Molecular characterisation of the Bacillus subtilis SpbK antiphage defence system

Domain: chemistry
Published: 2025-12-29
Editorial outcome: major_revision

Abstract
Bacteriahaveavarietyofmechanismsforlimitingpredationbyphages.SpbK
isaToll/interleukin-1receptor(TIR)domain-containingantiphagedefence
proteinfromBacillussubtilisthatprovidesprotectionagainstthetemperate
phageSPβviaabortiveinfection.HerewestructurallycharacteriseSpbKand
itsinteractionwiththeSPβproteinYonE.WedemonstratethatSpbKisan
NADasethatproducesbothADP-ribose(ADPR)andcanonicalcyclicADPRwith
aN1-glycosidicbond(cADPR,alsoreferredtoasN1-cADPR).Combiningcryo-
EM,insilicopredictions,site-directedmutagenesis,andphageinfection
assays,weshowthatformationoftwo-strandedhead-to-tailassembliesof
SpbKTIRdomainsisrequiredforbothNADaseactivityandantiphagedefence.
WealsodemonstratethatYonEisadodecamericportalproteinthatactivates
theNADasefunctionofSpbKbyfacilitatingTIRdomainclustering.Collec-
tively,ourresultsprovideinsightintohowbacterialTIRNADasesrecognise
phageinfection.

Introduction
Bacteria have highly diverse and sophisticated immune systems to
protectthemselvesfrompredationbyphages.Restrictionmodifica-
tionandCRISPR-Cassystemsarewellknown,butoverthepastdecade,
manynewdefencemechanismshavebeendescribed.Theseinclude
immune systems that disrupt membrane integrity1,2, produce small
molecule inhibitors of phage propagation3–5, rely on reverse tran-
scriptionofsmallRNAs6–8orproteolyticcleavage9,modifynucleotides
and viral DNA10,11, and deplete bacteria of nucleotides required for
phagereplication12,13orenergymetabolism14–19.

Acommonlyusedstrategyinmanyoftheseimmunesystemsis
abortiveinfection,inwhichtheinfectedcellcommitssuicidebefore
thephagecancompleteitsreplicationcycle,therebyprotectingthe
bacterial community by preventing the spread of phages to neigh-
bouringcells20.Severalbacterialimmunesystems,includingThoeris,
CBASS (cyclic-oligonucleotide-based antiphage signaling system),
Pycsar (pyrimidine cyclase system for antiphage resistance), Avs
(antiviralSTANDs),DSR(defence-associatedsirtuin),SIR2-HerA,and
SPARTA(shortprokaryoticargonauteTIR-APAZ),havebeenshownto
triggerabortiveinfectionbydepletingthecellsoftheessentialenergy
metabolitenicotinamideadeninedinucleotide(NAD+)14,16–19,21,22.These
systems feature either SIR2 (silent information regulator 2) or TIR
domains that can rapidly hydrolyse NAD+ into nicotinamide (NAM)
andADPR.

TIR domains are found in animal, plant, and bacterial immune
systems,andfunctionthroughself-associationandhomotypicinter-
actions with other TIR domains23,24. They were first described as
protein-proteininteractionmodulesmediatingsignallingdownstream
of the Toll-likereceptor (TLR)and interleukin-1 receptor families in
animals25, where they induce signalling via higher-order
oligomerisation26–30.However,studies ofthe neurodegenerative dis-
easetherapeutictargetSARM1(sterilealphaandTIRmotifcontaining
1),plantimmunereceptors,andmanybacterialTIRdomain-containing
proteins revealed thatTIRdomains have self-association-dependent
NADase activity15,17,31–35. SARM1 is the founding member of the TIR
NADasefamilyandcatalysesthehydrolysisofNAD⁺toproduceADPR
andN1-cADPR33.ThisactivityleadstorapidNAD⁺depletion,triggering
axondegeneration.Inbacterialimmunesystems,TIRdomainNADases
can either serve as effectors that rapidly deplete cellular NAD+ or
producers of immune signalling molecules such as histidine con-
jugated ADPR (His-ADPR)36,37, and the cADPR isomers 3’cADPR18,32,38,
2’cADPR32,39andN7-cADPR9,thatdifferfromN1-cADPRinthenatureof
thecycliclinkagebetweentheanomericpositionofthedistalribose
andtheadenosinemoiety(Fig.S1a).

ManystrainsofBacillussubtilisarelysogenicforthetemperate
phage SPβ40. Additionally, the integrative and conjugative element
ICEBs1 encodes a TIR domain NADase, SpbK, that is necessary and
sufficient for protection against SPß41. The anti-SPß phenotype and
NADaseactivityofSpbKareactivatedbytheproductoftheSPßgene
yonE.YonEishomologoustoportalproteinslikeGp6ofphageSPP142.
PortalproteinsarerequiredforprocapsidassemblyandcorrectDNA
packagingandarethusessentialforphageproduction41.

SpbKcausesdepletionofNAD+inB.subtiliscellsviainteraction
withYonE43.Here,wepresentastructuralanalysisoftheSpbKanti-
phage defence system. We show that SpbK has self-association-
dependentNADaseactivityandproducesbothADPRandN1-cADPR.
SpbKexistsasastabledimerinsolutionthatcanfurtherself-associate
into filaments. A 3.3Å resolution cryoEM structure of the filament
revealedatwo-strandedhigher-orderassemblyarrangementofSpbK
TIR domains that resembles the TIR domain assembly formed by
STING-TIR41. Mutagenesis studies demonstrate that the interfaces
withintheseassembliesarerequiredforbothSpbKNADaseactivity
andantiphagedefence.WealsodeterminedthecryoEMstructureof
dodecamericYonEat3.3ÅresolutionandfoundthatYonEinteracts
directly with SpbK and activates its NADase function. AlphaFold
modellingcombinedwithmutagenesisandgel-filtrationassaysfurther
demonstratethattheclipdomainsinthedodecamericportalserveas
docking stations for the N-terminal domain of SpbK, driving SpbK
clustering and activation of its NADase function. Collectively, our
studies provide a detailed structural model for SpbK activation
byYonE.

Results
SpbKisanNADasethatproducesADPRandN1-cADPR
SpbKconsistsofanN-terminal4-helixbundledomain(residues1–94;
SpbKNtd), an 18-residue flexible linker, and a C-terminal TIR domain
(residues113–266;SpbKTIR)(Fig.1a).Weexpressedandpurifiedfull-
lengthSpbKasanMBP(maltose-bindingprotein)fusionprotein(MBP-
SpbK).Real-timeNMR-basedNADaseassays confirmed thatSpbKis
enzymaticallyactiveandproducesNAM,ADPRandN1-cADPR(Figs.1b,
candS2).SimilartoN1-cADPRproducedbythemammalianNADase
CD3844, N1-cADPR produced by SpbK was unstable in the reaction
mixtureandeventuallyconvertedtoADPR.Consistentwiththeenzy-
mology of TIR domain NADases32,37,45, SpbK also catalysed base-
exchangereactionswithvariousheterocyclicbases,includingimida-
zole, IP6C46, 5-iodo-isoquinoline (1), 1,2-dihydro-2,7-naphthyridin-1-
one(2),and8-amino-isoquinoline(3)(Fig.S1b,c).

