Dimerization of Plant Defensin NaD1 Enhances Its Antifungal Activity

13 Pages·2012·2.87 MB·English

THEJOURNALOFBIOLOGICALCHEMISTRYVOL.287,NO.24,pp.19961–19972,June8,2012
©2012byTheAmericanSocietyforBiochemistryandMolecularBiology,Inc. PublishedintheU.S.A.
Dimerization of Plant Defensin NaD1 Enhances Its Antifungal
Activity*
Receivedforpublication,December7,2011,andinrevisedform,April16,2012 Published,JBCPapersinPress,April17,2012,DOI10.1074/jbc.M111.331009
FungT.Lay‡§1,GrantD.Mills‡§1,IvanK.H.Poon‡,NathanP.Cowieson¶,NigelKirby¶,AmyA.Baxter‡§,
NicoleL.vanderWeerden‡§,ConDogovski‡,MatthewA.Perugini‡,MarilynA.Anderson‡§,MarcKvansakul‡2,
andMarkD.Hulett‡§3
Fromthe‡DepartmentofBiochemistry,LaTrobeInstituteforMolecularScience,LaTrobeUniversity,Melbourne,
Victoria3086,§HeximaLimited,Melbourne,Victoria3000,andthe¶AustralianSynchrotron,800BlackburnRoad,Clayton,
Victoria3168,Australia
Background:NaD1isapotentantifungalplantdefensinfromNicotianaalataflowers.
Results:NaD1formsdimersasdeterminedbyx-raycrystallographic,biophysical,andbiochemicalapproaches.
Conclusion:DimerizationofNaD1enhancesitsfungalcellkilling.
Significance:UnderstandingthemolecularbasisofNaD1antifungalactivityhelpsdefinedefensinfunctionandhaspotential
applicationforimprovingplantresistanceagainstagronomicallyimportantfungalpathogens.
The plant defensin, NaD1, from the flowers of Nicotiana wall to the constitutive or inducible expression of chemical
alata,isamemberofafamilyofcationicpeptidesthatdisplays compounds, including secondary metabolites and defensive
growth inhibitory activity against several filamentous fungi, proteins.
includingFusariumoxysporum.TheantifungalactivityofNaD1 Of the defense proteins that are produced by plants, the
has been attributed to its ability to permeabilize membranes; defensinsrepresentoneofthelargestfamilies.Theyaresmall
however, the molecular basis of this function remains poorly ((cid:1)5 kDa), basic, and cysteine-rich and are abundantly
defined.Inthisstudy,wehavesolvedthestructureofNaD1from expressedinvariousplanttissues.Asaresultofrecentadvances
two crystal forms to high resolution (1.4 and 1.58 Å, respec- in whole-genome sequencing and proteomics, coupled with
tively),bothofwhichcontainNaD1inadimericconfiguration. improvedbioinformaticanalyses,therehasbeenahugeexpan-
Usingproteincross-linkingexperimentsaswellassmallangle sioninthenumberofdescribeddefensins,manyofwhichare
x-rayscatteringanalysisandanalyticalultracentrifugation,we encodedbymultigenefamilies(1–5).
showthatNaD1formsdimersinsolution.Thestructuralstudies Plantdefensinsaredistinguishedfromotherproteinfamilies
identifiedLys4ascriticalinformationoftheNaD1dimer.This basedonthenumberandarrangementofcysteines,theconfig-
wasconfirmedbysite-directedmutagenesisofLys4thatresulted urationofthedisulfidebridges(C–C ,C –C ,C –C ,and
I VIII II V III VI
in substantially reduced dimer formation. Significantly, the C –C ),andadistinctstructuralfoldconsistingofatriple-
reducedabilityoftheLys4mutanttodimerizecorrelatedwith stIrVandeVdIIantiparallel(cid:1)-sheettetheredviathreedisulfidebonds
diminished antifungal activity. These data demonstrate the to an (cid:2)-helix in a configuration known as the cysteine-stabi-
importanceofdimerizationinNaD1functionandhaveimplica- lized(cid:2)(cid:1)(CS(cid:2)(cid:1))motif.ThefourthdisulfidebondlinkstheN-
tionsfortheuseofdefensinsinagribiotechnologyapplications
andC-terminalregions,producingapseudocyclicproteinthat
such as enhancing plant crop protection against fungal
renderstheproteinhighlystabletochemicalandthermaldena-
pathogens.
turation (6, 7). Interestingly, the Petunia hybrida floral
defensinsPhD1andPhD2haveafifthdisulfidebond(8–10).
Apartfromthecysteines,onlyalimitednumberofotherresi-
Plants are continually exposed to a myriad of potentially
dues are conserved among members of this family. They
damaging microorganisms, phytophagous insects, and envi-
includeaserine(position8),twoglycines(position13and34),
ronmental stresses. As a counter-measure, plants have devel-
anaromaticresidue(position11),andaglutamicacid(position
opedsophisticatedinnatedefensemechanismsforprotection.
29)(numberingrelativetotheradishdefensinRs-AFP2)(7).An
Theserangefrompreformedphysicalbarrierssuchasthecell
aminoacidsequencealignmentofselectrepresentativeexam-
plesofplantdefensinsisgiveninFig.1.
*ThisworkwassupportedbyBalmoralAustraliaPtyLtd.,HeximaLtd.,Aus- Despitethelowprimarysequenceidentitybetweendifferent
tralianResearchCouncilProjectGrantDP0881732(toM.A.A.),andthe
plantdefensins,theirthree-dimensionalstructuresareremark-
National Health and Medical Research Council of Australia Fellowship
637372(toM.K). ablysimilar.Todate,thestructuresof13defensinshavebeen
Theatomiccoordinatesandstructurefactors(codes4aazand4ab0)havebeen elucidated(8,10–20).Allofthesedefensinstructures,withthe
depositedintheProteinDataBank,ResearchCollaboratoryforStructural
exception of one, were solved by solution 1H NMR spectros-
Bioinformatics,RutgersUniversity,NewBrunswick,NJ(http://www.rcsb.org/).
