAuthorsJournalcompilation(cid:3)c 2009BiochemicalSociety
Structuralinsightsintohumanotubain1 381
pH7.4,and2mMdithiothreitol),lineardi-ubiquitin,Lys48-/Lys63- Moleculargraphics
linked tetra-ubiquitin, di-SUMO2 or di-SUMO3 (250nM final
All structural Figures were generated using PyMOL v.99vc6
concentration) was incubated with recombinant OTUB1 or
(DeLanoScientific;http://pymol.sourceforge.net/).
OTUB2 proteins (50nM final concentration) or 5μg of cell
extract(preparedasdescribedbelow)inafinalreactionvolumeof
20μlfortheindicatedtimesat37◦C.Thereactionswerestopped
RESULTS
byaddingreducingSDSsamplebuffer,followedbyseparationon
Tris/TricineSDS/PAGE[23]andvisualizationbyanti-ubiquitinor Overallstructure
anti-SUMOimmunoblotting.FortheanalysisbyMS,1.5μgof
OTUB1 is an OTU family member with a conserved ovarian
Lys48- or Lys63-linked tetra-ubiquitin was incubated with 1μg
tumour domain between residues 85 and 271. Removal of
of recombinant OTUB1 for the indicated times and subjected
the N-terminal sequence allowed OTUB1 (residues 40–271) to
to analysis by infusion electrospray MS (Agilent 1100 series readilycrystallizeanddiffractto1.7Å(1Å=0.1nm)resolution.
LC/MSD-TOF spectrometer). Ubiquitin-AMC cleavage assays
A molecular replacement strategy using co-ordinates from the
wereperformedessentiallyasreportedin[21]andfurtherdetails
OTUB2 structure was employed to determine the OTUB1
aredescribedintheSupplementaryOnlineData.
structure, revealing all residues in the construct except for the
first five and Asp238 in the β3–β4 loop. As with other papain-
like proteases, the active site is formed at the interface of an
IdentificationofOTUB1interactorsbyMS/MS(tandemMS) α-helicallobe(α3–α10)andaβ-sheetlobe(β1–β5),centredon
HEK (human embryonic kidney)-293T cells were grown to thecatalyticcysteine(Cys91),whichissituatedattheC-terminal
confluence in DMEM (Dulbecco’s modified Eagle’s medium) poleoftheα3helix(Figure1A,left-handpanel).TheP-sideofthe
containing 10% (v/v) FBS (fetal bovine serum) and 1% activesiterunsalongonesideoftheinterface,andtheP(cid:2)-sideruns
streptomycin/penicillin.Cellswerethentransferredto150-mm- along the opposing side (Figures 1A and 1C). Interestingly, the
diameter, 100-mm-diameter, 50-mm-diameter or six-well tissue conformationofthecatalytictriadhistidineresidue(His265)and
culture dishes at a concentration of 0.4×106/ml and grown its distance from the catalytic cysteine (Cys91, 5.5Å) and cata-
overnightat37◦C.Thecellswerethenwashedandthetransfection lytic aspartate (Asp267, 4.6Å) residues is incompatible with
was performed using SuperFect reagent (Qiagen), according to catalysis (Figure 1B, top panel). Instead, His265 is sandwiched
themanufacturer’sprotocol,followedbyincubationovernightat betweentheguanidiniumandprolylsidechainsofArg262andPro87
37◦C.Approx.109cellspersamplewerelysedusingglassbeads respectively,interactingwiththecatalyticAsp267throughapairof
(Sigma)in150mMNaCl,5mMCaCl ,50mMTris/HCl(pH7.4) watermolecules.Inaddition,thecatalyticCys91istied-upthrough
2
and 250mM sucrose containing protease inhibitor cocktail hydrogenbondswiththebackbonecarbonyloxygenofArg86and
(RocheAppliedScience)asdescribedin[22].Alternatively,cells a water molecule that also interacts with Glu214. The conserved
werelysedin0.1%NP-40(NonidetP40),150mMNaCl,20mM Glu214 from the α9–α10 loop is inserted into the P1 pocket,
CaCl and 50mM Tris/HCl (pH7.4). For immunoprecipitation, forminghydrogenbondswiththeC-terminalpoleoftheα3helix
2
protein lysates (30mg per sample) were first diluted in NET andthebackboneofthecatalyticCys91,therebyblockingaccess
buffer(50mMTris/HCl,5mMEDTA,150mMNaCland0.5% to the active site (Figure 1B, top panel). This unique structure
NP-40, pH7.4) to a protein concentration of 1mg/ml, and pre- corresponds to a non-productive conformation of the catalytic
cleared with Protein A–agarose beads for 1h at 4◦C. Immuno- centrenotobservedinanyothercysteineproteases(Figure1B).
