Structure of the Absent in Melanoma 2 (AIM2) Pyrin Domain Provides Insights into the Mechanisms ...

12 Pages·2013·3.68 MB·English

THEJOURNALOFBIOLOGICALCHEMISTRY VOL.288,NO.19,pp.13225–13235,May10,2013
PublishedintheU.S.A.
Structure of the Absent in Melanoma 2 (AIM2) Pyrin Domain
Provides Insights into the Mechanisms of AIM2
Autoinhibition and Inflammasome Assembly*□S
Receivedforpublication,March8,2013Published,JBCPapersinPress,March25,2013,DOI10.1074/jbc.M113.468033
TengchuanJin,AndrewPerry,PatrickSmith,JianshengJiang,andT.SamXiao1
FromtheStructuralImmunobiologyUnit,LaboratoryofImmunology,NIAID,NationalInstitutesofHealth,
Bethesda,Maryland20892-0430
Background:AIM2bindsdsDNAandassociateswithASCthroughtheirPYDstoformaninflammasome.
Results:TheAIM2PYDstructureillustratesdistinctchargedistributionandauniquehydrophobicpatch.
Conclusion:TheAIM2PYDmaybindtheASCPYDandtheAIM2HINdomainthroughoverlappingsurface.
Significance:ThesefindingsprovideinsightsintothemechanismsofAIM2autoinhibitionandinflammasomeassembly.
Absentinmelanoma2(AIM2)isacytosolicdouble-stranded vate proinflammatory cytokine processing (1–4). The AIM2
(dsDNA)sensoressentialforinnateimmuneresponsesagainst inflammasomeisessentialforimmuneresponsesagainstDNA
DNAvirusesandbacteriasuchasFrancisellaandListeria.Upon virusessuchasvacciniavirusandmousecytomegalovirusand
dsDNA engagement, the AIM2 amino-terminal pyrin domain bacteriasuchasFrancisellatularensisandListeriamonocyto-
(PYD) is responsible for downstream signaling to the adapter genes(5–9).AIM2wasalsoreportedtoplayaroleinautoim-
protein apoptosis-associated speck-like protein containing a munedisorderssuchaspsoriasisthroughrecognitionofhost
caspase recruitment domain (ASC) through homotypic PYD- DNA (10). It belongs to the pyrin and HIN protein (PYHIN)
PYD interactions and the assembly of an inflammasome. familyofproteinsthatincludesinterferon(cid:1)-inducibleprotein
TowardabetterunderstandingoftheAIM2signalingmecha- 16 (11), IFN-inducible protein X/PYHIN1, and myeloid cell
nism,wedeterminedthecrystalstructureofthehumanAIM2 nucleardifferentiationantigeninthehumangenome(12,13).
PYD.Thestructurerevealsadeathdomainfoldwithashort(cid:1)3 AIM2 contains a carboxyl-terminal HIN domain that binds
helixthatisbuttressedbyahighlyconservedlysineresidueat dsDNA and an amino-terminal PYD that is responsible for
the(cid:1)2helix,whichmaystabilizethe(cid:1)3helixforpotentialinter- downstream signaling to the adapter protein ASC. Previous
action with partner domains. The surface of the AIM2 PYD phylogeneticanalysisofthePYDsequencesrevealedtwosepa-
exhibits distinct charge distribution with highly acidic (cid:1)1-(cid:1)2 ratecladesamongthemammalianPYHINproteins,onecon-
helices and highly basic (cid:1)5-(cid:1)6 helices. A prominent solvent- tainingtheAIM2orthologsandtheothercontainingallother
exposed hydrophobic patch formed by residues Phe-27 and PYHINproteins(14).Inagreementwithitsdistinctsequence,
Phe-28atthe(cid:1)2helixresemblesasimilarsurfaceinvolvedinthe the AIM2 PYD was shown to be the only PYHIN PYD that
deatheffectordomainhomotypicinteractions.Dockingstudies associateswithASCtoformaninflammasome(1,2).However,
suggestthattheAIM2PYDmaybindtheAIM2hematopoietic itremainsunclearhowthedistinctsequenceoftheAIM2PYD
interferon-induciblenuclear(HIN)domainorASCPYDusing contributestothisuniquefunction.
overlapping surface near the (cid:1)2 helix. This may ensure that The pyrin domains were found in both PYHIN proteins
AIM2 interacts with the downstream adapter ASC only upon and Nucleotide-binding oligomerization domain (NOD)-like
release of the autoinhibition by the dsDNA ligand. Our work receptors(NLRs)assignaltransductionmodulesthatadoptthe
thusunveilsnovelstructuralfeaturesoftheAIM2PYDandpro- six-helix bundle fold typical of the death domain superfamily
videsinsightsintothepotentialmechanismsofthePYD-HIN (15–19).BesidesPYD,thedeathdomainsuperfamilyincludes
andPYD-PYDinteractionsimportantforAIM2autoinhibition the death domain (DD), death effector domain (DED), and
andinflammasomeassembly. caspase recruitment domain (CARD). Many of them are
involvedintheassemblyofoligomericmultiproteinsignaling
complexes such as the PIDDosome (DD) (20), MyDDosome
AIM22 is a major cytosolic dsDNA sensor that forms an (DD)(21),apoptosomes(CARD)(22,23),andinflammasomes
inflammasomewiththeadapterASCandprocaspase-1toacti- (PYD)(24).Despiteprogressinthestructuralcharacterization
oftheDD,DED,andCARDsignalingcomplexes,themolecular
*Thisworkwassupported,inwholeorinpart,byaNationalInstitutesof mechanismsofPYD-mediatedsignalingeventsremainpoorly
HealthgrantfromtheDivisionofIntramuralResearch,NIAID(toT.S.X.).
□S ThisarticlecontainssupplementalFigs.1–4. understoodlargelybecauseofthelackofstructuralinformation
Theatomiccoordinatesandstructurefactors(code3VD8)havebeendepositedin
theProteinDataBank(http://wwpdb.org/).