1Bothauthorscontributedequallytothiswork. copyasmonomericproteins(7).Morerecently,thestructureof
2To whom correspondence may be addressed. Tel.: [phone]; Fax: theSPE10defensinfromPachyrrhizuserosusseedswassolved
61-3-9479-1266;E-mail:[email protected].
byx-raycrystallographyandpresentedasadimerintheasym-
3To whom correspondence may be addressed. Tel.: [phone]; Fax:
61-3-9479-1266;E-mail:[email protected]. metricunit(20).
JUNE8,2012•VOLUME287•NUMBER24 JOURNALOFBIOLOGICALCHEMISTRY 19961
This is an Open Access article under the CC BY license.
DimerizationofNaD1andAntifungalActivity
FIGURE1.AminoacidsequencealignmentofrepresentativematureplantdefensinsfromclassIandII.Thecysteineresiduesthatareinvariantinall
membersoftheplantdefensinfamilyarehighlightedandareincludedintheconsensussequence,togetherwithaserine,anaromaticresidue(denotedaso),
twoglycines,andaglutamicacidthatareconservedinmostofthesequences.Thedisulfidebondsareshownbelowtheconsensussequencebyconnecting
lines.Zistheone-lettercodeforpyroglutamicacid.TheaminoacidsareshadedinblackorgraytoindicateidentityorsimilaritytoNaD1,respectively.Spaces
havebeenintroducedtomaximizethealignment.Theaccessionnumbersforeachproteinaregiveninparentheses.Sourcesofproteinareasfollows:NaD1(N.
alata),FST(Nicotianatabacum),NpThio1(Nicotianapaniculata),PhD1andPhD2(P.hybrida),TPP3(Solanumlycopersicum),CcD1(Capsicumchinense),SPE10
(Pachyrrhizuserosus),Cp-thionin(Vignaunguiculata),Rs-AFP1(Raphanussativus),Hs-AFP1(Heucherasanguinea),Dm-AMP1(Dahliamerckii),andCt-AMP1
(Clitoriaternatea).
Importantly, plant defensins display a variety of biological brane and entry of NaD1 into the cytoplasm. It has been
functions despite their conserved three-dimensional struc- proposed that NaD1-mediated membrane permeabilization,
tures.Bestcharacterizedistheirabilitytoinhibitfungalpatho- togetherwiththerecognitionofintracellulartargets,leadsto
gens (9, 21–25). A number of defensins have also been fungalcelldeath(24,25).However,themolecularbasisofthis
described with antibacterial activity (26–28) or can act as processhasnotbeendefined.
inhibitors of protein synthesis (29–32), (cid:2)-amylases (33, 34), Previously, the structure of monomeric NaD1 was deter-
proteases(35,36),orionchannels(37,38).Itisinterestingto minedbysolution1HNMRspectroscopy(10).Here,wereport
notethatthelatterfunctionsaremoreconsistentwitharolein thehighresolutioncrystalstructuresofNaD1fromtwocrystal
defenseagainstinsectpests.Furthermore,somedefensinshave forms,bothofwhichcontainNaD1inadimericconfiguration.
been implicated in plant tolerance to environmental stresses Using cross-linking experiments as well as small angle x-ray
suchashighlevelsofzinc(39),salt(40,41),orcold(42).Hence, scatteringanalysisandanalyticalultracentrifugation,wedem-
the varied biological functions of plant defensins make them onstratethatNaD1formsdimersinsolution.Dimerizationof
attractive candidates for exploitation in agribiotechnological NaD1iscriticalforantifungalactivityasamutantthatdoesnot
applications to confer enhanced resistances to pathogens, dimerize has a greatly reduced ability to kill the filamentous
insect herbivory, or environmental stresses in commercially fungusF.oxysporum.
importantcrops.
EXPERIMENTALPROCEDURES
Insolanaceousplants,defensinsaredividedintotwoclasses.
ClassIdefensinsencodeaprecursorproteinwithanendoplas- Protein Purification—NaD1 was purified from whole N.
mic reticulum signal peptide and a mature defensin domain, alataflowersuptothepetalcolorationstageofflowerdevelop-
whereas class II defensins encode a precursor with an addi- mentasdescribedinvanderWeerdenetal.(25).
tional C-terminal propeptide (7, 9). At least for three class II Crystallization and Data Collection—NaD1 crystals were
defensins,namelyNaD1,PhD1,andPhD2(wheretheprotein obtainedbythesittingdropvapordiffusionmethodin20%PEG
hasbeencharacterized),theC-terminalpropeptideisremoved 1500 and 10% succinate/phosphate/glycine buffer, pH 9, and
fromthematurebiologicallyactiveprotein(8–10). nativedatawerecollectedfromtwodifferentcrystalforms,A
NaD1 is a defensin from the flowers of the ornamental andB,asdescribedpreviously(43).
tobacco,Nicotianaalata(9).Ithasbeensuggestedthatitspri- HeavyAtomDerivatizationandDerivativeDataCollection—
mary function is to protect the reproductive tissues against Initial attempts to solve the structure of NaD1 by molecular
damagefrompotentialfungalpathogens(9,24,25).Thisiscor- replacement using either the NMR structure of NaD1 (PDB4
roboratedbyinsituhybridizationstudiesonN.alataflowers code 1MR4) or other plant defensin structures failed. Conse-
demonstrating accumulation of NaD1 transcripts in tissues quently, NaD1 crystals from crystal form A were soaked in
surrounding the reproductive organs. Immunoblot analysis motherliquorsupplementedwith0.2M5-amino-2,4,6-triiodo-
withNaD1-specificantibodiesalsorevealedhighlevelsofthe isophthalicacid(44)and10%ethyleneglycolfor5min.Deriv-
proteininfloraltissues(9).Furthermore,NaD1exhibitspotent ativediffractiondatawerecollectedat100Kusingawavelength
invitroantifungalactivityagainstanumberofpathogenicfila- of 1.5 Å at the Australian Synchrotron (beamline 3ID1) and
mentous fungi, including Fusarium oxysporum f. sp. dianthi processedusingXDS(45).