N1-cADPRisproducedinSpbK-containingcellsafterSPβphage
infectionandYonEexpression
SinceN1-cADPRwasnotpreviouslyreportedasaproductofSpbKin
cells,weaskedwhetheritwasanartefactoftheinvitroassayorifN1-
cADPRisactuallyproducedinSpbK-expressingcellsfollowinginfec-
tion with SPβ. We examined the cell lysate by liquid
chromatography–tandemmassspectrometry(LC-MS/MS).Cellshar-
bouring SpbKgenerateda peakwith m/zconsistentwithN1-cADPR
(Fig.2a).ThesamepeakwasobservedincellsexpressingbothYonE
andSpbK,intheabsenceofphageinfection(Fig.2b).Asobservedfor
N1-cADPR in vitro, these peaks were short-lived. The MS/MS frag-
mentationpatternofthesepeaksmatchedthatofN1-cADPR(Fig.2c).
Unexpectedly,thematerialelutedastwopeakswithidenticalm/zand
fragmentationpatterns(Fig.2a–c).Wehypothesizedthateithertwo
cADPRvariantswereproducedbythecell,orcomponentsinthecell
lysate (e.g., divalent metal cations) caused the N1-cADPR to adopt
differentconfigurationswithdifferentretentiontimes.Todiscriminate
between these possibilities,we added purifiedN1-cADPRtothe cell
lysatethathadbeenfilteredtoremove>3kDacomponentsandlacked
endogenous N1-cADPR. If components in the lysate cause different
configurationstoform,thenthisexogenousN1-cADPRshouldeluteas
two peaks. Indeed, we observed two peaks (Fig. 2d). Therefore, we
concludedthatanunknowncomponentofthecelllysateinfluences
themigrationoftheanalyte,splittingitintotwoseparatepeaks.As
further confirmation of this hypothesis, we pre-purified the lysate
beforerunningonLC-MS/MSandfoundthatthetwopeaksconverted
toone(Fig.2e).Importantly,co-injectionofthepureN1-cADPRstan-
dard with this purified induced cell lysate revealed no new peaks,
indicatingthattheobservedpeaksmatchedN1-cADPR(Fig.2e).

SpbKassemblesintofilaments
We found that MBP-SpbK exists as a dimer in solution (Fig. 3a,
TableS2).SpbKNtdismonomeric(Fig.3b,TableS2),suggestingthatthe
TIRdomainisrequiredforMBP-SpbKdimerisation.Wealsoobserved
thatMBP-SpbKsolutionsbecometurbidwhentheMBP-tagisremoved
via TEV protease cleavage, suggesting higher-order oligomerisation.
Sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-
PAGE)analysisrevealedthatmorethan90%ofSpbKwaspresentinthe
insoluble fraction when MBP-SpbK (40µM)was incubated with TEV
protease(Fig.3c).Negative-stainelectronmicroscopy(EM)ofMBP-
SpbKincubatedwithTEVproteaserevealedthepresenceoflongfila-
mentswithadiameterof15–20nm(Fig.3d).Wealsoobservedthat
MBP-SpbKhassignificantlyhigherNADaseactivityinthepresenceof
TEV protease (Fig. 3e), indicating that filamentation enhances this
activity.

ThecryoEMstructureofSpbKrevealsthemolecularmechanism
underlyingitsself-association-dependentNADaseactivity
WesolvedthestructureoftheSpbKfilamentusingcryoEM(Figs.4a,b
and S3;Table S3).The three-dimensionalreconstruction of the fila-
ment,atanoverallresolutionof3.3Å,hasD1symmetry,ahelicalriseof
8.9Åandtwistof79.3°persubunit.Thefilamentisahollowtubewith
outerandinnerdiametersof~210Åand~160Å,respectively(Fig.4b).
TheSpbKcryoEMdensitymapallowedustobuildamodelcovering
residues111-266,whichcorrespondtotheTIRdomainofSpbK(Fig.4c,
d). No density was observed for the N-terminal domain and linker
region,whichpresumablyadoptsadynamicconformation.Eachpro-
tomer of the filament shows the canonical TIR domain structure,
consistingoffiveβ-strandssurroundedbyfiveα-helices;theloopsare
named based onthe elements of secondarystructure they connect
(Fig.4d).TheTIRdomainofSpbKalsohasanadditionalhelix,αF,that
foldsbackontothesurfacerealregionencompassingtheαAandαEheli-
ces. Among the known TIR domain structures, Maribacter poly-
siphoniaeSPARTA(PDB:8SPO)displaysthehighestsimilaritytoSpbK
(RMSDof2.6Åfor133Cαatoms)(Fig.4d).

TheSpbKfilamentiscomposedoffiveprotofilaments;eachhav-
ingtwoantiparallelstrandsofTIRdomainsinasimilararrangementto
theSfSTINGfilament47(Fig.4c,e).TheSpbKprotofilamentshavetwo
majorinterfaces(Fig.4f).Thefirstinterfaceistheintrastrandhead-to-
tail“BE”interface,withaburiedsurfaceareaof771Å2.TheBEinterface
isasymmetricandinvolvesresiduesintheBBloopofoneprotomer
(TIR-A)interactingwithresiduesoftheDDloop,βEstrandandαE-helix
oftheother(TIR-B)protomer(Fig.4f).Atthecoreofthisinterface,
D158oftheBBloopformshydrogenbondswithS204intheβDstrand
andK240intheαE-helix.Theinterfaceisalsostabilisedbythreemain
chain hydrogen bonds (D158-K224, E159-K224 and I161-N222). This
head-to-tail association creates the composite active site of TIR
NADases, consisting of two TIR subunits (TIR-Aand TIR-B)26,32,45. An
AlphaFold3modelofaSpbK:NAD+hexamerisalmostidenticaltothe
protofilament structure (Fig S3e–g), and the NAD+ binding mode is
similartothatof1ADand3ADincomplexwithSARM1TIRandAbTirTIR,
respectively32,45. The model suggests that residues S121, S122, T148,
I155, I161, F162, L165, Y188, and E192 in TIR-A may be involved in
coordinating binding to the nicotinamide base, the nicotinamide
riboseandthediphosphategroupofNAD+(Fig.4g).IntheSARM1TIR
andAbTirTIRcomplexstructures,theadeninebaseformsinteractions
withatryptophansidechaininTIR-B.Therearenoaromaticresidues
in this region of SpbK TIR-B, but the AlphaFold 3 model of the
SpbK:NAD+ hexamer suggests that K224 may be involved in the
interactionwiththeadeninebase.

Thesecondinterfaceistheinterstrand“CC”interfacebetweenthe
two strands of the protofilament, with a buried interface area of
1902Å2(Fig.4f).LiketheSfSTINGandonetypeofMyD88filaments47,48,
thisinterfaceissymmetricandhighlyconservedresiduesintheαC
helix(N191,G194andW197)formextensivepackinginteractions.The
SpbK interstrand interaction involves only one molecule from each
strandanddoesnotcontributetostabilisingtheintrastrandBEinter-
face.ThiscontrastswiththeSARM1TIRandAbTirTIRassemblies,where
the interstrand interface involves one molecule engaging with two
moleculesfromtheoppositestrand,therebystabilisingtheintrastrand
BEinterface(Fig.4e).

TheinterfacesbetweentheSpbKprotofilamentsinvolveresidues
predominantlylocatedintheαEandαFhelices(“EF”interface)and
haveaburiedsurfaceareaof774Å2(Fig.4f).Wespeculatethatthese
inter-protofilament interactions are most likely equivalent to non-
biologicalcrystalcontacts27,30,49.

Overall,ourstructuraldatasuggestthat,likeotherTIRNADases
such as SARM1, AbTIR, STING-TIR, SAVED-TIR, and plant
NLRs15,32,45,47,50,51,SpbKTIRdomainsformtwo-strandedassemblieswith
compositeactivesitesattheintrastrandBEinterfaces.