precipitation was then carried out either for 2h or overnight at Ourstructureoftheligand-freeOTUB1mightthereforerepresent
4◦C.MaterialwaselutedfromthebeadsusingtheTEVprotease an autoinhibited state, which is probably involved in a ligand-
(Invitrogen) for 2h at 4◦C and separated by SDS/PAGE and dependentinduced-fitmechanism.
visualized using silver staining. Gel bands that were unique to
lanes containing OTUB1 as well as the corresponding areas in StructuralcomparisonofOTUB1andOTUB2
the control lane were excised and subjected to in-gel digestion Despite being highly homologous with OTUB2 {RMSD (root
withtrypsinandanalysisbynano-LC(liquidchromatography)– meansquaredeviation)valueof1.68,Figure1and[14]},OTUB1
MS/MSasdescribedin[24].
displays a number of different structural features (Figures 1C
and 1D), particularly in the vicinity of the catalytic triad and
the P1/P1(cid:2) pockets. Importantly, in contrast with OTUB1, the
Gelfiltrationandimmunoblotting
structureofOTUB2revealsamorecanonicalactivesite,where
Fractionation of crude cell extracts prepared from HEK-293T the catalytic His224 is within hydrogen-bonding distance of the
cellsexpressingcontrolvector,wild-typeorC91SmutantOTUB1 catalyticcysteineandthepK ofHis224 isenhancedbyhydrogen
a
was performed using size-exclusion chromatography with an bondswiththesidechainsofadjacentresidues,includingthose
HPLC system (Agilent HP1200). Sample protein concentration of Asn226 and Thr45 [14] (Figure 1B, middle panel). But, unlike
was determined using a Lowry assay (Bio-Rad Laboratories). OTUB1, the P1/P2 pocket of OTUB2 is partially encumbered
Samples of 300μl (2.5mg/ml) were loaded and separation by the presence of Ser223 from the β3–β4 loop. Differences in
was achieved using a Zorbax G450 gel-filtration column theOTUB1β4–β5andOTUB2β3–β4loopsuggestthatitmay
(9.6mm×250mm;Agilent)ataflowrateof0.7ml/mininlysis undergoconformationalchangesuponubiquitinbindingthatthen
buffer (0.5% NP-40, 150mM NaCl, 5mM CaCl and 50mM allowrearrangementofHis265forcatalyticcysteineactivation.
2
Tris/HCl,pH7.4).Fractionsof0.5or0.7mlwerecollected,and TheP(cid:2)-sideofOTUB1,wheretheleavinglysylgroupfroma
proteinmaterialwasprecipitatedusingchloroform/methanol[25], targetsubstrateorubiquitylgroupfromachainedsubstratewould
followed by SDS/PAGE-based separation and immunoblotting be expected to bind, is significantly different from OTUB2. In
using anti-FUS [fusion involved in t(12;6) in malignant lipo- addition to a dissimilar surface charge property in this region,
sarcoma; also known as TLS (translocation in liposarcoma) or theα2helixofOTUB2iskinked,afeaturethatisnotobserved
CHOP(CCAAT/enhancer-bindingproteinhomologousprotein)] in the OTUB1 structure (Figure 1C). An additional structural
(SantaCruzBiotechnology)oranti-OTUB1antibodies. restriction of the catalytic cleft in OTUB1 is the presence of
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Figure1 OTUB1crystalstructureandacomparisonbetweenOTUB2andyeastOTU1boundtoubiquitin-Br3
(A)MolecularsurfaceandoverlayingribbonmodelofOTUB1(residues40–271,left-handpanel),OTUB2(middlepanel)andyeastOTU1–ubiquitin-Br3(Ub)(right-handpanel,ubiquitinindark
grey,OTU1inblue).Theconservedaminoacidsexpectedtobepartofthecatalyticcentreareindicatedinred,blue,greenandpurplerespectively.(B)Astereoviewofthecatalyticsiteregionsof
OTUB1(toppanel),OTUB2(middlepanel)andyeastOTU1–ubiquitin(lowerpanel)illustrateshydrogen-bondingnetworksbetweenresidueswithinthecatalyticsite.Ubiq.,ubquitin.(C)Display
ofOTUB1(pink)andOTUB2(yellow)asribbonmodels,inwhichtheα-helicesandβ-sheetsarenumbered.(D)OverlaidribbonmodelsofOTUB1(pink)andyeastOTU1–ubiquitin-Br3(grey)
showingthecatalyticcentre.Catalyticsiteresiduesareindicatedasin(B),andTrp175(yeastOTU1,grey)andGlu214(OTUB1,pink)areshown.