(Race2),F.oxysporumf.sp.vasinfectum,andBotrytiscinerea
(9,24,25).Recentstudieshavedemonstratedthattheantifun-
4Theabbreviationsusedare:PDB,ProteinDataBank;BS3,bis(sulfosuccinimidyl)-
galactivityofNaD1involvesspecificinteractionswiththefun-
suberate;BS(PEG) ,bis-N-succinimidyl-(pentaethyleneglycol)ester;SAXS,
5
galcellwall,followedbypermeabilizationoftheplasmamem- smallanglex-rayscattering.
19962 JOURNALOFBIOLOGICALCHEMISTRY VOLUME287•NUMBER24•JUNE8,2012
DimerizationofNaD1andAntifungalActivity
TABLE1
X-raycrystallographicdatacollectionandrefinementstatistics
I3Cis5-amino-2,4,6-triiodoisophthalicacid.
CrystalformA I3Cderivative CrystalformB
Datacollection
Spacegroup P2 P2 P321
1 1 2
Celldimensions
a,b,c(Å) 32.70,32.69,41.98 32.64,32.57,42.06 33.09,33.09,128.77Å
(cid:2),(cid:1),(cid:4)(°) 90.00,100.83,90.00° 90.00,100.81,90.00 90.00,90.00,120.00
Wavelength(Å) 0.9537 1.5 0.9537
Resolution(Å)a 41.23to1.4(1.47to1.40) 41.32to1.58(1.66to1.58) 64.39to1.64(1.72to1.64)
R orR a 0.089(0.404) 0.079(0.366) 0.081(0.479)
I/s(cid:5)ymIa merge 18.1(4.6) 16.6(5.0) 16.4(3.1)
Completenessa(%) 98.6(90.5) 99.0(93.3) 99.1(93.7)
Redundancya 6.9(4.8) 6.7(5.0) 9.4(5.5)
Refinement
Resolution(Å) 41.23to1.4 42.94to1.64
No.ofreflections 17,174 10,549
R /R 0.119/0.137 0.201/0.232
work free
No.atoms
Protein 1474 1400
Ligand/ion 1PO,6EDO 2PO
4 4
Water 369 78
B-factors
Protein 8.9 20.9
Ligand/ion 19.5 36.3
Water 21.0 32.6
Rootmeansquaredeviations
Bondlengths(Å) 0.011 0.006
Bondangles(°) 1.543 1.3°
aValuesinparenthesesareforthehighestresolutionshell.
Model Building and Refinement—5-Amino-2,4,6-triiodo- platingontoMDagar(13.4g/literyeastnitrogenbase(YNB),
isophthalicacidsiteswerefoundwithShelX(46)andrefined 400(cid:3)g/literbiotin,10g/literdextrose,and15g/literagar).A
usingPhenix(47).Clearandcontinuouselectrondensitywas singleHis(cid:2)colonywasusedtoinoculate200mlofBMG(100
obtainedforallresiduesoftheentiremolecule.Twochainsof mMpotassiumphosphate,pH6.0,13.4g/literYNB,400(cid:3)g/liter
NaD1werebuiltforcrystalformA.NaD1crystalformBwas biotin,1%(v/v)glycerol)andincubatedwithconstantshaking
solvedbymolecularreplacementusingmonomericNaD1from at 30°C until the A reached (cid:1)3.0. The cell mass was col-
600
crystalformAasasearchmodelwithPHASER(48).Thefinal lectedbycentrifugation(1,500(cid:3)g,10min)andresuspended
models for both crystal forms were built with Coot (49) and into1literofBMM(100mMpotassiumphosphate,pH6.0,13.4
refinedwithPhenixtoaresolutionof1.4Å(formA)and1.58Å g/literYNB,400(cid:3)g/literbiotin,0.5%(v/v)methanol)toinduce
(formB).Alldatacollectionandrefinementstatisticsaresum- expression. Expression was continued for 72 h with constant
marizedinTable1.RefinementyieldedR andR valuesof shakingat30°Cafterwhichtimethecellmasswasremovedby
work free
11.9and13.7%forcrystalformA,and20.1and23.2%forcrystal centrifugation(10,000(cid:3)g,10min)andthesupernatantcollected.
form B, respectively. The coordinates have been deposited in rNaD1, rNaD1(E2A), and rNaD1(K4A) were isolated from the
theProteinDataBank(accessioncodes4aazand4ab0,respec- supernatantusingion-exchangechromatographyusingSP-Sep-
tively).FigureswerepreparedusingPyMol(DeLanoScientific). haroseasdescribedforisolationofNaD1fromplanttissue.
Expression of NaD1 and NaD1 Mutants in Pichia pastoris— Fungal Growth Inhibition Assays—The ability of NaD1,
NaD1andNaD1mutantswererecombinantlyexpressedinthe rNaD1,rNaD1(E2A),andrNaD1(K4A)toinhibitthegrowthof
methylotrophic yeast P. pastoris essentially as described by F.oxysporumf.sp.vasinfectumwasassessedasdescribedpre-
Cabraletal.(50).Briefly,DNAencodingthematureregionof viously(25).