SpbKfilamentationcontrolsNADaseactivityandantiphage
defence
Using site-directed mutagenesis, we made altered residues in the
interstrand and intrastrand interfaces. The interstrand aC helix
mutantsN191R,G194R,andW197Aweredefectiveinformingfilaments
basedonnegative-stainEMofallthreemutants(Fig.5a,S4a).Mass
photometryandMALSanalysesalsoshowedthattheN191RandW197A
mutants were monomeric while the G194R mutant formed both
monomers(P1)anddimers(P2)(Figs.5bandS4b,TableS2).Impor-
tantly, these three mutants were also defective in NADase activity
(Fig.5c).TheD158RintrastrandBBloopmutantformedstabledimers
andassembledintofilaments,althoughthesewereshorterthanthose
ofwild-typeSpbK(Fig.5a).ThismutantwasalsodefectiveinNADase
activity(Fig.5c),indicatingthattheD158Rsubstitutionperturbsthe
intrastrandinterface,resultinginanalternativemodeofoligomerisa-
tion.Thisalterationlikelycompromisesthestructuralintegrityofthe
active site, which resides at the intrastrand BE interface, thereby
abrogatingitscapacitytobindandhydrolyseNAD⁺.Wealsotestedthe
D158R, N191R, G194R, and W197A mutants in B. subtilis. All four
mutationsabolishedantiphagedefenceagainstSPβphages(Fig.5d–g).
SPβwasabletogrowandmakeplaquesoncellsexpressingthesespbK
mutants,incontrasttotheabsenceofphagegrowthincellsexpressing
wild-typespbK(Fig.5d,e).Allofthemutantsexhibitedgrowthcom-
parableto wild-type spbK(Fig. 5f),and consistentwith their lossof
NADaseactivity,theydidnotinducegrowtharrestinthepresenceof
YonE(Fig.5g).Collectively,ourmutagenesisdatademonstratethatthe
observed arrangement of TIR domains within the SpbK filament is
importantforbothitscatalyticactivityandphagedefence,andthatan
intactBBloopisrequired.

YonEisadodecamericportalprotein
TheSPβphageproteinYonEactivatestheantiphagedefenceactivityof
SpbK and is predicted to be a portal protein43. We expressed and
purified a truncated YonE construct lacking the first 45 residues
predictedtobedisordered52.Negative-stainEMrevealedring-shaped
YonE particles with a diameter of 20nm (Fig. 6a), and mass photo-
metry analysis showed thatYonE exists asa dodecamer in solution
(Fig.6b).InitialcryoEManalysisconfirmedthatYonEformsadode-
camericassemblyfeaturingacentralchannelcharacteristicofportal
proteins(Fig.S5a).However,thedatasetexhibitedpreferentialparticle
orientation, which prevented detailed structure determination. To
addressthislimitation,weacquiredanadditionaldatasetusinga30°
tilt angle. Merging the tilted and untilted datasets led to a recon-
struction atan overall resolution of 3.3Å (Figs.6c and S5b–e). The
resulting cryoEM map, although moderate in quality, allowed us to
buildamodelencompassingresidues46–248and369–466ofYonE
(Fig.6d).Theseregionscorrespondtothewing(residues60–240and
369–414) and crown (residues 415–465) domains. Densities for the
stemandclipdomainsandthetunnelloop(residues249–368)were
absent. We speculate that this lack of density arises from both the
preferredorientationissueandincreasedflexibilityofthestemand
clip domains when YonE is not embedded within the procapsid or
associatedwiththeterminaseortailmachinery.

Togainstructuralinsightintothemissingregions,wemodelled
thedodecamericassemblyusingAlphaFold2(Figs.6eandS6).The
predictedwingandcrowndomainscloselymatchthecryoEMstruc-
ture.Thestemdomaincomprisestwolonghelicesconnectedbythe
clipdomain,whichconsistsofthreeβ-strandsandoneα-helix(Fig.6e),
formingthebaseoftheportalassembly.Themodelsuggeststhatall
four subdomains (wing, stem, clip, and crown) contribute to inter-
subunitinteractionswithinthedodecamer.AccordingtoDALIstruc-
turecomparisoncalculations53,closestructuralrelativesincludeportal
proteins from the PhiT myophage, siphophage lambda and bacter-
iophageG20C(Fig.6f).

YonEactivatestheNADasefunctionofSpbK
IncubationofMBP-SpbKwithYonEresultedinsignificantlyenhanced
NADaseactivity(Fig.7a),demonstratingthatYonEinteractsdirectly
with SpbK to activate its NADase function. The D158R intrastrand
interfacemutantandtheN191R,G194R,andW197Ainterstrandinter-
facemutantsdidnotdisplayNADaseactivityinthepresenceofYonE,
suggestingthatYonE-inducedSpbKclusteringcannotovercomethe
destabilisingeffectsofthesemutantsonTIRdomainoligomerisation
(Fig.S7a).

LoyoandGrossmanrecentlyreportedthattheN-terminaldomain
of SpbK is required for co-immunoprecipitation of YonE and SpbK
fromB.subtilislysate43.TheyalsofoundthataYonEclipdomainmutant
(A291P),whichfacilitatesSPβescape,didnotco-immunoprecipitate
withSpbK.Weusedanalyticalgel-filtrationassaystoconfirmthatYonE
interactsdirectlywithSpbKNtd(Fig.7bandS7b,c).BecauseSpbKexists
asadimerinsolution,wetestedSpbKNtdbyitselfandfusedtoglu-
tathione S-transferase (SpbKNtd-GST) and the tandem sterile alpha
motif(SAM)domainsofhumanSARM1(SpbKNtd-hSARMSAM).GSTand
hSARM1SAMexistasastabledimerandoctamer,respectively31,54.SDS-
PAGEanalysisofgel-filtrationfractionsshowsthatallthreevariantsof
theSpbKN-terminaldomainco-elutewithYonE.Wealsoexpressed
andpurifiedtheYonEA291Pmutant.MBP-SpbKincubatedwithYonE
A291P exhibits weak NADase activity, compared to MBP-SpbK incu-
batedwithwild-typeYonE(Fig.7c).TheA291Psubstitutionalsowea-
kenedtheinteractionwithSpbKNtd-hSARMSAM(Fig.7d).Theseresults
areconsistentwithLoyoandGrossman’sobservationthattheA291P
mutant only partially escapes SpbK-mediated defence43. Together,
these results demonstrate that YonE activates SpbK by interacting
directlywithitsN-terminaldomain.

ToprovidestructuralinsightintohowSpbKNtdinteractswithYonE,
wemodelledtheirinteractionusingAlphaFold3(Figs.7e,fandS8).In
themodel,SpbKNtdinteractswiththeclipdomainsofYonEandformsa
dodecameric ring at the base of the portal (Fig. 7e). The YonE clip
domainhelixpacksagainsttheα3-α4loopandtheendoftheα1helixin
SpbKNtd.BothF69andF71intheα3-α4loopofSpbKNtdformextensive
hydrophobiccontactswiththeclipdomain(Fig.7f).TheSpbKNtdmole-
culesalsoformasymmetricinteractionswitheachother;theα1andα4
helicesofoneprotomerinteractwiththeα2andα3helicesoftheother
protomer(Fig.7f).A291islocatedintheclipdomainhelixofYonE,and
substitution of this residue with proline is likely to induce structural
changesinthishelixandalterinteractionswithSpbKNtd,consistentwith
our NADase and gel-filtration data. To further validate the AlphaFold
model, we tested the interaction between YonE and two SpbKNtd
mutantswithdoublealaninesubstitutions(F69,F71andF9,Y48)ingel-
filtrationassays(Fig.7g).Bothmutantsdisruptedtheinteractionwith
YonE, suggesting that both YonE clip domain:SpbKNTD and
SpbKNTD:SpbKNTDinteractionsarerequiredforstablecomplexformation.
Together,theseresultsdemonstratethattheYonEclipdomainservesas
aplatformforrecruitmentandoligomerisationofSpbK.