1To whom correspondence should be addressed. E-mail: xiaot@niaid. nuclear;PYHIN,pyrinandhematopoieticinterferon-induciblenuclear;NLR,
nih.gov. NOD-likereceptor;NOD,nucleotide-bindingoligomerizationdomain;DD,
2The abbreviations used are: AIM2, absent in melanoma 2; PYD, pyrin deathdomain;DED,deatheffectordomain;MBP,maltose-bindingprotein;
domain;ASC,apoptosis-associatedspeck-likeproteincontainingaCARD; ITC,isothermaltitrationcalorimetry;POP,PYD-onlyprotein;FADD,Fas-
CARD,caspaserecruitmentdomain;HIN,hematopoieticinterferon-inducible associatedviadeathdomain.
MAY10,2013•VOLUME288•NUMBER19 JOURNALOFBIOLOGICALCHEMISTRY 13225
This is an Open Access article under the CC BY license.
TheStructureoftheAIM2PyrinDomain
on PYD-PYD complexes. To date, the structures of eight minal GB1 tag. Expression and purification were carried out
humanPYDsandtwomousePYDshavebeenexperimentally similarly to the AIM2 PYD except that the GB1 tag was
characterized, largely through nuclear magnetic resonance removedbytobaccoetchviruscleavage.
(NMR) spectroscopy. These include the PYDs from human Crystallization—Purified MBP-PYD protein was concen-
ASC (Protein Data Bank codes 1UCP (25) and 2KN6 (26)), tratedto20mg/mlbeforesettinguphangingdropsforvapor
NLRP1(ProteinDataBankcode1PN5)(27),NLRP3(Protein diffusion crystallization. Multiple commercial crystallization
DataBankcode3QF2)(28),NLRP4(ProteinDataBankcode screens were tested using the Mosquito crystallization robot
4EWI) (29), NLRP7 (Protein Data Bank code 2KM6) (30), (TTPLabtech,UK).Rod-shapedcrystalsgrewatroomtemper-
NLRP12(ProteinDataBankcode2L6A)(31),andPOP1/ASC2 aturein1weekusingawellsolutioncontaining20%polyethyl-
(ProteinDataBankcode2HM2)(32)andmouseNLRP10(Pro- eneglycol(PEG)3350and0.1MpotassiumacetateatpH4.0.
teinDataBankcode2DO9)aswellasthePYHINfamilymem- 20%ethyleneglycol(v/v)wasaddedtothereservoirsolutionas
bersmyeloidcellnucleardifferentiationantigen(ProteinData thecryoprotectanttofreezethecrystalsinliquidnitrogenfor
Bankcode2DBG;human)andp205b(ProteinDataBankcode x-raydiffractiondatacollection.
2YU0;mouse).Interestingly,allknownPYDstructureshavea X-rayDiffraction,StructureDetermination,andRefinement—
uniquelyshort(cid:2)3helixcomparedwithotherDDsuperfamily X-ray diffraction data were collected at the General Medical
members(33). Sciences and Cancer Institutes Collaborative Access Team
Previously, we reported the crystal structure of the AIM2 (GM/CA-CAT) of the Advanced Photon Source, Argonne
HINdomainincomplexwithdsDNAandfoundthattheHIN NationalLaboratory.DatawereprocessedwiththeHKL2000
domainbindsdsDNAthroughelectrostaticattraction,andthe programsuite(37).Thesolventcontentwas53.2%foronemol-
AIM2PYDandHINdomaininteractionmaintainsthereceptor eculeofthefusionproteinperasymmetricunit.Thestructure
in an autoinhibited state in the absence of dsDNA (34). To was determined by molecular replacement with Phaser (38)
examinethestructureandfunctionoftheAIM2PYD,wecar- fromtheCCP4programsuite(39).AnMBPstructurefromthe
riedoutcrystallographicstudiesoftheAIM2PYD.TheAIM2 ProteinDataBank(code3DM0)wasusedasthesearchmodel
PYDwascrystallizedasafusionwithmaltose-bindingprotein (35). Electron density maps calculated with phases from the
(MBP).Thestructurerevealsatypicaldeathdomainfoldforthe MBP search model clearly showed positive densities for the
PYD with distinct surface charges and hydrophobic patches. AIM2PYD.ThePYDmodelwasmanuallybuiltwithCoot(40)
We identify a highly conserved lysine residue at the (cid:2)2 helix and refined with Phenix.refine (41). The final structure con-
that stabilizes the short (cid:2)3 helix, a common feature for all tains467residuesofwhichresiduesMet-371toLys-467corre-
knownPYDstructuresthathasnotbeendescribedpreviously. spond to the first 97 residues of the AIM2 receptor (NCBI
Ourdockingandbindingstudiessuggestpotentialmodesofthe accessionnumberNP_004824).ValidationbytheMolProbity
PYD-PYD and PYD-HIN associations through overlapping server(42)andResearchCollaboratoryforStructuralBioinfor-
surfaceattheAIM2PYDsuchthattheAIM2receptorsignal matics ADIT validation server (43) showed that 97.5% of all
transductiononlyoccursuponligandengagement. proteinresidueswereinthefavoredregionsoftheRamachan-
dran plot with no outliers. Electrostatic surfaces were calcu-
EXPERIMENTALPROCEDURES
latedwithprogramDelphi(v4)(44)anddisplayedwithPyMOL
Protein Expression and Purification—The pyrin domain of (Schrödinger,LLC).Calculationofthesolvent-accessiblearea
human AIM2 (NCBI accession number NP_004824; residues wasperformedwiththeprogramAreaimolfromtheCCP4pro-
1–107) was cloned into a pET30a-derived vector with a non- gramsuite(39,45).
cleavable amino-terminal MBP tag and a carboxyl-terminal SequenceAlignmentandCalculationofConservationScores—
hexahistidine tag. The MBP tag harbors mutations (D82A/ SequencealignmentofthePYDswasperformedwiththepro-
K83A/E172A/N173A/K239A)designedtoenhanceitscrystal- gram MEGA5 (46) with minor adjustments. The phylogenic
lization propensity (35, 36). Transformed BL21(DE3) Codon treewascalculatedusingthemaximumlikelihoodmethodin
Plus RIPL cells (Stratagene, Santa Clara, CA) were grown at MEGA5, and the reliability of the tree was tested with boot-
37°C, induced with 0.3 mM isopropyl 1-thio-(cid:3)-D-galactopy- strapping for 1000 replications. The sequence conservation
ranosideat18°Cfor4h,harvested,andlysedinbufferA(20mM scores were calculated by the ConSurf server (47) using the
Tris-HCl,pH8.0,100mMNaCl)plus5mMimidazole,DNase sequencealignmentinFig.1.