NaD1wasclonedintothepPIC9expressionvector(Invitrogen) Protein Cross-linking—NaD1 at 125–500 (cid:3)g/ml was cross-
directly in-frame with the (cid:2)-mating factor secretion signal linkedthroughprimaryaminogroupsbytheadditionof6.25
usingtherestrictionenzymesXhoIandNotI.Analaninewas mM bis[sulfosuccinimidyl] suberate (BS3) (Thermo Scientific)
addedtotheNterminusoftheNaD1sequencetoensureeffi- in a buffer containing 20 mM sodium phosphate and 150 mM
cientcleavageofthesignalattheKex2cleavagesite.Theglu- NaCl,pH7.1,atroomtemperaturefor30min.Thesampleswere
tamic acid at position 2 and the lysine at position 4 of the reduced with dithiothreitol, denatured, and subjected to SDS-
matureNaD1sequenceweremutatedtoalanineusingthePhu- PAGEpriortoCoomassieBrilliantBluestaining.NaD1,rNaD1,
sion site-directed mutagenesis kit (Finnzymes) to generate rNaD1(E2A),andrNaD1(K4A)werealsocross-linkedwith6.25
mutants rNaD1(E2A) and rNaD1(K4A), respectively. After mMBS3orwith10mMbis-N-succinimidyl-(pentaethyleneglycol)
transformation into Escherichia coli TOP10 cells, the pPIC9- ester(BS(PEG) )(ThermoScientific)atfinalproteinconcentra-
5
NaD1, pPIC9-NaD1(E2A), and pPIC9-NaD1(K4A) plasmids tionsof250or375(cid:3)g/ml,respectively,asdescribedabove.
wereisolatedandlinearizedusingSalItoallowintegrationat SmallAngleX-rayScattering—SAXSdatawerecollectedat
thehis4locusoftheP.pastorisgenome.LinearizedDNAwas the SAXS/WAXS beamline at the Australian Synchrotron.
transformed into electrocompetent yeast as described by NaD1at0.125,0.25,0.5,1,and2mg/mlindistilledwateratpH
Changetal.(51),andHis(cid:2)transformantswereselectedforby 7.0wasmeasuredinaQrangebetween0.035and0.6Å(cid:4)1at12
JUNE8,2012•VOLUME287•NUMBER24 JOURNALOFBIOLOGICALCHEMISTRY 19963
DimerizationofNaD1andAntifungalActivity
keV and with a 1.6-meter camera length. Normalization was
achievedviaanintegratingbeamstop.Dataweremeasuredon
aPilatus1Mcamera(Dectris)andabsolutescaledusingdis-
tilledwater.Tocontrolforradiationdamage,thesampleswere
measured in a 1.5-mm quartz capillary and flowed past the
beam while 10 1-s exposures were measured on samples and
blanks. The exposures were compared for agreement before
beingaveraged.Theradiusofgyrationwascalculatedfromthe
highresolutionmodelsusingtheprogramMoleman2(Uppsala
SoftwareFactory).
AnalyticalUltracentrifugation—Sedimentationvelocityexper-
iments were conducted in a Beckman model XL-A analytical
ultracentrifuge at a temperature of 20°C. Samples of rNaD1
andrNaD1(K4A)werestudiedataconcentrationof0.5mg/ml
(94(cid:3)M)solubilizedinPBS,pH7.4.380(cid:3)lofsampleand400(cid:3)l
of reference solution were loaded into a conventional double
sectorquartzcell,whichweresubsequentlymountedinaBeck-
man4-holeAn-60Tirotor,andcentrifugedatarotorspeedof
40,000 rpm overnight. Data were collected at a single wave-
length(237nm)incontinuousmodeat15-minintervalsusinga
stepsizeof0.003cmwithoutaveraging.Solventdensity(1.0053
g/mlat20°C),viscosity(1.0184centipoise),andanestimateof
the partial specific volume of rNaD1 (0.727 ml/g) and
rNaD1(K4A) (0.725 ml/g) were computed using the program
SEDNTERP(52).Sedimentationvelocitydataatmultipletime
points were fitted to a continuous sedimentation coefficient
FIGURE2.CrystalstructureofNaD1.A,schematicrepresentationofNaD1
(c(s)) distribution and a continuous mass (c(M)) distribution fromcrystalformA.Disulfidebondsareshownasballandstick.B,superim-
model(53–55)usingtheprogramSEDFIT,whichisavailable positionoftheNaD1NMRstructure(gray,PDBcode1MR4)withNaD1from
crystalformA(green).C,superimpositionofasingleNaD1monomer(green)
on-line. fromcrystalfromAontotheNMRensembleofPDBcode1MR4(gray).
RESULTS
sufficiently stable to be observed in solution. In contrast, the
CrystalStructureofNaD1—ThestructureofNaD1(crystal dimericconfigurationofcrystalformBisformedbytheasso-
formA)wassolvedbysingleheavyatomisomorphousreplace- ciationofthe(cid:1)1-strandsfromthetwoparticipatingmonomers
mentwithanomaloussignalandrefinedtoaresolutionof1.4Å. (Fig.3B).Thiscreatesanextended(cid:1)-sheetleadingtothefor-
A second crystal form (form B) was solved by molecular mationofamorecompactandsymmetricaldimer.Thedimer
replacementusingmonomericNaD1fromthepreviousexper- interfacehasacombinedburiedsurfaceareaof678Å2andis
imentallydeterminedstructureofformA.Asexpected,NaD1 stabilized by three hydrogen bonds, two from Lys4–Lys4 and
adopts the cysteine-stabilized (cid:2)(cid:1)(CS(cid:2)(cid:1)) motif formed by a one from Ser35–Cys47. Additionally, a salt bridge is found
triple-strandedantiparallel(cid:1)-sheetandasingle(cid:2)-helixthatis betweenCys47–Arg40(Fig.3C).
tethered to the sheet via three disulfide bonds (Fig. 2A). The Electrostatic Surfaces of the NaD1 Dimers—The biological
fourthdisulfidebondreinforcestheN-andC-terminalregions activityofNaD1hasbeenlinkedtoanactivityatthefungalcell
of the molecule. Superimpositions of monomeric NaD1 from wall (24, 25). To investigate the possibility of NaD1 surface
crystalformAwithchain1orall20chainswithintheensemble interactionswithcomponentsofthefungalcellwall,weexam-
oflowestenergyNMRstructuresforNaD1(PDBcode1MR4) ined the electrostatic surface potential of the two different
areshowninFig.2,BandC,respectively.AsevidentfromFig. dimerconfigurations(Fig.3,C–F).