Methods
Cloning
The cDNA of YonE (residues 46–506), MBP-SpbK, SpbK N-terminal
domain (residues 1–94, SpbKNtd), SpbKNtd—SARM SAM domain (resi-
dues 409–548) fusion protein (SpbKNtd-SARMSAM) and SpbKNtd—GST
fusionprotein(SpbKNtd-GST)weresynthesisedasgBlocks(Integrated
DNA Technologies). Mutants of YonE (A291P), MBP-SpbK (D158R,
N191R,E192A,G194RandW197A)andSpbKNtd-SARMSAM(F69AF71Aand
F9AY48A)werealsosynthesisedasgBlocks(IntegratedDNATechnol-
ogies).AllprimersusedforcloningwereobtainedfromIntegratedDNA
Technologies.YonE(wild-typeandmutants),SpbKNtd,SpbKNtd-SARMSAM
(wild-typeandmutants)andSpbKNtd-GSTwereamplifiedbyPCRand
clonedintothepMCSG7vectorusingligation-independentcloning68.
MBP-SpbK(wild-typeandmutants)wasclonedintothepET28bvector
using the Gibson Assembly reaction69. Pure plasmids were prepared
usingtheQIAprepSpinMiniprepKit(Qiagen),andthesequenceswere
confirmedbytheAustralianGenomeResearchFacility.

Proteinproductionandpurification
Wild-typeandmutantSpbKandYonEproteins(TableS4)werepro-
duced inE.coliBL21(DE3)cells,usingtheautoinductionmethod70.
Theywerepurifiedtohomogeneity,usingacombinationofimmobi-
lised metal-ion affinity chromatography (IMAC) and size-exclusion
chromatography(SEC).Thecellsweregrownat37°C,untilanOD600
of0.6–0.8wasreached.Thetemperaturewasthenreducedto20°C,
andthecellsweregrownovernightforapproximately16h.Thecells
wereharvestedbycentrifugationat5000× gat4°Cfor15min,and
the cell pellets were resuspended in 2–3mL of lysis buffer (50mM
HEPESpH8.0,500mMNaCl)pergofcells.Theresuspendedcellswere
lysed using a digital sonicator and clarified by centrifugation
(15,000×g for 30minutes). The clarified lysate was supplemented
withimidazole(finalconcentrationof30mM)andthenappliedtoa
nickelHisTrapcolumn(Cytiva)pre-equilibratedwith10CVsofwash
buffer(50mMHEPESpH8.0,500mMNaCl,30mMimidazole)ata
rateof4mL/min.Thecolumnwaswashedwith10CVsofwashbuffer,
followed by elution of bound proteins using elution buffer (50mM
HEPES,pH8,500mMNaCl,250mMimidazole).Theelutionfractions
wereanalysedbySDS-PAGE,andthefractionscontainingtheprotein
ofinterestwerepooledandfurtherpurifiedoneitheraS75HiLoad26/
600column(SpbKNtd)oraS200HiLoad26/600column(YonE,MBP-
SpbK, SpbKNtd-SARMSAM and SpbKNtd-GST) pre-equilibrated with gel-
filtrationbuffer(10mMHEPESpH7.5,150mMNaCl).Thepeakfrac-
tions were analysed by SDS-PAGE, and the fractions containing the
protein of interest were pooled, concentrated (SpbKNtd, 31mg/mL;
wild-type and mutant YonE, 29-42mg/mL; wild-type and mutant
MBP-SpbK, 2–10mg/mL; wild-type and mutant SpbKNtd-SARMSAM,
40-59mg/mL;SpbKNtd-GST,39mg/mL),flash-frozenas10μLaliquots
inliquidnitrogen,andstoredat−80°C.

NMR-basedenzymaticassays
NMRsampleswerepreparedin175μLHBSbuffer(50mMHEPES,pH
7.5,150mMNaCl),20μLD O,and5μLDMSO-d6,resultinginatotal
2
volume of 200μL. Each sample was subsequently transferred to a
3mmBrukerNMRtube.All1HNMRspectrawereacquiredwitheithera
BrukerAvanceNeo600MHzNMRspectrometerequippedwithaQCI-
F cryoprobe, a Bruker Avance III HD 800MHz NMR spectrometer
equippedwithaTCIcryoprobe,oraBrukerAvanceIIIHD600MHz
NMRspectrometerequippedwithaTCIcryoprobeat298K.Tosup-
press resonance from H O, a water suppression pulse programme
2
(P3919GP)usinga3-9-19pulse-sequencewithgradients71wasimple-
mentedtoacquirespectrawithanacquisitiondelayof2sand32scans
persample.Foreachreaction,spectrawererecordedatmultipletime
pointssuchas10min,2h,4h,8h,and16h,dependingoninstrument
availability.AllspectrawereprocessedbyTopSpin™4.4.0(Bruker)and
MNova 14.3.0 (Mestrelab Research). The reaction progression was
calculated based on the integration of non-overlapping resonance
peaks,whichvarydependingonsamplecomposition,frompairsof
reactant and product, such as NAD+ and NAM, respectively. The
detectionlimit(signal-to-noiseratio>2)wasestimatedtobe10μM.

ProductionandpurificationofN1-cADPRfromSpbK
ToconclusivelyverifytheidentityofSpbK-producedN1-cADPR,pro-
duction reactions for N1-cADPR were performed using conditions
similartothe1HNMRNADaseassay.Asolventvolumeof4mLwas
usedforeachreaction,consistingofHBSbuffer(50mMHEPES,pH7.5,
150mMNaCl)with0.5μMMBP-SpbKand5mMNAD+.Thesereactions
wereperformedatroomtemperatureandmonitoredintermittentlyby
1HNMR.Tostopthereactions,theHis -taggedproteinwasremoved
6
byincubatingthemixturewith200μlofHisPurnickel–nitrilotriacetic
acid resin for 30–60min. The resin was subsequently removed by
centrifugationat1500xgfor5min,andthesupernatantwassubjected
to HPLC-based separation to purify the base-exchange products. A
ShimadzuProminenceHPLCequippedwithaSynergi4-μmHydro-RP
80-Åcolumnwasusedforseparation.Themobilephaseconsistedof
phase A (0.04% v/v TFA in water) and phase B (0.04% v/v TFA in
acetonitrile). Different gradients, flow rates, and run times were
applied depending on prior optimisation with individual reaction
mixtures.Productpeakswereconfirmedbycomparisonwithindivi-
dual chromatograms of NAD+, NAM, and ADPR. Fractions corre-
sponding to the N1-cADPR product peaks were collected,
concentrated,lyophilisedandstoredat−20°C.

NMR spectroscopy and high-resolution mass spectrometry
(HRMS)wereemployedtocharacteriseN1-cADPRproducedbySpbK
and compare it with a commercial sample of N1-cADPR purchased
fromMerck(C7344)forstructuralvalidation.About2mgofpurified
N1-cADPRwasdissolvedin200μLofD O.Thesamplewastransferred
2
toa 3mmNMR tube. The Bruker Avance Neo600MHz NMR spec-
trometerwasutilisedtoacquire1H(Fig.S2a),13C,1H-1HCOSY,1H-13C
HSQC,and1H-13CHMBCspectraat298K,whichinformedtheassign-
mentsof1Hand13Cpeaksandcorrelations(TableS1),especiallythose
linking the adenine ring and the two ribose rings (Fig. S2c). HRMS
showedSpbK-producedN1-cADPRhasthesamem/zratioandasimilar
fragmentationpatterntothecommercialsample(Fig.S2b).