(Biomatik, Wilmington, DE), and protease inhibitors (Roche ProteinStructureDocking—DockingoftheAIM2PYD(Pro-
AppliedScience).Solubleproteinfromthecelllysatewaspuri- teinDataBankcode3VD8)withtheASCPYD(ProteinData
fied by Hisprep IMAC column (GE Healthcare) and further Bankcode1UCP)ortheAIM2HINdomain(ProteinDataBank
purifiedusingaXK26/60Superdex200sizeexclusioncolumn code3RN2)wasperformedwiththewebserverClusPro(48),
inbufferAsupplementedwith5mMmaltose(ResearchProd- takingintoaccountbothshapecomplementarityandelectro-
ucts International Corp., Mount Prospect, IL). The mutant staticchargeinteractions.Aninteractivedockingprogram,Hex
AIM2PYDconstructswereproducedusingthePhusionsite- (v6.3)(49),wasalsousedforcomparison.Nopreidentification
directed mutagenesis kit (Thermo Scientific, Waltham, MA), ofresidueswithinoroutsidethecontactsurfacewasspecified.
andtheexpressionandpurificationwerecarriedessentiallythe Thechoiceofeitherdomainasthestationaryreceptorresulted
sameasforthewildtypeprotein.ThehumanASCPYD(resi- inasimilarmodeofinteractionsfortheAIM2PYD(seeFigs.5
dues1–91)codingsequencewasclonedintoapET30a-derived and6):thetopdockingmodelsconsistentlyplacedthe(cid:2)2helix
vectorwithatobaccoetchvirusproteasecleavableamino-ter- of the AIM2 PYD at the PYD-PYD and PYD-HIN interfaces.
13226 JOURNALOFBIOLOGICALCHEMISTRY VOLUME288•NUMBER19•MAY10,2013
TheStructureoftheAIM2PyrinDomain
FIGURE1.SequencealignmentofthehumanPYDs.Thesix(cid:2)helicesofthePYDswithknownstructuresareunderlinedandmarked.Thehighlyconserved
residues((cid:2)80%)areshadedinyellow.Theconservedlysineresidueatthe(cid:2)2helixisindicatedwithamagentadiamond.Theacidicresiduesatthe(cid:2)1,(cid:2)2,and
(cid:2)6helicesoftheAIM2PYDareshadedinred,thebasicresiduesatthe(cid:2)5and(cid:2)6helicesareshadedinblue,thehydrophobicresiduesatthe(cid:2)2-(cid:2)3helicesare
shadedingreen.Similarly,theacidicresiduesatthe(cid:2)1and(cid:2)4helicesandthebasicresiduesatthe(cid:2)2and(cid:2)3helicesoftheASCPYDareshadedredandblue,
respectively.ConservedresiduesamongthePYHIN-onlyproteinsaremarkedwithorangeboxes.TheAIM2-specificresidueswithinthePYHINproteinsare
markedwithblackboxes.
ThetoprankeddockingmodelforthePYD-HINcomplexwas theinteractionbetweentheAIM2PYDandtheASCPYD.The
subjectedtoenergyminimizationusingtherelaxmodeofthe PYDcodingsequenceswereclonedintothepGBKT7plasmid
Rosetta(v3.4)program(50). encoding DNA-binding domain and the pGADT7 plasmid
MBPPulldown—PurifiedMBPorMBP-taggedwildtypeor encoding activation domain. Combinations of DNA-binding
mutantAIM2PYDsamples(200(cid:4)g)wereimmobilizedwith50 domainandactivationdomainplasmidpairsweretransformed
(cid:4)l of amylose beads (New England Biolabs, Ipswich, MA) in intoyeaststrainAH109.Thecellswereplatedonagarplatesof
bufferB(20mMHEPES-Na,pH7.4,100mMNaCl).Afterthree minimumsyntheticdropoutmediumwithoutleucineandtryp-
washingstepsusingbufferB,200(cid:4)gofAIM2HINproteinwas tophan((cid:1)Leu/(cid:1)Trp)toselectfortransformants.Singlecolo-
incubatedwiththebeadsfollowedbythreewashingstepswith nieswerethenpickedandreplicatedonto(cid:1)His/(cid:1)Leu/(cid:1)Trp
bufferB.Theboundproteinswereelutedwith50(cid:4)lofbufferB plates.Growthofthecoloniesat30°Cwasrecorded72hlater.
plus10mMmaltoseandanalyzedbySDS-PAGE. Isothermal Titration Calorimetry (ITC)—The association
Yeast Two-hybrid Assay—The GAL4-based Matchmaker betweentheAIM2PYDandHINdomainwasmeasuredusingan
yeast two-hybrid system from Clontech was used to examine ITC200calorimeter(Microcal,Piscataway,NJ)at288K.Allpro-
MAY10,2013•VOLUME288•NUMBER19 JOURNALOFBIOLOGICALCHEMISTRY 13227
TheStructureoftheAIM2PyrinDomain
teinsamplesweredialyzedextensivelyinbufferB.Thewildtypeor TABLE1
mutantMBP-AIM2PYDat0.3mMwastitratedinto(cid:3)200(cid:4)lof X-raydiffractiondatacollectionandstructuralrefinementstatistics
purifiedAIM2HINdomainat30(cid:4)M.Thebindingisothermdata r.m.s.d.,rootmeansquaredeviation.