Pro87 that is located in close proximity to Cys91 (Figure 1B). In ubiquitin C-terminal fusions, a trait that is not observed
the OTUB2 structure, this position is occupied by Gly47. The for OTUB2 [14]. A similar argument was made for explaining
presenceofamorebulkysidechaincouldpotentiallycontribute the specificity of the SUMO-specific protease SENP2 towards
to OTUB1’s cleavage preference for isopeptide bonds over lysinedeconjugation[26].
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Structuralinsightsintohumanotubain1 383
Figure2 OTUB1andOTUB2reactivitytowardsubiquitinprobeswithdifferentC-terminalchemicalgroups
(A)HA-taggedubiquitinequippedeitherwithaC-terminalBr2(HA–ubiquitin-Br2),Cl2(HA–ubiquitin-Cl2)orVMEmoiety(HA–ubiquitin-VME)wasusedtotestthefinespecificityofOTUB1and
OTUB2.(B)CrudeextractspreparedfromHEK-293TcellsoverexpressingOTUB1–HAorOTUB2–HA(C)wereincubatedwithHA–ubiquitin-Br2,HA–ubiquitin-Cl2orHA–ubiquitin-VME,analysed
bySDS/PAGEandanti-HAimmunoblotting.Ub,ubiquitin.
StructuralcomparisonofOTUB1andOTUB2withyeastOTU1bound ure2).OTUB2reactedwithallthreeprobesequallywell,whereas
toubiquitinindicatesconformationalchangesuponligandbinding OTUB1 reacted with the alkyl halides, but not the VME probe
(Figures2Band2C).TheseresultshintthatOTUB1andOTUB2
YeastOTU1hasbeencrystallizedrecentlyinacovalentcomplex
may have slightly different specificities, probably expressed
withaubiquitinC-terminalBr3derivative[15].Althoughthereis
by structural differences proximal to their catalytic centres.
minimalprimarysequencehomologybetweentheOTUdomains
ofyeastOTU1andhumanOTUB1(<15%identity),theyshare Furthermore,thissupportsthenotionthatOTUB1isautoinhibited
and that specific substrates, or interactors, are required for its
similarstructuralfeatures,andacomparisonmayprovideinter-
activation,probablythroughaninduced-fitmechanism.
esting insights into the conformational changes that occur upon
ubiquitinbinding(Figures1Aand1B).Inadditiontotheubiquitin
tail, which makes typical residue-specific peptide interactions OTUB1specificitytowardsubiquitinandUbls
withtheactivesite(classicalP1–P4substrateresiduesandthecor- In order to test the deubiquitination properties of OTUB1, we
repondingS1–S4proteasepockets),onefaceofthecoredomain examined its ability to cleave linear di-ubiquitin as well as
ofubiquitinisalsorecognizedbyOTU1.Thisareaoftheenzyme, Lys48- and Lys63-linked tetra-ubiquitin by immunoblotting and
herein referred to as the SD site [the subsite of the enzyme MS.OTUB1didnotcleavedi-ubiquitin,consistentwithprevious
that docks with the core globular domain of ubiquitin (residues observations[5](Figure3A).Atanenzyme/substrateratioof1:5,
1–70)],mediatesnumerousinteractions,includingthosecentred OTUB1 clearly showed a preference for cleaving Lys48-linked
onHis192 andIle221,andislikelytocontributetotheinduced-fit tetra-ubiquitin when assessed by anti-ubiquitin immunoblott-
mechanism of theenzyme, theKm of the ubiquityl substrate, or ing (Figure 3B). This preference was confirmed by MS-based
the Kd of the ubiquityl inhibitor. OTUB2 contains a discordant analysis(Figure3C).Theuseofahigherenzyme/substrateratio
loop(residues198–204)thatwouldprobablyclashwithubiquitin (1:1.5) in the latter analysis indicated further that OTUB1 may
binding. This region of OTUB1 forms a well-defined β-strand also slowly cleave Lys63-linked ubiquitin chains (Figure 3C).