2C,therearenoappreciabledifferencesbetweenthecrystaland TheNaD1dimerincrystalformAhastwodistinctnegatively
NMRstructuresforNaD1,withthecrystalstructurefittingwell chargedpocketsattheendsofthedimer,withtwosmallareas
withintheNMRensemble.Moreover,superimpositionofthe oflocalpositivecharge(Fig.3,CandE).Incontrast,thecharge
monomersfromcrystalformAorBwiththeNMRstructureof distributiononthesurfaceoftheNaD1dimerfromcrystalform
NaD1 yields root mean square deviation values of 1.0–1.3 Å Bismarkedlydifferent.Acontinuoussurfaceofpositivecharge
overall47C(cid:2)backboneatoms. is present on one side of the dimer that begins on the first
TwoAlternativeDimericForms—NaD1ispresentasadimer monomer and continues to the end of the second monomer
intheasymmetricunitofbothcrystalforms(Fig.3).Incrystal (Fig.3F).ThetwoLys4residuesmakekeycontributionstothe
formA,the(cid:2)1-(cid:1)2loopofonemonomerfacesthe(cid:2)-helixofthe middle of the positively charged face of the dimer (Fig. 3G).
othermonomer(Fig.3A).However,therearenoprotein-pro- Flanking either side of the positive charge are two well sepa-
teincontactsatthedimerinterface,withallinteractionsbeing rated negatively charged pockets (Fig. 3F). Furthermore, the
mediatedbyorderedwatermolecules.Consequently,itispos- oppositesideofthisdimerpresentsaweakeracidicandhydro-
siblethatthisdimericformonlyexistsinthecrystalandisnot phobicsurface(Fig.3D).
19964 JOURNALOFBIOLOGICALCHEMISTRY VOLUME287•NUMBER24•JUNE8,2012
DimerizationofNaD1andAntifungalActivity
FIGURE3.TwoalternativedimericconfigurationsforNaD1.AandB,schematicrepresentationsofNaD1crystalformsAandB.Residuesinvolvedinthe
dimerinterfacefordimerformBareshownassticks,andhydrogenbondsandsaltbridgesareshowninblackasdottedlines.CandD,electrostaticsurface
representationofNaD1crystalformsAandB.TheviewsareconservedfromAandB.E,electrostaticsurfacerepresentationofNaD1crystalformA.Theviewis
rotatedby180°throughthehorizontalaxisfromC.F,electrostaticsurfacerepresentationofNaD1crystalformB.Theviewisrotatedby180°throughthe
horizontalaxisfromD.G,surfacerepresentationofNaD1dimerformB.TheviewisasinF.TheNaD1surfaceisshowningray;Lys4residuesareshowninmagenta
andcyanfromeachmonomericchain.
ProteinCross-linkingStudies—Toinvestigatethepossibility absenceofBS3.WhentheBS3cross-linkerisadded,anaddi-
that NaD1 forms a dimer in solution, we utilized chemical tionalproteinbandappearswithincreasedrelativemolecular
cross-linkingstudiestodirectlyassesstheconfigurationofthe massthatisconsistentwiththatofaNaD1dimer((cid:1)10kDa)
dimer in solution using BS3. BS3 contains an amine-reactive (Fig. 4A). The protein band corresponding to the dimer
N-hydroxysulfosuccinimide ester at each end of an 8-carbon becomesmorepronouncedwithincreasingamountsofNaD1
(11.4 Å) spacer arm. Once the N-hydroxysulfosuccinimide beingusedinthecross-linkingreaction.Atthehigherconcen-
estersreactwithprimaryamines(presentinthesidechainof trations(e.g.500(cid:3)g/mlNaD1(cid:2)BS3),multimersof15and20
lysineresiduesandtheNterminusofeachpolypeptide)atpH kDaarealsoapparent,consistentwiththeexpectedsizesofa
7–9, it forms stable amide bonds, along with release of the trimerandtetramerofNaD1,respectively.
N-hydroxysulfosuccinimideleavinggroup. Basedonourstructuralstudiesthatsuggestedapivotalrole
AsillustratedinFig.4A,NaD1migratesasasingleprotein forLys4inNaD1dimerization,wegeneratedaK4Amutantof
band at (cid:1)5 kDa (monomer) on reducing SDS-PAGE in the NaD1inP.pastoristoexaminewhetherdimerizationwouldbe
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DimerizationofNaD1andAntifungalActivity
FIGURE4.Chemicalcross-linkingofNaD1insolution.A,abilityofNaD1toformdimersinsolutionasdeterminedbyprotein-proteincross-linkingwithBS3.
NaD1at125,250,and500(cid:3)g/mlwereincubatedatroomtemperaturefor30minintheabsenceorpresenceofBS3(6.25mM)priortoSDS-PAGEanalysisunder
reducinganddenaturingconditionsandstainingwithCoomassieBrilliantBlue.B,NaD1,rNaD1,rNaD1(E2A),andrNaD1(K4A)at250(cid:3)g/mlcross-linkedwith
BS3.C,rNaD1andrNaD1(K4A)at375(cid:3)g/mlintheabsenceorpresenceofthecross-linkerBS(PEG) .SamplesinBandCweresubsequentlytreatedasperA.
5
affected.Ascontrols,rNaD1andrNaD1(E2A)(glutamicacidat
position2isnotinvolvedatthedimerinterface)werealsopro-
duced.TheseproteinsweresubjectedtoBS3cross-linking.As
shown in Fig. 4B, rNaD1 or rNaD1(E2A) was able to form
dimers. In contrast, dimerization was abolished in the
rNaD1(K4A)mutant.
WhenusingBS3,threepotentialcross-linkscanbeformedin
dimerformA(Lys17–Lys22,Lys28–Lys45,andLys17–Lys28)and
one in dimer form B (Lys4–Lys4). Because mutation of Lys4
couldpreventcross-linkingwithBS3indimerformBandthus
leadtoanapparentlossofcross-linkingthatisnotcorrelated
with loss of dimerization, we also performed chemical cross-
linkingusingBS(PEG) ,whichhasaspacerarmof21.7Å(Fig.