wereanalyzedwiththeprogramOriginusingasinglesitebinding Unitcell
a,b,c(Å) 61.6,91.9,100.3
modelaftersubtractingthebufferdilutionbackground. (cid:2),(cid:3),(cid:1)(°) 90,90,90
FluorescencePolarizationAssay—A5(cid:4),6-fluorescein-labeled Resolution(Å) 50-2.07(2.10-2.07)a
20-mer DNA oligo with a sequence of 5(cid:4)-ccatcaaagagagaaag- NReod.uonfdreafnleccytions(total/unique) 274.05(,866.05)/a35,164
agc-3(cid:4)(IntegratedDNATechnologies,Coralville,IA)wasdis- Completeness(%) 99.0(97.9)a
I/(cid:5)(I) 12.4(2.2)a
solved in buffer B and annealed with its reverse complement R (%)b 11.9(64.6)a
merge
DNAoligo.A4nMconcentrationoftheprobewasmixedwith Rpim(%)c 4.9(28.9)a
100nMpurifiedAIM2HINdomain,andincreasingconcentra- Refinement
Resolution(Å) 50-2.07
tionsofthewildtypeormutantMBP-AIM2PYDwereaddedto
No.ofproteinatoms 3,688
the above DNA-HIN complex. The mixtures were then ali- No.ofsolvent/heteroatoms 390
r.m.s.d.bondlengths(Å) 0.003
quotedintripletsintoblack96-wellplates,andthefluorescence
r.m.s.d.bondangles(°) 0.694
polarization was measured with a Paradigm spectrometer R (%)d 18.21
work
R (%)e 22.12
(MolecularDevices,Sunnyvale,CA).Datawereanalyzedusing free
Ramachandranplotfavored/disallowedf 97.5/0.0
theprogramPrism(GraphPad,SanDiego,CA). ProteinDataBankcode 3VD8
aNumbersinparenthesescorrespondtothelastresolutionshell.
RESULTS bR (cid:5)(cid:6)(cid:6)(cid:1)I(h)(cid:1)I(h)(cid:1)/(cid:6)(cid:6)I(h)whereI(h)andI(h)aretheithandmean
merge h i i h ii i
measurementoftheintensityofreflectionh.
SequenceComparisonoftheHumanPYDs—Thereareatotal cRpim(cid:5)(cid:6)h[(1/n(cid:1)1)1⁄2](cid:6)i(cid:1)Ii(h)(cid:1)I(h)(cid:1)/(cid:6)h(cid:6)iIi(h)whereIi(h)andI(h)aretheith
of 22 PYD-containing proteins in the human genome (13), andmeanmeasurementoftheintensityofreflectionh,andnistheredundancy
ofreflectionh.
including the PYHIN proteins, the NLR family members dR (cid:5)(cid:6)(cid:2)F (h)(cid:1)(cid:1)(cid:1)F (h)(cid:2)/(cid:6)(cid:1)F (h)(cid:1)whereF (h)andF (h)arethe
NLRP1–14, the pyrin protein encoded by the Mediterranean owbosrekrvedahndobcsalculatedcsatlrcucturehfacotbosrs,respectivoeblys.NoI/(cid:5)ccaultcoffwas
applied.
fever gene, the adapter protein ASC, and PYD-only proteins eR istheRvalueobtainedforatestsetofreflectionsconsistingofarandomly
free
(POPs)POP1/ASC2andPOP2.Asequencealignmentofthese selected5%subsetofthedatasetexcludedfromrefinement.
fValuesfromMolProbityserver.
PYDsguidedbytheknownstructuresrevealsthatseveralcon-
servedresiduesaresharedbetweenthePYHINPYDsandother maystabilizetherelativepositionsoftheMBPandPYD,thus
PYDs(Fig.1).Theseresiduesaremostlyhydrophobicwiththe facilitatingcrystallization(supplementalFig.2B).
exception of Lys-26 at the (cid:2)2 helix (see below). Divergent TheAIM2PYDiswellresolvedintheelectrondensitymaps
sequencesoutsideoftheseconservedresiduesplacethePYHIN (supplementalFig.2C).Ithasaglobularstructureofasix-helix
proteinsataseparatecladeinaphylogenetictree(supplemen- bundle similar to other members of the death domain super-
talFig.1).Forexample,thePYHINPYDscontainshorter(cid:2)2–3 family(Figs.1and2A).TypicaloftheknownPYDstructures,
loops compared with the other PYDs, and their amino- and the AIM2 PYD has a short (cid:2)3 helix (33). This helix and the
carboxyl-terminal regions harbor conserved residues among connectingloopspossesshighertemperaturefactorscompared
thePYHINproteinsonly(Fig.1,orangeboxes).Furthermore, with the bulk of the domain, suggesting higher static or
the AIM2 PYD sequence further diverges from those of the dynamicmobility(Fig.2B).Asearchofstructuralhomologues
otherPYHINproteinswiththeAIM2-specificresidueslocated usingtheDaliserver(52)revealedthattheAIM2PYDismost
throughoutthePYDsequence(Fig.1,blackboxes).Notably,the similartothatfromASCandNLRP3withZ-scoresof12.4and
AIM2-specificresiduesatthe(cid:2)2helixcontributetothedistinct 12.3,respectively(Table2).SuperpositionofthesethreePYD
patternofsurfacechargeandhydrophobicitypatchatthePYD structures illustrates that the six helices from each can be
as described below. To understand the contribution of these superimposedwellwithvariationsintheorientationandlength
AIM2-specificresiduestoitsdistinctstructureandinturnits ofthehelices(Fig.2,CandD).Inparticular,the(cid:2)6helixofthe
uniqueabilitytoassociatewiththeadapterASC,weinitiated AIM2 PYD is the longest among the known PYD structures
structuralstudiesoftheAIM2PYD. withthemostvariablesequences(Figs.1and2,AandC).The
The AIM2 PYD Adopts a Six-helix Bundle Structure—Our functionalsignificanceofthislong(cid:2)6helixiscurrentlyunclear.