Preparationofphage-infectedcelllysateforLC-HRMSanalysis
Lysates were prepared as described previously46, with minor mod-
ificationsdescribedbelow.AnovernightcultureofB.subtilisCMJ82
was diluted 1:50 into 200mL fresh LB. The diluted cultures were
incubatedat37°C,220rpmfor3~4hoursuntilOD ~0.6.40mL
600nm
of the culture was removed as a t=0min sample and immediately
centrifuged at 10,000×g, 4°C for 7min. The supernatant was dis-
carded, and the cell pellet was temporarily stored at −80°C. Then,
200mLofSPBetaclear-plaquephage(~7×109PFUs/mL)wasaddedto
thehostcells to reachMOI>10.Theinfected cellculture was incu-
batedat30°C,220rpm,and40mLoftheculturewasremovedevery
10mintobeimmediatelycentrifugedat10,000×g,4°Cfor7min.The
supernatantwasdiscarded,andthecellpelletwastemporarilystored
at −80°C. The cell pellets were thawed at room temperature and
resuspendedin600µLof100mMsodiumphosphatebuffer(pH=7)+
4mg/mLlysozyme.Afterincubationatroomtemperaturefor10min,
thecellsweretransferredinto2mLtubeswithLysingMatrixB(MP
Biomedicals#116911050)andlysedusinganOmniBeadRuptor12for
2×40sat6m/swithadwelltimeof4mininbetween.Afterlysis,the
tubeswerecentrifugedat20,000×g,4°Cfor5min.Then,500µLof
eachsupernatantwastransferredtoAmiconUltra-0.5CentrifugalFil-
ter Units 3kDa (EMD Millipore #UFC500396) and centrifuged for
20minat13,500×g,4°C.Thefiltratewascollected,and10µLofeach
wasusedforLC-MSanalysis.

PreparationofIPTG-inducedcelllysateforLC-HRMSanalysis
Lysates were prepared as described previously46 with minor mod-
ificationsdescribedbelow.OvernightcultureofB.subtilisCMJ685was
diluted 1:100into 400mL freshLB. The diluted cultures wereincu-
batedat37°C,220rpmfor3~4hoursuntilOD ~0.3.40mLof
600nm
theculturewasremovedasat=0minsampleandimmediatelycen-
trifugedat10,000×g,4°Cfor7min.Thesupernatantwasdiscarded,
and the cell pellet was temporarily stored at −80°C. Then, 3.6mL
100mMIPTGwasaddedtothehostcellstoreachafinalconcentration
of1mM,inducingtheexpressionofYonE.Theinducedcellculturewas
incubatedat30°C,220rpm,and40mLoftheculturewasremoved
every 15min to be immediately centrifuged at 10,000×g, 4°C for
7min. The supernatant was discarded, and the cell pellet was tem-
porarilystoredat−80°C.Thecellpelletswerethawedatroomtem-
perature and resuspended in 600µL of 100mM sodium phosphate
buffer (pH=7)+4mg/mL lysozyme. After incubation at room tem-
perature for10min,the cells were transferredinto2mLtubeswith
LysingMatrixB(MPBiomedicals#116911050)andlysedusinganOmni
Bead Ruptor 12 for2 × 40s at 6m/s with a dwell timeof 4minin-
between.Afterlysis,thetubeswerecentrifugedat20,000×g,4°Cfor
5min. Then,500µLofeachsupernatant wastransferred to Amicon
Ultra-0.5CentrifugalFilterUnits3kDa(EMDMillipore#UFC500396)
and centrifuged for 20min at 13,500×g, 4°C. The filtrate was col-
lected,and10µLofeachwasusedforLC-MSanalysis.

LC-MS/MSanalysisofN1-cADPRinthephage-infectedcelllysate
&IPTG-induced-celllysate
LiquidchromatographyanalysiswasperformedonanACQUITYUPLC
I-ClassPLUSSystemusingaLunaOmega5-μmPolarC18100Åcolumn
(250×4.6mm).ThemobilephaseAwaswater+0.1%(v/v)formicacid,
andthemobilephaseBwasacetonitrile+0.1%(v/v)formicacid.The
flowratewaskeptat0.7mLmin–1andthegradientwasasfollows:0%B
(0–10min), increase to 2.5% B (10–15min), increase to 5% B
(15–16min), increase to 95% B (16–17min), hold 95% B (17–27min),
decreaseto0%B(27–28min),hold0%B(28–38min).High-resolution
electrosprayionisation (HR-ESI) massspectra with collision-induced
dissociation(CID)MS/MSwereobtainedusingaWatersSynaptG2S
QuadrupoleTime-of-Flight(QTOF).The instrumentwasoperatedin
negative ionisation mode. The MS spectrawere obtained onthe Time-
of-Flightanalyserwithascanrangeof300–800Daandanalysedusing
MassLynx4.1software.Them/zofinterestwasfilteredthroughthe
quadrupole,subjectedtoCID(energyramp34–44V),andanalysedon
theTime-of-Flightanalyserwithascanrangeof50–750Da.

Pre-purificationofIPTG-induced-celllysate
The liquid chromatography pre-purification was performed on an
Agilent1260InfinityIIHPLCsystemusingaLunaOmega5μmPolar
C18 100Å column (250×4.6mm). The mobile phase A was water
+0.1%(v/v)formicacid,andthemobilephaseBwasacetonitrile+
0.1%(v/v)formicacid.Theflowratewaskeptat0.7mLmin–1andthe
gradient was as follows: 0% B (0–15min), increase to 100% B
(15–16min),hold100%B(16–26min),decreaseto0%B(26–27min),
hold 0% B (27–37min). 9—12min fractions were collected, dried
underlyophilisationandredissolvedwith100μLwaterforfurther
MSanalysis.

Analyticalsize-exclusionchromatography
Afterincubation,500μLofeachsamplewasloadedontoaS20010/
300column(Cytiva)usinganAktaPuredevice(Cytiva)at0.5mL/min.
Analysisofchromatogramsandimagepreparationwereperformedin
Unicorn7software(Cytiva)andExcel(Microsoft).Peakfractionswere
analysedbySDS-PAGE.

Massphotometry
Massphotometry datawerecollectedonaRefeynOneMP system
(Refeyn Ltd, Oxford, UK). High precision glass coverslips (Mar-
ienfeldLtd)werethoroughlywashedthreetimesusingMilli-Q®H O,
2
ethanol and 2-propanol, followed by drying under a stream of
nitrogengas.Forsampleloading,asilicongasket(RefeynLtd)was
placedontheslide.Tofocustheobjective,16µLofbuffer(10mM
HEPESpH7.5,150mMNaCl)wasplacedinawell.Thereafter,4µLof
the test sample was loaded and mixed by pipetting the volume
up–down five times, followed by immediate measurement. Mass
photometrydatawereacquiredandanalysedusingAcquireMPand
DiscoverMP software (Refeyn Ltd), respectively. A Gaussian curve
wasfittedtotheresultingpeaksinthecontrasthistogram(depict-
ing the number of detected molecules and their corresponding
contrast value), where each peak represented a subpopulation of
molecules with a particular molecular weight (MW). The contrast
valuesofobservedcountswereconvertedintoMWbycalibration
using a control protein of known MW and size; in-house bovine
serumalbumin(BSA),withamonomerat66kDa,adimerat122kDa,
andatrimerat198kDa.