5
4C).BS(PEG) enablesadditionalcross-linksindimerformB
5
betweenLys28–Lys45andLys45–Lys45.WhenusingBS(PEG) ,
5
rNaD1 again revealed cross-linked dimers on SDS-PAGE,
whereas rNaD1(K4A) showed substantially reduced amounts
FIGURE5.AntifungalactivityofNaD1,rNaD1,andrNaD1variantsE2A
of dimer. Overall, our chemical cross-linking results suggest andK4A.GrowthinhibitionofF.oxysporumf.sp.vasinfectumbyNaD1com-
thatrNaD1(K4A)hasasignificantlylowerpropensitytoform paredwithrNaD1andrNaD1variantsE2AandK4A.Hyphalgrowthisplotted
relativetoanoproteincontrol.Errorbarsrepresentmean(cid:6)S.E.(threeinde-
dimerscomparedwiththewild-typerNaD1.
pendentexperimentsperformedatleastintriplicate).
Circular dichroism analysis of NaD1, rNaD1, rNaD1(E2A),
and rNaD1(K4A) indicated that there was no major spectral thespectraacquisition(atpH4)(10),wesoughttoestablishif
differencesbetweentheproteinssuggestingthattheyweresim- NaD1couldbepresentasadimerinsolutionundermorephys-
ilarly folded (data not shown). Therefore, the reduced iologically relevant conditions. For this purpose, we utilized
dimerizationofrNaD1(K4A)isnotaresultofincorrectfolding SAXSanalysis.TheNaD1proteinwasmeasuredatfiveconcen-
and provides evidence to support the importance of Lys4 in trations,0.125,0.25,0.5,1,and2mg/ml.Thescatteringcurves
NaD1dimerization. showasimilarshapeateachconcentration(Fig.6A).Thelow-
Antifungal Activity of NaD1, rNaD1, and rNaD1 Mutants— estangleregionsofthescatteringcurvesconformtoastraight
TodeterminewhetherdimerizationofNaD1contributestoits line on a Guinier plot (Fig. 6B), and the calculated radius of
antifungal activity, the ability of rNaD1(K4A) to inhibit the gyration does not vary significantly across the concentration
growth of F. oxysporum f. sp. vasinfectum was assessed and range,indicatinganabsenceofsignificantconcentrationeffects
comparedwithrNaD1andrNaD1(E2A)(Fig.5).Theinhibitory (Table2).
activityofrNaD1andrNaD1(E2A)wassimilartonativeNaD1 TwoelementsofthescatteringdataindicatethatNaD1was
(IC value of (cid:5)1.15 (cid:3)M). In contrast, there was a significant largelyadimerinsolution.First,themolecularmass,calculated
50
decreaseintheabilityofrNaD1(K4A)toinhibitfungalgrowth, fromI(0)ontheabsolutescatteringscaleacrosstheconcentra-
with an approximate 5-fold increase in its IC value (5 (cid:3)M) tionrange,wasaround8.9kDa.Thiscorrespondedtoanolig-
50
relativetorNaD1andrNaD1(E2A). omerizationstatearound1.7,suggestingthatmoreofthepro-
SAXS Analysis—As the solution NMR structure of NaD1 teinisinadimericstatethanamonomericstate.Inaddition,
indicatedthatthemoleculewaspresentasamonomerduring theradiusofgyrationwascalculatedfromthehighresolution
19966 JOURNALOFBIOLOGICALCHEMISTRY VOLUME287•NUMBER24•JUNE8,2012
DimerizationofNaD1andAntifungalActivity
1.26S);however,rNaD1(K4A)showssignificantlylesspropen-
sitytodimerizerelativetorNaD1,whichpredominantlyexists
asadimer.
DISCUSSION
The formation of dimers and/or higher order oligomers in
antimicrobialpeptideshasbeenproposedasacontributingfac-
tortotheirabilitytodisruptbiologicalmembranes(56–61).In
thisstudy,weemployedanumberofbiophysicalapproachesto
examine the three-dimensional and quaternary structures of
NaD1, both in solid state (i.e. crystals) and in solution. In all
examined environments, NaD1 formed dimers and smaller
amountsofhigherorderoligomers.Thisisconsistentwithpre-
viousobservationsmadeforvariousotherplantdefensins.For
example, Terras et al. (22) demonstrated using SDS-PAGE
analysis that unreduced radish defensins Rs-AFP1 and
Rs-AFP2hadapparentmolecularmassesof15(trimer)and/or
20 kDa (tetramer). This was in contrast to the reduced and
S-pyridylethylatedderivativesofbothproteinsthatmigratedas
singlebandswitharelativemolecularmassof(cid:1)5kDa.Thiswas
similarlydemonstratedforotherplantdefensins,namelyDm-
AMP1, Dm-AMP2, Hs-AFP1, Ah-AMP1, and Ct-AMP1 (21).
Melo et al. (35) also demonstrated that the cowpea defensin,
Cp-thionin,couldexistasdimersalongwithsmallerquantities
ofothermultimersbyMALDI-TOFmassspectrometry.
More recently, Song and colleagues (20, 62, 63) provided
FIGURE6.SAXSanalysisofNaD1.A,log-logplotshowingrawSAXSdatafor
NaD1atfiveconcentrations(0.125,0.25,0.5,1,and2mg/ml;frombottomto experimentalevidence,includingacrystalstructuretosupport
top).B,GuinierplotsofNaD1SAXSdataat0.125,0.25,0.5,1,and2mg/ml adimericconfigurationfortheplantdefensinSPE10.Similarto
(frombottomtotop)withstraightlinefits.
ourstudy,theauthorsobservedadimerofSPE10intheasym-
TABLE2 metricunit(20).However,unlikethe(cid:1)-sheetto(cid:1)-sheetcon-
Smallanglex-rayscatteringdata figuration of NaD1 crystal form B (Fig. 8A), the two SPE10
Oligomerization monomersarearrangedinasidebysidemannerwiththe(cid:2)-he-
Guinier (monomeris5.3 lixofonemonomercontactingthe(cid:1)-sheetofthesecondmono-
Concentration I(0) R range Mass kDa)
g
mer (Fig. 8B), resulting in an extended and slightly twisted
mg/ml absolute Å Å kDa
calibration molecularsurface.Dimerizationburies606Å2ofsolventacces-
0.125 0.00071 12.6 0.015–0.11 9.02 1.70 siblesurfaceareaandresultsintheformationofintermolecular
0.25 0.0014 12.9 0.015–0.11 8.81 1.66
0.5 0.0029 14.1 0.015–0.11 9.23 1.74 hydrogen bonds involving Arg36–Asp21, Arg40–Asp22, Glu4–
1 0.0056 13.4 0.015–0.11 8.87 1.67 Lys25,Asn5–Asn26,Asn17–Asp37,andGly18–Asp37(20).