initialeffortstocrystallizetheAIM2PYDwerehamperedbyits The Highly Conserved Lys-26 Residue Stabilizes the (cid:2)3
tendencytoformsevereaggregatesuponoverexpression,per- Helix—Amongthedeathdomainsuperfamilymembers,PYDs
hapspartiallyreflectingitsfunctionasaprotein-proteininter- haveuniquelyshort(cid:2)3helicesconnectedtoflexibleloops(33).
action module. We overcame this challenge using an MBP Wethereforewonderedhowtheconformationofthe(cid:2)3helices
fusion strategy reported previously to be successful for other is stabilized. We noticed that the (cid:2)2 helices of PYDs contain
recalcitrantcrystallizationtargets(35,36,51).TheMBP-PYD highlyconservedlysineresidues(Figs.1and2A).Thesidechain
fusion protein was expressed, purified to high homogeneity, ofthisresidueintheAIM2PYDpointstowardthe(cid:2)3helix(Fig.
andcrystallized.Crystallographicdatacollection,modelbuild- 2A),resultinginexposureofonly8%ofitssurfacetothesol-
ing, and refinement statistics are presented in Table 1. One vent.ThepoorsurfaceexposuresuggeststhatLys-26maynot
MBP-PYD fusion protein is present in the crystallographic beinvolvedindomain-domaininteractions.Whatthenisthe
asymmetricunit(supplementalFig.2A).Therearefourhydro- functionofthisconservedlysineresidue?AnalysisofthePYD
genbondsand10vanderWaalscontactsbetweenthe(cid:2)1and structures from AIM2 (Protein Data Bank code 3VD8), ASC
(cid:2)6helicesoftheAIM2PYDandtheMBP-linkerregion,which (Protein Data Bank code 1UCP), NLRP3 (Protein Data Bank
13228 JOURNALOFBIOLOGICALCHEMISTRY VOLUME288•NUMBER19•MAY10,2013
TheStructureoftheAIM2PyrinDomain
FIGURE2.OverallstructureoftheAIMPYD.A,ribbondiagramoftheAIM2PYDstructurecoloredbasedonsequenceconservationscorescalculatedbythe
ConSurfserver(47).Thecolorisfromblue(conserved)tored(non-conserved).ResiduesLys-26,Phe-27,andPhe-28atthe(cid:2)2helixandLys-87,Lys-90,Lys-93,
andLys-97atthe(cid:2)6helixareshownasball-and-stickmodels.Thesix(cid:2)helicesandbothterminiarelabeled.B,aplotofthetemperaturefactorsfortheAIM2
PYDresidues.Theaveragetemperaturefactorofthedomainisindicatedasareddottedline,andthesix(cid:2)helicesaremarkedabovetheplot.C,superposition
ofthePYDstructuresfromAIM2(orange),ASC(green),andNLRP3(cyan)witheachhelixrepresentedasacylinder.D,a90°rotationoftheviewinC.
TABLE2 staticcharges(Fig.4,A–C)similartootherknownPYDstruc-
ComparisonoftheAIM2PYDstructurewithotherPYDstructuresby tures.However,thepatternofchargedistributionisdistinctfor
theDaliserver theAIM2PYDwithdominantacidicresiduesatthe(cid:2)1-(cid:2)2heli-
ProteinDataBankcodesareinparentheses.r.m.s.d.,rootmeansquaredeviation;
MNDA,myeloidcellnucleardifferentiationantigen. cesincludingresiduesGlu-7,Asp-15,Asp-19,Glu-20,Glu-21,
Aligned andAsp-23(Figs.1and4,AandB)andprimarilybasicresidues
Z-score r.m.s.d. residues Identity Lys-64,Arg-67,Lys-71,Lys-79,Arg-80,Lys-85,Lys-87,Lys-90,
Å % Lys-93, and Lys-97 at the (cid:2)5-(cid:2)6 helices (Figs. 2A and 4C). In
hASCa(1UCP) 12.4 1.6 83 19
hNLRP3a(3QF2) 12.3 1.8 82 27 agreementwiththisdistinctpatternofchargedistribution,resi-
hPOP1a(2HM2) 12.2 1.5 80 23 duesLys-64andLys-85areconservedamongthePYHINproteins
hMNDAa(2BDG) 12.1 3.8 92 28
hNLRP4a(4EWI) 11.1 1.6 78 27 only(Fig.1),andresiduesGlu-20andGlu-21areAIM2-specific
hNLRP7a(2KM6) 10.8 2.4 85 20 residues(Fig.1andsupplementalFig.3,AandB).Incomparison,
mNLRP10b(2DO9) 10.7 3.6 89 24
hNLRP12a(2L6A) 10.3 1.6 78 23 theASCPYDdisplaysadifferentchargedistributionwithacidic
mP205b(2YU0) 9.5 4.1 84 24 (cid:2)1and(cid:2)4helicesandbasic(cid:2)2-(cid:2)3helices(Fig.4,DandE).
hNLRP1a(1PN5) 8.1 2.4 76 20 AdjacenttotheacidicresiduesattheAIM2PYD(cid:2)2helixare
aHumanprotein.
bMouseprotein. several hydrophobic and non-charged residues that are
exposedtothesolvent.TheseincluderesiduesPhe-27,Phe-28,
Phe-33, Ile-35, Thr-37, and Thr-42 (Figs. 1 and 4, B and C).