(β4), extending the β-sheet lobe, but not interfering with the Ubiquitin-cleavage assays performed with a truncated form of
putativeubiquitin-bindingregion(Figure1C).Theflexiblenature OTUB1 lacking the first 40 amino acids did not affect the
ofasingleresidueintheβ3–β4loop(Arg238)inOTUB1hintsat proteolyticpropertiesofOTUB1(resultsnotshown),indicating
the possibility that OTUB1 might adopt a conformation similar thattheN-terminalregionofOTUB1doesnotinfluencecleavage
tothatofOTUB2duringaregulatorycycle,andthattheOTUB1 preferenceinvitro.
andOTUB2structuresmayrepresenttwoconformationalstates WeexaminedfurthertheabilityofOTUB1tocatalysegeneral
inaregulatorypathway.Alternatively,thesestructuraldifferences deubiquitinationreactionsbyexaminingcrudeextractsprepared
maysuggestdiverseubiquitin-bindingmodes. from HEK-293T cells overexpressing either wild-type or C91S
inactivemutantOTUB1.Weobservedonlyminoreffectsonthe
level of polyubiquitinated material in vitro and in living cells
Differentialproteolyticfinespecificitiesbetween
(Figure3D).Thisobservationisconsistentwithpreviousfindings
OTUB1andOTUB2
[5,6]andsuggeststhatOTUB1mayactonlyonadiscretesetof
To profile potential differences in substrate specificity between substrates.
OTUB1 and OTUB2, we subjected these enzymes to reaction In contrast with OTUB1, OTUB2 appears to readily cleave
with various ubiquitin-based active-site probes that have subtle Lys63-linked tetra-ubiquitin in preference to Lys48-linked tetra-
differences in their reactive electrophiles. Probes with a C- ubiquitin (Figure 4A). We therefore evaluated structural differ-
terminal Cl2, Br2 or VME group were incubated with enzyme encesbetweenOTUB1andOTUB2thatmightexplaintheirdif-
and analysed by electrophoresis and immunoblotting (Fig- ferentialreactivitytowardstheactive-siteprobeubiquitin–VME
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384 M.J.Edelmannandothers
Figure3 OTUB1hasapreferenceforcleavingLys48-linkedtetra-ubiquitinoverLys63-linkedtetra-ubiquitin
(A)RecombinantOTUB1orcellextractwasincubatedwithlineardi-ubiquitinfor1or15hat37◦C,separatedbySDS/PAGEandanalysedbyanti-ubiquitinimmunoblotting.(B)RecombinantOTUB1
wasincubatedwitheitherLys48-linkedtetra-ubiquitin(K48Ub;upperpanels)orLys63-linkedtetra-ubiquitin(K63Ub;lowerpanels),andcleavagewasmonitoredbySDS/PAGEandanti-ubiquitin
4 4
immunoblotting.Asacontrol,thetetra-ubiquitinsubstrateswereincubatedeitherwithcrudecellextractornoenzyme.MolecularmassesareindicatedinkDa.(C)Ub K48(upperpanels)or
4
Ub K63(lowerpanels)wereincubatedwithrecombinantOTUB1foreither2or18hfollowedbyTOF(time-of-flight)–MSanalysis.(D)EffectofOTUB1onpolyubiquitination.HEK-293Tcontrol
4
cellsortransfectedwithwild-typeOTUB1orC91SmutantweretreatedornotwiththeproteasomeinhibitorZLVS(50μMfinalconcentration)for4hat37◦C,andcrudeextractswereanalysed
3
bySDS/PAGEandanti-ubiquitinimmunoblotting(toppanel).Alternatively,crudecellextractspreparedfromHEK-293TcontrolorcellsexpressingOTUB1orC91Smutantwereincubatedwith
ZLVS(50μMfinalconcentration)for30minat37◦CandanalysedbySDS/PAGEandanti-ubiquitinimmunoblotting(α-UbIB).Asaloadingcontrol,anti-tubulinimmunoblottingwasperformed.
3
Ctrl,control;wt,wild-type.
andthedifferentubiquitinchains.Inparticular,wenotedthatthe We next examined the possibility that OTUB1 recognizes
P1(cid:2) siteofOTUB1wasstericallyrestrictedbythepresenceofa otherubiquitin-likemodifiers.Forthispurpose,anN-terminally
prolineresidueatposition87,whichisaglycineresidue(Gly47)in biotinylated seven-amino-acid peptide was conjugated to ubi-
thecorrespondingpositionintheOTUB2structure(Figure1B). quitin, NEDD8, ISG15 or SUMO1 in order to create substrates
OTUB1P87GandOTUB2G47Pmutantproteinsweretherefore with an isopeptide bond. These were incubated with either
generatedandtestedfortheirabilitytocleavedifferentubiquitin OTUB1 or a positive control (Figure 5A). Our results indicate
chains. Interestingly, we observed that the steric restriction thatOTUB1hasaclearpreferenceforubiquitinand,atalower
imposedbyaprolineresidueinthisareaoftheP1(cid:2)siteinOTUB2 rate, for NEDD8 conjugates. In addition, OTUB1 was unable
affected the ability of cleaving Lys63-linked tetra-ubiquitin, to cleave di-SUMO2 and di-SUMO3 substrates (Figure 5B).