Size-exclusionchromatography–coupledmultianglelight
scattering
ADAWNHELEOSII10-anglelightscatteringdetectorcoupledwithan
OptilabrEXrefractiveindexdetector(WyattTechnology)combined
withaSuperdex2005/150Increasesize-exclusioncolumn(Cytiva)and
connectedtoaProminenceHPLC(Shimadzu),wasusedforSEC-MALS.
Thecolumnwasequilibratedingel-filtrationbuffer,and30μLofthe
purifiedproteinwasrunthroughthecolumnat0.25mL/min.Mole-
cularmasseswerecalculatedusingAstra6.1(WyattTechnology).

Negative-stainEM
4μLofsamplewasplacedonacarbon-coatedcoppergridandincu-
batedfor60s.ThegridwasthenwashedwithMilli-Q®H Oandstained
2
with1%uranylacetatefor60sandair-dried.Theimageswerecollected
on a JEOL JEM-1400Flash TEM transmission EM at ×20,000-60,000
magnificationat80keV.

CryoEMdatacollection
YonE: Purified YonE (0.75mg/mL; 12 μM) was loaded onto a glow-
discharged Quantifoil grid (R1.2/1.3 300-mesh holey carbon film
coated,ProSciTech),blottedfor5.5sunder95%humidityat4°C,and
plunged into liquid ethane, using a FEI Vitrobot Mark 2 automatic
plungefreezer(ThermoFisherScientific).Fordatacollection,movies
(0° and 30° tilt) were acquired on a JEM-Z300FSC (Cryo-ARM300,
JEOLLtd.)operatingatanaccelerationvoltageof300keVequipped
with an in-column Omega energy filter set with a slit opening of
20eV. Movies were recorded using a K3 Summit direct electron
detector(Gatan)atamagnificationof×100,000correspondingtoa
pixelsize0.48Åatthespecimenlevel.Allmovieswereexposedusing
atotaldoseof40e−/Å2over40framesandadefocusrangebetween
−0.5and−2.5µm.
SpbK:FilamentswereobtainedbyincubatingpurifiedMBP-SpbK
(1mg/mL; 10 μM) with TEV protease (5 μM) in HBS buffer supple-
mentedwith10%glycerolfor1hat25°C.Thefilamentsamplewasthen
loadedontoaglow-dischargedQuantifoilgrid(R2/1300-meshholey
carbonfilmcoated,ProSciTech),blottedfor8.5sunder95%humidity
at4°C,andplungedintoliquidethane,usingaLeicaEMGP2Automatic
PlungeFreezer(LeicaMicrosystems).Fordatacollection,movieswere
acquired ona JEM-Z300FSC(Cryo-ARM300,JEOLLtd.)operating at
300keVequippedwithanin-columnOmegaenergyfilter.Movieswere
recordedwithaK3Summitdirectelectrondetector(Gatan)operating
at×100,000magnification(0.48Åperpixel).Allmovieswereexposed
using a total dose of 40 e-/Å2 over 40 frames and a defocus range
between−0.5and−2.5µm.

CryoEMdataprocessing
All processing steps were performed using CryoSPARC v4.5.1 and
v4.6.272,andthecryoEMprocessingworkflowsaresummarisedinFigs.
S3andS5.
YonE:Atotalof4089movieswerecollectedandimportedinto
CryoSPARC.Alignmentofmovieframeswasperformedusingpatch-
basedmotioncorrection.Fittingofthecontrasttransferfunctionand
defocusestimationwasperformedusingpatch-basedCTFestimation.
Particlepicking wasperformedusingtheBlobpickerinCryoS-
PARC, and 246,156particles wereextracted with a 700 px box size
(Fouriercroppedto256px).Severalroundsof2Dclassificationwere
performedtoremoveinferiorparticles.22,320particlesrepresenting
thebest2Dclasseswerethenusedforab-initioreconstructionusing
C1symmetry,followedbyhomogeneousandnon-uniformrefinement
with C12 symmetry imposed. The final resolution of the 3D recon-
struction was 3.3Å, based on the gold standard FSC 0.143 criteria.
Local resolution distribution was evaluated using CryoSPARC. The
statisticsarelistedinTableS3.
SpbK:Atotalof11,993movieswerecollectedandimportedinto
CryoSPARC.Alignmentofmovieframeswasperformedusingpatch-
basedmotioncorrection.Fittingofthecontrasttransferfunctionand
defocusestimationwasperformedusingpatch-basedCTFestimation.
Filament segments were auto-picked by “Filament Tracer” using a
filament diameter of 100Å and a separation distance of 50Å. The
resultingfilamentsegmentswereextractedwithan800pxboxsize
(Fouriercroppedto400px).Iterativeroundsof2Dclassificationwere
performed to remove junk classes and poor-quality filaments. 3D
reconstructions of the final particle set containing 116,455 filament
segmentswereperformedusingHelicalRefinement.Areconstruction
withrecognisableproteinfeatures(α-helices,β-strandsandsidechain
densities)wasobtainedusingC1symmetry,ahelicalriseof8.9Åand
twistof79.3°persubunit.Inspectionofthisreconstructionrevealed
that the filament contains 5 protofilaments, each consisting of two
antiparallel TIR domain strands with dihedral (D1) symmetry. The
qualityofthemapwasfurtherimprovedbyapplyingD1symmetryand
non-uniform regularisation to the Helical Refinement protocol. The
finalresolutionofthe3Dreconstructionwas3.3Å,basedonthegold
standardFSC0.143criteria.Thefinalvolumewassharpenedwithlocal
resolutionfilteringautomaticallyestimatedinCryoSPARC.Thestatis-
ticsarelistedinTableS3.

Modelbuildingandrefinement
YonE:ModelAngelo73suppliedwiththeYonEsequencewasusedto
build an initial model of the entire dodecamer into the map.
An AlphaFold 2 model of a YonE monomer was subsequently
fitted into the map by structural alignment to the ModelAngelo
model. The fitted model was subjected to iterative rounds of
model building and refinement using Coot74 and phenix.real_spa-
ce_refinefromthePHENIXsuite75,76.Thefullmodelwasgeneratedby
applying symmetry operators and refined further using Coot and
phenix.real_space_refine.
SpbK:ModelAngelo74suppliedwiththeSpbKsequencewasused
tobuildaninitialmodelintothehelicalrefinementmap.Tocaptureall
protein-protein interfaces in the filament, 10 chains from the Mod-
elAngelomodelweresubjectedtoiterativeroundsofmodelbuilding
and refinement using Coot74 and phenix.real_space_refine from the
PHENIXsuite75,76.