code 3QF2), NLRP4 (Protein Data Bank code 4EWI), NLRP7
SimilartotheacidicresiduesGlu-20andGlu-21ofthe(cid:2)2helix,
(ProteinDataBankcode2KM6),andPOP1(ProteinDataBank
code2HM2)revealsthatthisconserved(cid:2)2helixlysineresidue the hydrophobic residues Phe-27 and Phe-28 are conserved
buttressesthe(cid:2)3helixthroughhydrogenbondswithtwomain amongtheAIM2proteinsfromdifferentspeciesbutnotamong
chaincarbonyloxygens(i.e.thoseofLeu-40andAla-43from theotherPYHINproteins(Fig.1andsupplementalFig.3,Aand
AIM2),therebystabilizingtheconformationof(cid:2)3anditscon- B),suggestingthattheymaycontributetoAIM2-specificfunc-
nectingloops(Fig.3,A–F).Inaddition,theonlynon-conserved tions.Intriguingly,similarhydrophobicresiduesatthe(cid:2)2helix
residuesatthisposition(GlninNLRP1andArginNLRP6)are oftheFADDDEDwereshownpreviouslytobeessentialforits
all capable of forming similar hydrogen bonds. Because the bindingofthecaspase-8DED(53,55),andarecentreportonthe
(cid:2)2-(cid:2)3helicesoftheDD,DED,andCARDhaveallbeenimpli- NLRP3PYDstructuresuggestedthathydrophobicandcharged
cated in homotypic interactions (20, 21, 53, 54), it is possible residuesmaymediatetheNLRP3inflammasomeassembly(28).
that the conserved Lys-26 residue may indirectly facilitate CouldtheseAIM2-specifichydrophobicandchargedresiduesalso
PYD-PYDinteractionsthroughstabilizingthe(cid:2)3helix. playaroleinmediatingPYD-PYDinteractions?
SurfaceoftheAIM2PYDExhibitsDistinctPatternofElectro- PolarandHydrophobicInteractionsMayMediatetheAIM2
staticChargesandHydrophobicity—BesidestheLys-26residue PYD-ASCPYDAssociation—Toinvestigatethepotentialroles
thatislargelyburied,mostofthechargedAIM2PYDresidues of the (cid:2)2 helix residues in mediating AIM2-ASC association,
areexposedandcontributetoabipolardistributionofelectro- weperformeddockingstudiesoftheAIM2PYDandASCPYD
MAY10,2013•VOLUME288•NUMBER19 JOURNALOFBIOLOGICALCHEMISTRY 13229
TheStructureoftheAIM2PyrinDomain
FIGURE4.ElectrostaticsurfaceofthePYDs.A,electrostaticchargesurface
oftheAIM2PYDisdisplayedonascaleof(cid:1)5kT/e(red)to5kT/e (blue).The
acidicresiduesarelabeledinblack.TheviewisthesameasinFig.2Awiththe
(cid:2)1helixfacingtheviewer.B,a90°rotationoftheviewinAwiththe(cid:2)2-(cid:2)3
helicesfacingtheviewer.Theacidicresiduesarelabeledinblack,andthe
non-chargedresiduesarelabeledingreen.C,electrostaticchargesurfaceof
theAIM2PYDwithits(cid:2)5helixfacingtheviewer.Theviewisrotated(cid:3)180°
FhIeGlUixR.ET3h.eThreegcioonnsneeravretdhleyscionnesreervsiedduelysaitntehree(cid:1)si2duheelaixndbutthtere(cid:2)s3sehsetlhixea(cid:1)r3e tfrhoemsatmhaetivnieAw.Da,sthineAelwecitthrotshteat(cid:2)ic1chhealrigxefascuirnfgactehoefvtiheewAeSr.CTPhYeDacisidshicorwesnidwuieths
arelabeledinblack.E,a90°rotationoftheviewinDandthesameasinBwith
shownfortheAIM2PYDinA(orange),ASCPYDinB(green),NLRP3PYDinC the(cid:2)2-(cid:2)3helicesfacingtheviewer.Thebasicresiduesarelabeledinyellow.
(cyan),NLRP4PYDinD(wheat),NLRP7PYDinE(salmon),andPOP1PYDinF
(yellow).Hydrogenbondsareindicatedwithgraydottedlines.
anAIM2PYDmodelbuiltfromtheASCPYDstructuresug-
using the ClusPro protein-protein docking server V2.0 (48). gestedthatintheabsenceofthedsDNAligandtheAIM2recep-
Theresultsshowthatthe(cid:2)2helixoftheAIM2PYDisindeed tor resides in an autoinhibited state with its PYD and HIN
locatedatthePYD-PYDinterfacewiththe(cid:2)2helixacidicres- domainforminganintramolecularcomplex.Tofurtherchar-
idues Asp-19, Glu-20, and Asp-23 and hydrophobic residues acterizethisintramoleculardomaininteraction,wesoughtto
Phe-27 and Phe-28 within contact distance of the ASC PYD investigatethePYD-HINinteractionthroughdockingoftheir
(Fig.5,AandB).Thissuggeststhatthe(cid:2)2helixoftheAIM2 crystalstructuresusingtheClusProdockingserverandcom-
PYDmaybeinvolvedinmixedpolarandhydrophobicinterac- pare the results with those from the interactive docking pro-
tionswiththeASCPYD. gramHex(49)thatweusedpreviously.
ToexaminetheroleoftheAIM2PYD(cid:2)2helixinmediating The top 10 solutions from the ClusPro server placed the
itsinteractionwiththeASCPYD,weperformedanMBPpull- AIM2PYDattheconcavesurfaceoftheHINdomainoverlap-
down assay. Although the wild type MBP-AIM2 PYD associ- pingwithitsDNA-bindingsurface(Fig.6,AandB).Mostofthe
ated with the ASC PYD as expected, mutation of either the dockingmodelsplacedthenegativelycharged(cid:2)2helixofPYD
hydrophobicresidues(F27AandF28A;designated“Mut1”)or atthecenterofitsinterfacewiththeHINdomain,whereasthe
acidicresidues(D19A,E20A,E21A,andD23A;“Mut2”)abol- positivelycharged(cid:2)6helixwaslocatedawayfromtheinterface
ished this association (Fig. 5C). This result was further con- inagreementwithourpreviousdockingandmutagenesisstud-
firmedwithayeasttwo-hybridassay(Fig.5D),suggestingthat ies (34). Importantly, such docking models were essentially
both hydrophobic and acidic residues at the AIM2 PYD (cid:2)2 recapitulatedregardlessofwhethertheHINorPYDwaschosen
helixareessentialforitsinteractionwiththeASCPYD. asthestationaryreceptormolecule(Fig.6,CandD)andfurther
ElectrostaticInteractionsMediatethePYD-HINAssociation— confirmedusingtheinteractivedockingprogramHex(supple-
TheAIM2receptoriscomposedofaPYDandaHINdomain, mental Fig. 4). The top ranked docking model from ClusPro
the latter responsible for the recognition of dsDNA in a wasenergy-minimizedusingtheprogramRosetta(v3.4)(50).