whereas cleavage slightly improved upon the introduction of a Since these experiments are based on the use of a mono- or
glycineresidueatthispositioninOTUB1(Figure4A).Consistent di-ubiquitin/Ubl-basedsubstrate,thespecificitymaybedifferent
with this, we observed that cleavage of ubiquitin-AMC was forpolyubiquitin/Ubls,particularlyinlivingcellswhereOTUB1
also altered, although to a greater extent by the G47P mutation may be present in multiprotein complexes in which co-factors
in OTUB2 compared with the inverse modification in OTUB1 couldinfluenceitscleavagespecificity.
(Figure 4B). Combined, these results support the notion that
OTUB1 has a more restricted specificity, mainly towards Lys48-
linked ubiquitin chains, owing to a narrower P1(cid:2) site, and that InitialanalysisofintracellularOTUB1-interactingproteins
OTUB1 and OTUB2 may act on a different set of substrates, Asshownpreviously,OTUB1wasfoundtobeincomplexwith
althoughtheoverallstructureoftheseenzymesissimilar. GRAILandUSP(ubiquitin-specificpeptidase)8inlymphocytes
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Figure4 ModificationoftheP1(cid:2)siteaffectscleavageofLys63-andLys48-linkedtetra-ubiquitinbyOTUB1andOTUB2
(A)ModificationoftheP1(cid:2)siteaffectscleavageofLys63-andLys48-linkedtetra-ubiquitinbyOTUB1andOTUB2.Recombinantwild-typeOTUB1,P87GmutantOTUB1,wild-typeandG47Pmutant
OTUB2wereincubatedwitheitherLys48-orLys63-linkedtetra-ubiquitin(Ub-K48andUb-K63respectively)fortheindicatedtimeat37◦C.SampleswereseparatedbySDS/PAGEandanalysedby
4 4
anti-ubiquitinimmunoblotting.Alongerexposureoftheexperimentperformedwithwild-typeandP87GmutantOTUB1isshowninthelower-left-handpanel.Anti-histidineimmunoblotting(α-HIS)
indicatesequalloadingofaddedenzyme.MolecularmassesareindicatedinkDa.(B)EffectofP1(cid:2)modificationsonubiquitin-AMCcleavagebyOTUB1andOTUB2.Ubiquitin-AMC(100nM)was
incubatedwithwild-typeormutantG47POTUB2,UCH-L3orOTUB1attheindicatedconcentrations(left-handpanel).Cleavageofubiquitin-AMCwithwild-typeormutantP87GOTUB1wasobserved
athigherconcentrationsonly(right-handpanel).AMCcleavagewasmeasuredasafunctionoftimebyfluorescence(380nmexcitation/460nmemission).a.u.,arbitraryunits;Ub,ubiquitin.
[6] and can interact with the enteropathogen Yersinia encoded context, we performed proteomics-based studies using co-
virulence factor YpkA (YopO) [8]. In order to identify immunoprecipitation and identification by MS/MS. HEK-293T
potential OTUB1-interacting proteins in a non-haemopoietic cells were transiently transfected with either wild-type or
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386 M.J.Edelmannandothers
Figure5 OTUB1cleavesubiquitinandNEDD8conjugates,butnotSUMO1/2/3orISG15
(A)Ubiquitin,NEDD8,ISG15andSUMO1wereconjugatedtoabiotinylated7-merpeptideandincubatedwitheitherrecombinantOTUB1orcontrolsincludingUCH-L3,crudelysateorSENP2.
CleavagewasmonitoredbySDS/PAGEandstreptavidin–HRP(horseradishperoxidase)immunoblotting.(B)Di-SUMO2anddi-SUMO3conjugateswereincubatedwithrecombinantOTUB1orcell
lysateasacontrol,andthecleavagewasmonitoredattheindicatedtimes,followedbySDS/PAGEandanti-SUMOimmunoblotting.Thepositionofthe20kDabandisindicated.
catalytically inactive (C91S) OTUB1–HA. The inactive mutant OTUB1,andthereforeexaminedtheextentofpolyubiquitinated
was used as a ‘substrate trap’, preventing potential substrates FUS/TLS present in cells. Transfection experiments with HA-
from being turned over and released [27]. Cell extracts tagged ubiquitin, subsequent anti-HA immunoprecipitation and
were prepared after 24h, either with or without detergent anti-FUSimmunoblottingindicatedthatasmallfractionofFUS
solubilization, and subjected to anti-HA immunoprecipitation, isindeedubiquitinated(Figure7C).Combined,thesedatasuggest
followed by cleavage of bound material using TEV protease. thatthecatalyticactivityofOTUB1mayaffecttheubiquitination
This approach was used previously to reduce background stateofasmallsubsetofFUS/TLSprotein.
arising from non-specific protein binding to beads [28].