StructurepredictionofYonEandYonE:SpbKcomplex
YonE:AmodelofdodecamericYonEwasgeneratedusingAlphaFold2
Multimer52,77implementedintheColabFoldinterface(v1.5.1),usinga
localColabFoldinstallationwithdefaultsettings.
YonE:SpbKcomplex:AlphaFold378(https://AlphaFoldserver.com)
wasusedtogenerateamodelofdodecamericYonEincomplexwith12
copiesofSpbKNtd.Onlytheclipdomainregion(residues270-325)of
YonE was used for modelling. The YonE clip domain:SpbKNtd and
SpbKNtd:SpbKNtd interfaces had ipTM scores of 0.56–0.60 and
0.71–0.73, respectively (Fig. S8). To accommodate modelling com-
plexesoffull-lengthSpbKandnearfull-lengthYonE(residue46–506),
weranalocalimplementationofAlphaFold3(commit:69b749c)with
pair_attention_chunk_sizesetto8,pair_transition_shard_specsetto8
andunifiedmemoryenabled.InferencewasperformedusinganNvidia
H200GPU(141GBVRAM).Tosamplecandidatepredictions,wepro-
duced10independentreplicateswith5diffusionsamplesperseed.
Oneofthepredictedmodelsdisplayedsimilarinterfacestoboththe
truncatedYonE:SpbKNtdmodelandtheSpbKTIRdomainfilament,but
thechainipTMscoreswereverylow(~0.15),andthemodelhasonly
beenusedforillustrativepurposesinFig.8.