sequence-independentmanner(34).Ourpreviousworkusing The resulting structure shows that the PYD-HIN interface is
13230 JOURNALOFBIOLOGICALCHEMISTRY VOLUME288•NUMBER19•MAY10,2013
TheStructureoftheAIM2PyrinDomain
FIGURE5.AnalysisoftheAIM2PYDandASCPYDassociation.A,superpositionofthetop10dockingmodelsoftheAIM2PYD-ASCPYDcomplexbythe
ClusProserverwiththeAIM2PYDasthestationaryreceptor.TheAIM2PYDandASCPYDarecoloredorangeandgreen,respectively.TheAIM2residuesAsp-19,
Glu-20,Asp-23,Phe-27,andPhe-28areshownasyellowspheres.B,superpositionofthetop10dockingmodelsoftheAIM2PYD-ASCPYDcomplexwiththeASC
PYDasthestationaryreceptor.The10modelsweresuperimposedontheirAIM2PYDs.C,MBPpulldownassayfortheWTandmutantAIM2PYDassociation
withtheASCPYD.“I”denotesinputsample,and“E”denoteselutionsample.D,yeasttwo-hybridanalysisoftheAIM2PYDandASCPYDinteraction.Yeastcells
co-expressingGAL4DNA-bindingdomain(BD)-ASCPYDfusionandGAL4activationdomain(AD)fusionsweregrownonagarplateslackingleucineand
tryptophan((cid:1)Leu/(cid:1)Trp)fortransformantgrowthandlackinghistidine,leucine,andtryptophan((cid:1)His/(cid:1)Leu/(cid:1)Trp)fordetectingPYD-PYDinteraction.Three
individualclonesforeachcombinationwereplated.(cid:1)denotesemptyvectorcontrol.
dominatedbyelectrostaticinteractionsbetweenbasicresidues helices.The(cid:2)2helixalsocontainssurface-exposedhydropho-
fromtheHINdomainDNA-bindingsurfaceandacidicresidues bicresiduesPhe-27andPhe-28thatareuniquetoAIM2PYD.
fromthe(cid:2)1-(cid:2)2helicesofPYD(Fig.6,E–G).Toconfirmthis A highly conserved Lys-26 residue at the (cid:2)2 helix is largely
interaction,weperformedITCstudiestoanalyzetheinterac- buried and forms hydrogen bonds with main chain carbonyl
tionoftheAIM2PYDandHINdomaininsolution.Thewild oxygens within and adjacent to the short (cid:2)3 helix. This may
typeAIM2PYDbindstheAIM2HINdomainwithadissocia- stabilizetheconformationofthe(cid:2)3helixtofacilitateitspartic-
tionconstant(Kd)of23.5(cid:4)M(Fig.7A).Mutationofthehydro- ipationinPYD-PYDinteractions.Suchhydrogenbonding-me-
phobic residues (Mut1) only marginally affected the binding diatedstabilizationofthe(cid:2)3helicesappearstobeuniquefor
withaKdof56.8(cid:4)M(Fig.7B).Incontrast,mutationoftheacidic thePYDsasmostofthereportedDD,DED,andCARDstruc-
residuesatthe(cid:2)2helix(Mut2)abolishedtheinteraction(Fig. turesusehydrophobiccontactsbetweenthe(cid:2)2and(cid:2)3helices
7C).Inagreement,Mut1retaineditsabilitytocompeteforHIN
(20,21,23,54).
domainbindingbyDNA,whereasMut2largelylostthisinhib-
Whataretheimplicationsoftheabovestructuralfeaturesfor
itoryfunction(Fig.7B).Thesedatasuggestthattheacidicres-
theAIM2PYD-ASCPYDinteractions?Threetypesofassocia-
idues are essential for the PYD-HIN interaction, whereas the
tions have been identified for the death domain fold, all of
adjacent hydrophobic residues may not be in direct contact
whichinvolveelectrostaticinteractions(56).ThetypeIinter-
withtheDNA-bindingsurfaceoftheHINdomain.