Analysis by gel electrophoresis, silver staining and LC–
DISCUSSION
MS/MS revealed a total of >80 proteins, of which 26 were
uniquely present when OTUB1–HA was expressed, but The structure of OTUB1 reveals a number of properties that
not detected when OTUB1–HA was absent (Figure 6 and addressactivationandsubstratespecificity.Inanalogytoautoin-
Supplementary Table S1 at http://www.BiochemJ.org/bj/418/ hibition observed in HAUSP (herpesvirus-associated ubiquitin-
bj4180379add.htm). The catalytically inactive OTUB1 mutant specific peptidase), USP8 and USP14 [32–34], where the
(C91S) showed a slightly faster migration profile, a feature unligandedactivesiteisblockedbysurfaceloops,theapoform
commonly observed with mutant proteins. We also detected ofOTUB1adoptsaconformationoftheactivesitethatappearsto
OTUB1 material that was of higher molecular mass, indicating beclosedandnotcompetentforcatalysis.Acomparisonbetween
thepresenceofpost-translationalmodifications. OTUB1andtheyeastOTU1–ubiquitin-Br3structuresuggestsfur-
Among the hits, two proteins, FUS/TLS and RACK1 [recep- therthataconformationalchangewouldactivatetheirrespective
tor for activated kinase 1; also known as GNB2L1 (guanine- catalytictriads(Figure1)[15].WeproposethatthetightOTUB1
nucleotide-binding protein β polypeptide 2-like 1)] were evalu- β4–β5loopimmediatelybeforeHis265 (Arg-Pro-Gly264)andthe
ated further with specific pull-down and follow-up biochemical α9–α10 loop, which influences the catalytic cysteine residue
experiments. Immunoprecipitation of OTUB1–HA followed by throughGlu214,couldplayarolebymediatingaconformational
anti-FUS/TLSandanti-RACK1immunoblottingconfirmedtheir change upon ubiquitin binding, allowing His265 to participate
presenceinOTUB1complexes,butnotincontrols(Figure7A). in the catalytic triad. However, analysis of the geometry of
Since FUS/TLS can exist in different forms [29] and also as the active sites revealed subtle variations between OTUB1 and
fusion proteins in the context of oncogenic transformations OTUB2, which were reflected by a differential reactivity with
[30,31], we examined the effect of OTUB1 on FUS/TLS alkyl halide and VME-based ubiquitin probes (Figure 2) [22],
stabilitybysize-exclusionchromatographyandimmunoblotting but also ubiquitin-AMC (Figure 4B). This may be explained
using an anti-FUS polyclonal antibody that detects multiple by subtle structural differences between the P1(cid:2) sides of those
molecular-mass forms of this protein (Figure 7B, left-hand enzymes.Inparticular,sterichindrancebythepresenceofPro87
panel). FUS/TLS appeared predominantly as high-molecular- in OTUB1’s catalytic centre may prevent the accommodation
mass species, but upon overexpression of OTUB1, a FUS/TLS of the AMC fluorescent group and the VME side chain of the
polypeptide of the expected molecular mass of ∼60kDa was probe,whereasthealkylhalidesareabletoreactwiththecatalytic
detected (Figure 7B, left-hand panel). Using a monoclonal cysteineresiduetoundergoanucleophilicsubstitutionreaction.