2 0.011 12.9 0.015–0.11 8.56 1.61
Todate,therelevanceofdimerformationinplantdefensins
hasnotbeenexamined.However,ithasbeensuggestedthatfor
structures of monomer and dimer. The R of the monomeric somecationicantimicrobialproteins,suchasthemammalian
g
structurewascalculatedtobe10Å,althoughthedimerwas15 defensins,dimericconfigurationsrepresentthefunctionalbio-
Å. The measured R from the data was 13.2 Å or somewhere logical units (56–61). Furthermore, the dimeric protein unit
g
betweenmonomeranddimer. may undergo orchestrated oligomerization to form larger
SedimentationVelocityAnalysisofrNaD1andrNaD1(K4A)— assembliesthatarepertinenttotheabilityofthegivencationic
SedimentationvelocitystudiesofrNaD1andrNaD1(K4A)were antimicrobialproteintokill.Thishypothesisiscertainlycentral
employed in the analytical ultracentrifuge to further validate tomodelsinwhichcationicantimicrobialproteinsoperatevia
theobservationsfromourchemicalcross-linkingexperiments thecreationofoligomericpores.
andSAXSanalysis.Theabsorbanceversusradialpositionpro- Consideringthatoligomerizationofdefensinsmayplayakey
filesatdifferenttimepointswereinitiallyfittedtoacontinuous role for their biological function and that their initial site of
c(s) distribution model (Fig. 7). The c(s) distribution analyses activityiseitheracellwallorplasmamembrane,ourobserva-
yielded excellent fits to the data as demonstrated by the low tion of a dimer coupled with an extended positively charged
root mean square deviation values of 0.00464 for rNaD1 and surfaceareaononesideoftheNaD1dimerissignificant.Fungal
0.00410forrNaD1(K4A),aswellasthelowRunstestZvaluesof cellwallsaredecoratedwithnegativelychargedglycoproteins
1.50 for rNaD1 and 0.86 for rNaD1(K4A). The resulting c(s) (64,65),anddimericNaD1maybeimportantforinitialbinding
distributionsshowthatbothrNaD1andrNaD1(K4A)existasa to these cell wall components as a precursor step for
mixtureofmonomers(s(cid:1)0.57–0.77S)anddimers(s(cid:1)1.05– oligomerization.
JUNE8,2012•VOLUME287•NUMBER24 JOURNALOFBIOLOGICALCHEMISTRY 19967
DimerizationofNaD1andAntifungalActivity
FIGURE7.SedimentationvelocityanalysisofrNaD1andrNaD1(K4A).Continuoussedimentationcoefficient,c(s)(Svedberg(cid:4)1),distributionplottedasa
functionofsedimentationcoefficient(Svedberg)forrNaD1(A)andrNaD1(K4A)(B).Thec(s)analysiswasperformedusingtheprogramSEDFIT(53–55)ata
resolutionof200,withs (cid:7)0.1S,s (cid:7)5S,andaconfidencelevel(pvalue)(cid:7)0.5.Insets,residualsplottedasafunctionofradialposition(cm)forthec(s)
min max
distributionbestfitsshownineachrespectivepanel.
FIGURE8.DimerassembliesforNaD1comparedwithSPE10plantdefensinandhumandefensinsHNP-3andHBD-2.Allviewsaredownthedimer
interfaces.ThePDBaccessionnumbersforSPE10,HNP-3,andHBD-2are3PSM,1DFN,and1FD3,respectively.
An important functional link between NaD1 dimerization fungalactivitywasdemonstratedastheK4Amutantdisplayed
andantifungalactivitywasstronglysuggestedbysite-directed agreatlyreducedcapacitytokillthefilamentousfungusF.oxy-
mutagenesisofLys4.Asdescribed,thesolvedstructureofNaD1 sporum with an approximate 5-fold increase in its IC value
50
crystal form B indicated that Lys4 plays a crucial role in the comparedwithnativeandrecombinantNaD1.Examinationof
monomer-monomer interaction to form the dimeric confor- theaminoacidsequencesofallclassIIdefensinsdescribedtodate
mation.Indeed,thesubstitutionofLys4withalanineresultedin (7)indicatesthatLys4istotallyconserved.Assuch,itistemptingto
amarkedlyreducedabilityofNaD1toformdimers,asshownby speculatethatallclassIIdefensinsmayhavetheabilitytodimerize
chemical cross-linking and sedimentation velocity analyses. andthatdimerizationmaybeimportantfortheirrespectivefunc-
TheimportanceoftheNaD1dimericconfigurationforitsanti- tions(e.g.byenhancingtheirantifungalactivity).