actionhasbeenreportedforDD(20,21),DED(53),andCARD
DISCUSSION (54), whereas type II and type III interactions have only been
reportedforDD(20,21,57).AsnoPYD-PYDcomplexstruc-
TheAIM2receptorisacriticalinnateimmunesensorinthe
ture has been reported, it is currently unknown which of the
cytosolthatrespondstoinvasionbycertainDNAvirusesand
bacteriaandplaysaroleintheautoimmunedisorderpsoriasis. threetypesoranoveltypeofinteractionmaymediatethePYD-
AfundamentalaspectoftheAIM2functionistheabilityofits PYD association. One mode of interaction predicted by our
PYD to interact with that of the adapter ASC and form an dockingstudiesillustratesthattheAIM2PYD(cid:2)2helixbinds
inflammasome. In this study, we determined the AIM2 PYD theASCPYDthroughacidicandhydrophobicresiduesunique
crystalstructure,whichrevealsasix-helixbundletypicalofthe to the AIM2 PYD, and this was confirmed by our MBP pull-
death domain superfamily. The AIM2 PYD features distinct downandyeasttwo-hybridstudies.Thisisalsoinagreement
chargedistributionswithacidic(cid:2)1-(cid:2)2helicesandbasic(cid:2)5-(cid:2)6 with reports that hydrophobic and/or charged residues may
MAY10,2013•VOLUME288•NUMBER19 JOURNALOFBIOLOGICALCHEMISTRY 13231
TheStructureoftheAIM2PyrinDomain
FIGURE6.DockingoftheAIM2PYDandHINdomains.A,superpositionofthetop10dockingmodelsoftheAIM2PYD-HINdomaincomplexwiththeHIN
domainasthestationaryreceptorbytheClusProserver.TheAIM2PYDandHINdomainarecoloredorangeandcyan,respectively.B,thestructureoftheAIM2
HIN-DNAcomplex(ProteinDataBankcode3RN2)isshownforcomparison.C,superpositionofthetop10dockingmodelsoftheAIM2PYD-HINcomplexwith
thePYDasthestationaryreceptorbytheClusProserver.The10modelsweresuperimposedontheirHINdomains.D,theoriginal10dockingmodelsforCare
superimposedontheirPYDswiththeAIM2residuesAsp-19,Glu-20,Asp-23,Phe-27,andPhe-28shownasyellowspheres.E,thetopscoringmodelfromAwas
energy-minimizedwithRosettaandisshowninribbons.ThePYDandHINdomainarecoloredorangeandcyan,respectively.ThePYDhelicesandterminiare
labeled.TheopenbookviewofthePYD-HINinterfaceisshownaselectrostaticsurfaceinF(HIN)andG(PYD).ThebasicresiduesinHINarelabeledinyellow,and
acidicresiduesinPYDarelabeledinblack.
mediateFADDDEDandcaspase-8DEDassociationaswellas (Fig.4D).Thesepredictedtypesofinteractionsarenotmutually
NLRP3PYDandASCPYDassociation(28,53,55). exclusive:oligomericdeathdomainsignalingplatformssuchas
Analysisofthecomplementarychargedistributionbetween the PIDDosome (20) and MyDDosome (21) are assembled
theAIM2PYDandASCPYDsuggestsotherpossiblemodesof throughseveralbindingsitesatmultipleinterfaces.Muchofthe
interaction: the acidic (cid:2)1 helix of the former (Fig. 4A) may details on the oligomeric PYD complexes will require future
interactwiththebasic(cid:2)2-(cid:2)3helicesofthelatter(Fig.4E),and experimentalcharacterizationoftheinflammasomestructures.
thepositivelycharged(cid:2)5helixoftheAIM2PYD(Fig.4C)may Structural studies of the AIM2 inflammasome have so far
associatewiththenegativelycharged(cid:2)4helixoftheASCPYD beenfocusedonitsHINdomain(34,58).Basedontheelectro-
13232 JOURNALOFBIOLOGICALCHEMISTRY VOLUME288•NUMBER19•MAY10,2013
TheStructureoftheAIM2PyrinDomain
FIGURE7.BindingoftheAIM2PYDandHINdomains.A,theassociationoftheAIM2HINandwildtype(A),Mut1(F27A/F28A;B),andMut2(D19A/E20A/
E21A/D23A;C)wereanalyzedbyITC.D,inhibitionoftheAIM2HIN-DNAinteractionbythewildtypeandmutantAIM2PYDsasanalyzedbyfluorescence
polarization.mP,millipolarizationunits.
static nature of the AIM2 HIN domain association with AIM2PYD-ASCPYDandAIM2PYD-AIM2HINcomplexes
dsDNA, we hypothesized that an intramolecular interaction revealthatoverlappingnegativelychargedsurfaceattheAIM2
betweentheHINdomainandPYDretainsthereceptorinan PYD may be involved in its association with both partner
autoinhibitedstateintheabsenceofligandbinding(34).The domains. Such mutually exclusive interactions may serve to
directinteractionofthePYDandHINdomainwasconfirmed ensurethatAIM2interactswiththedownstreamadapterASC
by an in vitro pulldown assay (34). In the current work, our onlyuponactivationbythedsDNAligand.
dockingstudiesusingthecrystalstructuresofthePYDandHIN
domain clearly demonstrated strong preference of the two
Acknowledgments—We thank Dr. Lars C. Pedersen at the NIEHS,
domainstointeractthroughtheirrespectivechargedsurfaces
NationalInstitutesofHealthfortheMBPfusionplasmids,Dr.David
(Fig.6).Wefurthervalidatedthisautoinhibitionmodelusing
S.WaughattheNCI,NationalInstitutesofHealthforthetobacco
ITC studies and fluorescence polarization inhibition assays.
etchvirusproteaseexpressionconstruct,andDr.GerhardWagnerat
BecausethePYDandHINdomainarecovalentlylinked,their
HarvardMedicalSchoolfortheGB1encodingplasmid.WethankDr.
higheffectivelocalconcentrationsmaypromotetheintramo-
TingheWuforhelpwiththeASCPYDexpressionconstructandDr.D.
lecular association that prevents undesirable activation of
Eric Anderson at the Mass Spectrometry facility of the NIDDK,
AIM2.Similarautoinhibitionmechanismshavebeenobserved
NationalInstitutesofHealthfortechnicalsupport.Wearegratefulto
forothermultidomainreceptorssuchasretinoicacid-inducible
the beam line scientists at the GM/CA-CAT, Advanced Photon
gene1(RIG-I)(59)andApaf-1(60).Itappearsthatautoinhibi-
Sourceforsupport,whichisfundedinwholeorinpartwithfederal
tion is a common regulatory mechanism for multidomain
funds from the NCI (Grant Y1-CO-1020) and NIGMS (Grant
immune receptors to safeguard against spurious activation of Y1-GM-1104),NationalInstitutesofHealth.
excessive inflammatory responses. Our docking models of the
MAY10,2013•VOLUME288•NUMBER19 JOURNALOFBIOLOGICALCHEMISTRY 13233
TheStructureoftheAIM2PyrinDomain
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