antibody that recognizes a C-terminal epitope of FUS/TLS, we Consistentwiththisnotion,theOTUB2structurecontainsGly47
observed a similar ∼60kDa form that appears to be stabilized at the equivalent position (Figure 1). Mutational analysis using
upon overexpression of OTUB1, but not in the presence of the recombinantOTUB1P87GandOTUB2G47Pmutantsconfirmed
mutantC91S(Figure7B,right-handpanel).Wenotedthatonlya that steric hindrance at this position compromises the ability
smallfractionofhigh-molecular-massFUS/TLSwasaffectedby to cleave ubiquitin-AMC and to react with the probes, but
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Figure6 MS/MS-basedscreenofOTUB1-containingproteincomplexes
(A)HEK-293TcontrolcellsorHEK-293TcellstransfectedwithOTUB1orC91SmutantwerelysedeitherusingglassbeadsorNP-40detergent-containingbuffer,followedbyananti-HA
immunoprecipitation.IsolatedmaterialwaselutedfrombeadsusingTEVproteaseandanalysedbySDS/PAGEandsilverstaining.ProteinbandspresentinOTUB1orC91Slanesandthe
correspondingregionsinthecontrolweresubjectedtoLC–MS/MSanalysis.Atotalof26outof>80proteinswerefoundtobepresentonlyinOTUB1/C91S-containinglanes.Molecularmassesare
indicatedinkDa.Ctrl,control;DDX,DEADboxpolypeptide;EBP2,EBNA1(Epstein–Barrvirusnuclearantigen1)-bindingprotein2;hc,heavychain;hnRNP,heterogeneousnuclearribonucleoprotein;
lc,lightchain;NPM,nucleophosmin;PCNA,proliferating-cellnuclearantigen;WT,wild-type.(B)MS/MSspectraofatrypticpeptidederivedfromRACK1(upperpanel)andatrypticpeptidederived
fromFUS/TLS(lowerpanel).
substitution of glycine for Pro87 in OTUB1 did not improve affectedtheabilityoftheseenzymestocleaveLys63-linkedtetra-
ubiquitin-AMCcleavage,neitherdiditrestorereactivitytowards ubiquitin in the opposite way (Figure 4). This result is fairly
theubiquitin–VMEprobe(Figure4Bandresultsnotshown).This surprising, since Lys63-linked polyubiquitin chains are in an
suggests that other structural constrains must exist, or that the extended conformation compared with Lys48-linked chains and
introductionofamutationinthispositionmayalsoleadtomore therefore would be expected to be less susceptible to structural
complex rearrangements of the P1(cid:2) site. Reactivity of OTUB1 constraintsintheP1(cid:2)pocket[35].Recentstructuralinformationas
towardstheubiquitin-Cl2probewasobservedinthepresentstudy, wellaspredictionssuggestthattheP1(cid:2)siteharbourstheisopeptide
butwasnotdetectedinapreviousstudy[22].Thismightbedue bondlysinesidechain,andthatthenatureofthebindingpocket
to overexpression of OTUB1 in the labelling assay (Figures 2B fordistalubiquitinispartiallyresponsiblefortheaffinitytowards
and2C),whereas,atphysiologicallevels,otherDUBspresentin differentubiquitinchains[36].Ourresultsprovideinsightsinto
cellextractsmayeffectivelycompeteforbindingtotheubiquitin– theroleofnon-catalyticsidechainsthatcaninfluenceproteolysis
chlorideprobe.Differentstructuralfeaturesanddiscrepanciesin ofdifferentubiquitinchains.However,otherstructuralelements,
the reactivity towards the active site ubiquitin probes between such as a kinked α2 helix present in OTUB2, but not OTUB1,
OTUB1 and OTUB2 may hint towards a divergence in their maybenecessarytodeterminelinkagepreference.
cleavageorsubstratespecificities.Thishypothesiswasconfirmed The oxyanion hole in OTUB1 appears to be formed by the
by the finding that OTUB1 has a preference for cleaving Lys48- backbone amide of the catalytic Cys91 and the backbone amide
linked tetra-ubiquitin over Lys63-linked tetra-ubiquitin, whereas of Asp88. This is very similar to the oxyanion hole geometry
the opposite appears to be the case for OTUB2. We observed observed in OTUB2, A20 and yeast OTU1, thereby confirming
that full-length OTUB2 retained cleavage activity which was that this may be an element specifically observed in OTUs, but
unchanged when deleting the C-terminal amino acids 229–234 notothercysteineproteasefamilies[12,14,15].
compared with a previous study ([5] and M. Akutsu and With the exception of virus-encoded OTU variants that have
S.Dhe-Paganon,unpublishedwork).ModificationoftheP1(cid:2)site acquiredanadditionalspecificityforISG15[3],ovarian-tumour-
in OTUB1 by a P87G and in OTUB2 by a G47P substitution domain-containing DUBs have a preference for ubiquitinyl
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388 M.J.Edelmannandothers
Figure7 OTUB1isincomplexwithRACK1andFUS/TLS
(A)OTUB1wasimmunoprecipitatedfromHEK-293TcontrolcellsorcellstransfectedwithOTUB1,followedbyanti-RACK1andanti-FUS/TLSimmunoblotting.(B)OTUB1affectsthemolecular-mass
distributionofFUS/TLS.EqualamountsofcellextractsderivedfromcontrolorHEK-293Tcellsexpressingwild-typeorC91SmutantOTUB1wereseparatedbysize-exclusionchromatography.