19968 JOURNALOFBIOLOGICALCHEMISTRY VOLUME287•NUMBER24•JUNE8,2012
DimerizationofNaD1andAntifungalActivity
Itisinterestingtocomparethefindingsdescribedhereinfor plausible to suggest that electrostatic attraction mediated by
NaD1dimerizationwithdefensinsfromotherspeciessuchas anionicsurfacecomponents(suchasphospholipids,sphingo-
those from mammals. The mammalian defensins are defined lipids,orglycoproteins),maybeimportant.
underthreedistinctstructuralsubfamilies,(cid:2),(cid:1),or(cid:6),basedon Itisknownthatfortheseeddefensinsfromradish(Rs-AFP2)
theirdifferencesinsize,theplacementandconnectivityoftheir anddahlia(Dm-AMP1),interactionswithspecificsphingolip-
(six)cysteines,thenatureoftheirprecursors,andtheirsitesof ids(glucosylceramideandmannose-(inositolphosphate) -cer-
2
expression(66–71).Itisworthnotingherethatplantdefensins amide,respectively)onfungalplasmamembranesisrequired
do not share primary or three-dimensional structures with fortheirantifungalactivity(83,84).Theyareabletopermeabi-
thesemammaliannamesakesexceptthattheyallhaveanimpli- lizethefungalmembraneandinduceCa2(cid:2)influxandK(cid:2)efflux.
catedroleininnatehostimmunity.Indeed,itwasinthecontext Giventhatfungigrowfromthetipandrequirethemaintenance
of their defense role that the name “defensin” was originally ofanintracellularCa2(cid:2)concentrationgradienttodrivepolar-
coined(72,73). ized growth (85, 86), Thevissen et al. (87) suggested that the
Human (cid:2)-defensins or human neutrophil peptides exhibit growth inhibition may be due to dissipation of this gradient.
broadantimicrobialactivityagainstGram-negativeandGram- Interestingly,otherplantdefensinsfromalfalfaandcornhave
positive bacteria, fungi, and enveloped viruses (74) and have beenreportedtoblockionchannels,althoughinteractionwith
alsobeenlinkedwithadaptiveimmunityasimmunostimulat- fungalCa2(cid:2)channelshasnotyetbeendemonstrated(37,38).It
ingagents(67,68).Thethree-dimensionalstructuresofseveral is noteworthy that scorpion toxins, which are bone fide ion
(cid:2)-and(cid:1)-defensinshavebeensolved(57–59,61,75–82).Inter- channelblockers,alsodisplaytheCS(cid:2)(cid:1)motifthatisconserved
estingly, the structures of several human defensins, including intheplantdefensins(88–90).
HNP-1,HNP-3,andHBD-2,indicatedthepresenceofdimers Numerous models have been proposed in the literature to
and/ormultimersintheasymmetricunits.ForHNP-1,Weiet accountforthepotentialmolecularmechanismsofactionfor
al. (61) recently suggested a functional link between multim- various cationic antimicrobial peptides on target membranes
erization and antibacterial activity as mutants with reduced (91). Based on the crystal structure of HNP-3, Hill et al. (57)
capacitytoself-associateexhibitedpoorbactericidalactivity. proposedthreepossiblemodelsforHNP-3-membraneinterac-
HNP-3 (PDB code 1DFN) consists of a triple-stranded tionasfollows:(i)awedgemodel,(ii)adimerporemodel,and
antiparallel(cid:1)-sheetthatformsasix-strandedsheetaspartofa (iii)ageneralporemodel.Inallproposedmodels,thedimeris
symmetricaldimer(57).Thedimerisstabilizedbyfourdirect the protomeric unit, building from a single dimer, to two
hydrogen bonds between the (cid:1)2-strands of the monomers, stacked dimers onto a large annular pore involving multiple
threehydrogenbondsmediatedthroughwatermolecules,and dimers. Intriguingly, these models (or variations thereof) are
several hydrophobic interactions (Fig. 8C). This is somewhat conceivableforHNP-3andotherdimer-formingcationicanti-
reminiscentofthe(cid:1)-sheetpackingobservedforNaD1crystal microbial proteins such as NaD1 and may indeed represent
form B. Moreover, HNP-3 was also able to form dimers and transitionstates.Assuch,factorsincludingproteinconcentra-
higheroligomersbyequilibriumcentrifugationinsolutioncon- tion,thelocalenvironmentofthetargetmembrane(e.g.lipid
ditions that matched that used for the crystallization (except composition),thevolumeofthemolecule,itsstructure,andits
thatPEG8000wasomitted)(57). oligomeric state in solution and in membranes would make
HBD-2consistsofatriple-strandedantiparallel(cid:1)-sheetand importantcontributionstodeterminingwhichmodeloperated.
an(cid:2)-helixbutinan(cid:2)(cid:1)(cid:1)(cid:1)arrangement((cid:1)(cid:2)(cid:1)(cid:1)forNaD1)(59), Insummary,NaD1adoptsadimericconfigurationinsolu-
whichwasobservedintwocrystalforms(PDBcodes1FD3and tionunderphysiologicalconditionsthatenhancesitsantifungal
1FD4). The orthorhombic form contained four monomers in activity.ThedimerizationofNaD1resultsintheformationofa
the asymmetric unit, formed by two topologically identical largecontinuouspositivelychargedsurface.Thisregionofthe
dimers (Fig. 8D). Its dimer interface is also mediated by two NaD1dimermaybeimportantformediatinginteractionswith
hydrogen bonds and several hydrophobic (van der Waals) targetcellsurfacessuchasthefungalcellwallandplasmamem-
interactionsfromthe(cid:1)1-strandsofthemonomers,resultingin brane, thereby promoting cell permeabilization and entry of
a six-stranded (cid:1)-sheet for the dimer as in NaD1 and HNP-3. NaD1intothecelltoreachintracellulartargets.
The monoclinic form contained two octameric assemblies
within the asymmetric unit, which were proposed to be the Acknowledgments—We thank the staff at the Bio21 Collaborative
stable,nativequaternarystructureofHBD-2foractivity(59), CrystallizationCentreforthesetupofcrystalscreens.Diffractionand
although no additional evidence for the presence of such an scatteringdataweremeasuredattheMXandSAXS/WAXSbeam-
oligomer in solution was presented. Intriguingly, subsequent linesattheAustralianSynchrotron.WethankthestafffromtheMX
dynamic light scattering experiments revealed that concen- teamforassistancewithx-raydiffractiondatacollection.
trated solutions of HBD-2 mainly formed dimers with other
aggregatedforms(59).ThisledHooveretal.(59)tosuggestthat
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