IndividualfractionswereanalysedbySDS/PAGEandimmunoblottingusingananti-OTUB1andapolyclonalanti-FUS/TLSantibodythatrecognizesmultipleforms(left-handpanel),oramonoclonal
anti-FUS/TLSantibodythatrecognizesa∼60kDaform(right-handpanel).Notethatthefractionnumbersarenotcomparablebetweentheleft-andright-handpanels.Thearrowindicatesthe
observed∼60kDaformofFUS/TLS.Oneoutofthreeindependentexperimentsisshown.MolecularmassesareindicatedinkDa.(C)HEK-293Tcellsweretransfectedwitheitheremptyvectoror
HA–ubiquitin.Cellextractswerepreparedafter24handsubjectedtoanti-HAimmunoprecipitation,followedbySDS/PAGEseparationandanalysisbyanti-HAandanti-FUSimmunoblotting.Ctrl,
control;MW,molecularmass(indicatedinkDa).
derivatives.Ourdataconfirmthisfindingandindicatefurtherthat validatedbyco-immunoprecipitationexperiments.FUS/TLShas
OTUB1mayhavesomereactivitytowardsNEDD8.Thisapparent been described as an RNA-splicing factor involved in chromo-
dualspecificityhasalsobeenobservedforotherDUBs,suchas somalaberrationsobservedinEwingtumours,erythroleukaemia,
UCH-L1, UCH-L3 [37,38], USP21 and the COP9 signalosome acute myelocytic/lymphoblastic leukaemia and fibrosarcoma
[39,40]. As observed with the OTU domains of A20 and yeast [42–44], and it was shown to be present in protein networks
OTU1, OTUB1 has a preference for Lys48-linked polyubiquitin containing CIP29 (cytokine-induced protein of 29kDa) and
overLys63-linkedpolyubiquitin(Figure3)[15],whereasOTUB2 DDX39 (DEAD box polypeptide 39) [45]. The major form of
prefers the latter (Figure 4). However, as observed for A20, the ∼60kDacanberecognizedwithananti-FUS/TLSmonoclonal
in vitro result may not necessarily reflect biological behaviour antibody (Figures 7A and 7B) [46]. Additional high-molecular-
in living cells, a trait that remains to be examined for OTUB1- massformscanalsobedetectedusingapolyclonalanti-FUS/TLS
specificsubstrates. antibody (Figure 7B). OTUB1, when overexpressed in HEK-
Asasteptowardsaddressingthisissue,proteomics-andMS- 293T cells, has an effect on the molecular size of a subset of
based screens for OTUB1 interactors and substrates identified FUS/TLS-containing material (Figure 7B). This appears to be
anumberofcandidates(seeSupplementaryTableS1).Wenoteda dependentonthecatalyticactivityofOTUB1,suggestingthata
considerableamountofco-immunoprecipitatingnuclearproteins, functionalOTUB1enzymeisrequiredforthisprocess.Usingco-
suchascellgrowthnucleolarproteins,DNAtopoisomerasesand transfectionexperimentswithHA-taggedubiquitin,wewereable
severalRNAhelicasessharingaDEAD(Asp-Glu-Ala-Asp)box toobservethatasmallfractionoftheFUS/TLS-containinghigh-
motif (Figure 6 and Supplementary Table S1). OTUB1 does molecular-massmaterialisubiquitinated(Figure7C).Alteration
containapredictedNLS(nuclearlocalizationsequence)(residues ofasmallsubsetdidnotleadtoanysignificantchangesintotal
69–85), and initial microscopy studies suggest that OTUB1 FUS/TLSproteinlevels,althoughthe∼60kDaformofFUS/TLS
subsets are present in the nucleus ([41] and A. Ipho¨fer, M. J. appears to be present in more chromatography fractions when
Edelmann and B. M. Kessler, unpublished work). On the other wild-typeOTUB1wasoverexpressed(Figure7B,left-andright-
hand,RNA-bindingproteinsareabundantandcommonlyfound hand panels). Attempts to address whether FUS/TLS may be a
ascontaminantsinMS-basedpull-downassays[22],althoughall direct substrate of OTUB1 turned out to be challenging, in part
proteins listed were not detectable in controls without the bait due to the fact that FUS/TLS may exist in multiple forms, and
protein(Figure6).Amongthehits,FUS/TLSandRACK1were thatamajorfractionofhigh-molecular-massmaterialreactiveto
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