TestingofSpbKmutantsinB.subtilis
Mediaandgrowthconditions:B.subtiliscellsweregrowninLBorS7
50
minimalmediumsupplementedwith0.1%glutamateand1%glucoseas
a carbon source. Isopropyl β-D-1-thiogalactopyranoside (IPTG) was
used at 1mM to induce expression from the LacI-repressible IPTG-
inducible promoter Pspank(hy). Antibiotics used for selection in B.
subtilis included: kanamycin (5µg/mL), tetracycline (10µg/mL) and
spectinomycin(100µg/mL).
Fortypicalexperiments,aculturewasstartedfromasinglecolony
(afterovernightgrowth)andgrownat37°Ctomid-lateexponential
phaseintheindicatedmedium.Cellsweredilutedintofreshmedium
for continued growth as indicated. For experiments with genes
expressedfromaninduciblepromoter,culturesweresplitatthetime
ofdilutionintomediumwithandwithoutinducer,asindicated.
TemperatephageswereinducedbytheadditionofmitomycinC
(MMC)to1µg/mLtoalysogengrowingexponentiallyinLBmediumat
37°C.AfterMMCinduction,cellsweregrownforoneadditionalhour
andpelletedat4000×gfor5minutesinatabletopcentrifuge.The
lysatewastransferredtoanewtube,1:100v/vchloroformwasadded
toinhibitcellgrowth,andstoredat4°C.
Efficiency ofplating assays: Phage stockswerediluted in phage
buffer(150mMNaCl,40mMTris-Cl,10mMMgSO )andmixedwith
4
300µLofcellsthatweregrowingexponentiallyinLBmedium.Cells
andphagewereincubatedfor5minutesatroomtemperaturetoallow
adsorption,transferredto3mLofmoltenLB+0.5%Bacto-agar,and
plated onto warm LB agar plates. Plates were incubated at 30°C
overnighttoallowplaqueformation.Serialdilutions(typically10-fold)
forspotassaysweremadeinphagebuffer,and2µLwerespottedonto
a lawn of cells on LB agar plates. Phage plaquing efficiencies were
determinedessentiallyasdescribed41.Briefly,100µLofvariousdilu-
tionsofaphagestockweremixedwith300µLofcellsthathadbeen
growntoOD600of~0.5(~3×107cells),incubatedatroomtempera-
turefor5minutes,mixedwith3mLLBtopagarandspreadontoanLB
agarplate.Plaqueswerecountedafterincubationovernightat30°C
and are presented as plaque-forming units per mL (PFU/mL) of
phagestock.
Strainsandalleles:B.subtilisstrainsusedinthisstudyarelistedin
Table S5. All strains are derivatives of PY7979 or CU105080, both of
whicharecuredofSPβandICEBs1.Indicatedalleleswereintroduced
by natural transformation81 and appropriate selection. The spbK
mutant alleles were constructed by PCR and Gibson isothermal
assembly with primer overhangs encoding the desired mutation.
GenomicDNAusedforconstructionwasderivedfromCMJ684(PY79,
lacA::{spbKkan}).B.subtiliscellsweretransformedvianaturaltrans-
formation,withGibsonassemblyreactionaddedtocompetentcells.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "There are two major criticism that this reviewer had regarding the manuscript.\n\nThe mechanistic details of the enzymatic reaction remains unclear despite the detailed cryoEM structure determination and the series of mutants used in this work. First, TIR domain is essential for enzymatic reaction of SPBK, which is clearly demonstrated in this work. However, the experimental design of the work is convoluted by polymerization of the TEV cleaved MBP-SPBK. While the MW of MBP-SPBK wild type and mutants D158R, N191R, E192A, W197A etc are similar (Figure 5), yet the enzymatic activity is present only for the TEv-cleaved WT. Does it imply that TEV-cleaved Mutants donot oligomerize ? Or does it oligomerize in a manner that is not conformationally competent for the reaction. This reviewer strongly suggest (a) Structural studies of NAD+ (non hydrolyzable analogue) or (b) mutant protein that has intermediate activity or (c) constitutively active monomer/dimer. This is especially pertinent since the Figure 3 demonstrates clearly that MBP-SPBK and TEV cleved MBP_SPBK has drastically different kinetics. While oligomerization could be essential , Is it possible that the mode of polymerization influence the kinetics of the reaction?\n\nSecond, the impact of YonE on the reaction mechanism of SPBK remains unclear to this reviewer. While the graph in The SEC profiles suggest that the two proteins interact, yet the relevance of this interaction in the NAD+ consumption is unclear to this reviewer. The graph is figure 7a and 3e suggests that kinetics of the control SPBK is different in two experiments atleast in 16 hrs. Unless the mechanistic details of the reaction mechanism is clarified , the utility of Fig 8 remains inconsistent with the claims of this paper.\n\nMinor point\nWhile NMR has been used to monitor the enzymatic reaction of NAD consumption, an orthogonal method might be required, since there can be contribution from NH signals of the protein side chain.", "reviewer_2": "The work in this paper is significant because it illustrates yet another mechanism in the arsenal of bacterial defense systems.\nThe authors successfully demonstrate that TIR-containing SpbK, when in the presence of the phage particle YonE, increases the NADase activity required to defend against pathenogenic activity.\nThe structural mechanism proposed could benefit from a few more experimental ideas: could cryoEM be performed on the stable dimer form of SpbK, and also, SpbK in complex with YonE? Both experiments could further illustrate the mechanistic story and may be simple to do since purification was already performed of each. Also, for the cryoEM reconstruction of YonE, I am curious to see the full computational processing workflow. Was any symmetry or 3D classification performed prior to the final reconstruction of this 12-mer density of YonE? If so, it would be ideal to inspect the C1 solution prior to any imposed symmetry, as well as any other resulting 3D classes, to see if the missing base units can be found. This could be included in the supplemental processing figure, but it would lend to a better understanding or interpretation of the missing density, which can potentially get averaged out if it does not follow an imposed symmetry rule.\nOverall the findings do support and elucidate SpbKs mechanism in the fight against SpB phage infection, and it adds to the expansive set of tools available to bacterial immune systems.", "reviewer_3": "SpbK is a TIR domain containing protein from Bacillus, shown previously to be an enzyme with NAD+ depletion activity which presumably leads to cellular growth arrest in response to phage infection. The authors have elucidated the structure of higher order filaments using cryo-EM and established structural similarity of these filaments to other known TIR-domain containing proteins. In addition, the authors describe interactions between YonE (a portal protein from phage SPbeta) and SpbK. Based on separate structural determinations of SpbK and YonE complemented with Alphafold predictions and mutagenesis experiments, the authors have determined residues/interfaces (namely, SpbK’s N terminal domain to YonE’s clip domain) which are key to interactions between SpbK and YonE.\n\nThe authors have presented us with a very thorough study that has layers of details which will of interest to biochemists, structural biologists, phage biologists, and those studying immune signaling. This represents one of the few research articles in recent memory that are explicit about the pitfalls of studying cyclic ADPR analogs and that provide clear and in-depth analysis of these molecules. The story is clear and the data and analysis are original and exciting. Minor comments mainly on the formatting of figures and suggestions for one or two additional experiments to solidify alphafold predictions can be found below.\n\n• First results subheading “an” NADase not “a”\n• For the double-peaks observed for N1-cADPR: Can the authors speculate more on what component in lysate could be causing this phenomenon? Metals perhaps? Could the second peak which disappears following pre-purification be a less-stable isomer which simply does not survive the lyophilization procedure?\n• When discussing alphafold prediction of the hexamer bound to NAD+, the authors should consider adding mutagenesis data to supplement the supposition. Mutants of residues suspected to form the composite active site are especially interesting (perhaps K224 stands out as the authors mention this residue specifically).\n• Can the authors please comment here and in the main text why they chose to remove the first 45 amino acids of the YonE construct used for cryo-EM analysis? The statement that the residues were truncated is made but no justification is provided.\n• In experiments mixing YonE and SpbK WT/mutants, why were those concentrations tested? (2uM and 0.2 uM, respectively)? If this is simply due to the expected stoichiometry of the final complex, then the authors should amend the manuscript with this detail for completeness and/or conduct additional experiment - titration of [YonE] into SpbK.\n• Can the authors share the SpbK NTD to YonE monomer to monomer alphafold prediction (possibly as a supplementary figure)? The statistics might look better and still provide useful information regarding the binding interactions.\n• Figures with SEC-MALS should clearly describe in the figure legends what is indicated by the orange trace observed in the graphs.\n• Figure 5f and 5g- The wildtype spbK + yonE trace needs to be presented as well for appropriate comparison and indeed it might be beneficial to combine both panels into one (which could look messier) or somehow redistribute the information to make the meaning behind the figure clearer.\n• Figure 7c- Adding a legend on the figure would be useful and for consistency among other figure panels in this manuscript.\n• Supplementary Figure 1b- It might also be important and useful to show the NMR spectra for the individual bases as controls (in absence of enzyme).\n• Explanation of what a portal protein is should likely come early on in the manuscript rather than in the discussion. “Portal proteins such as YonE are required for pro-capsid assembly and correct DNA packaging and are thus essential for phage production (41)”.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "Comment: There are two major criticism that this reviewer had regarding the manuscript.\nResponse: We have addressed these criticisms in the comments below. We hope our\nresponses help clarify these points and resolve the concerns raised.\nComment: The mechanistic details of the enzymatic reaction remains unclear despite\nthe detailed cryoEM structure determination and the series of mutants used in this work.\nFirst, TIR domain is essential for enzymatic reaction of SPBK, which is clearly\ndemonstrated in this work. However, the experimental design of the work is convoluted\nby polymerization of the TEV cleaved MBP-SPBK. While the MW of MBP-SPBK wild type\nand mutants D158R, N191R, E192A, W197A etc are similar (Figure 5), yet the enzymatic\nactivity is present only for the TEv-cleaved WT. Does it imply that TEV-cleaved Mutants\ndonot oligomerize ? Or does it oligomerize in a manner that is not conformationally\ncompetent for the reaction.\nResponse: In the MALS and mass photometry experiments, only the MBP-D158R mutant\nbehave similar to wild-type MBP-SpbK. Both the wild-type protein and the D158R mutant\nexist as stable dimers. The N191R and W197A mutants exists as monomers in solution,\nwhile the G194R mutant forms both monomers and dimers. We did not make or test the\nE192A mutant. We have now included molecular weights in both the mass photometry\nand MALS panels in Figs. 3a-b, 5b and S4a-b to make it clearer that the molecular weights\nare not similar for wild type SpbK vs the N191R, P194R and W197A mutants. Our data\ndemonstrate that the dimerisation defective N191R, P194R and W197A SpbK mutants\ncannot oligomerise into filaments. Hence there is strong correlation between NADase\nactivity and ability to dimerise and oligomerise into filaments. D158R still retains the\nability to oligomerise into filaments, and as the reviewer points out, the lack of NADase\nactivity for this mutant is likely due to oligomerisation in a manner that is not\nconformationally competent for the NADase reaction (described on page 10 of the\nmanuscript). In the revised manuscript, we have now more clearly highlighted that the\nD158R mutation likely promotes an alternate oligomerisation mode of SpbK:\n“The D158R intrastrand BB-loop mutant formed stable dimers and assembled into\nfilaments, although these were shorter than those of wild-type SpbK (Fig. 5a). This mutant\nalso lacks NADase activity (Fig. 5c), indicating that the D158R substitution perturbs the\nintrastrand interface, resulting in an alternative mode of oligomerisation. This alteration\nlikely compromises the structural integrity of the active site, which resides at the\nintrastrand BE interface, thereby abrogating its capacity to bind and hydrolyse NAD⁺.”\nComment: This reviewer strongly suggest (a) Structural studies of NAD+ (non\nhydrolyzable analogue) or (b) mutant protein that has intermediate activity or (c)\nconstitutively active monomer/dimer. This is especially pertinent since the figure 3\ndemonstrates clearly that MBP-SPBK and TEV cleved MBP_SPBK has drastically dieerent\nkinetics.\nNAD+ analog could be insightful, to understand in detail how the substrate engages with\nthe active site. We did attempt to obtain a structure of SpbK with the non-cleavable NAD+\nanalog Carba-NAD+ which we synthesized in-house. Carba-NAD+ is identical to NAD+\nexcept for the oxygen atom in the ribose ring, which is replaced by a carbon atom, forming\na carbocyclic ring. We prepared Spbk filaments in the presence of 1 mM carbaNAD and\ncollected ~5000 movies. After 2D classification, 8,791 filament segments were used for\n3D reconstruction, yielding a 4.4 A\" resolution reconstruction (Fig. R1 below) However, we\ndid not observe electron density for Carba-NAD+ in the active site, suggesting that this\nNAD+ analog either binds only transiently or that the carbocyclic ring is not favourable for\nbinding. As an alternative, we modelled the interaction between SpbK and NAD+ using\nAlphaFold 3 (Fig. 4g and S3e-g in manuscript). The results suggest that NAD+ engages the\nactive site in a manner similar to the SARM1 and AbTIR 1AD/3AD complexes (Manik et al.,\n2022; Shi et al., 2022).\nUnfortunately, we don't have a mutant with intermediate activity that can be used for\nstructural studies. Considering that TIR NADase activity requires two TIR domains to\ncome together to form an active site (Bhatt et al., 2024; Manik et al., 2022; Shi et al.,\n2022), we think it will be nearly impossible to design a constitutively active monomer, and\nbecause the active site is formed via an asymmetric head-to-tail interaction, designing a\nconstitutively active dimer would also be extremely dieicult and beyond the scope of this\nmanuscript.\nComment: While oligomerization could be essential , Is it possible that the mode of\npolymerization influence the kinetics of the reaction?\nResponse: As discussed in detail above, our D158R mutagenesis data suggest that\nperturbation of the oligomerisation/polymerisation mode, specifically through the head-\nto-tail interface, modulates the kinetics of the reaction.\n2\na 5,158 movies\nMotion correction\nCTF estimation Ab Initio (1 class)\nFilament tracing Helical refinement,\nD1 point symmetry 4.4 (cid:1344)\n4x 2D class\n133,642particles\nHelical twist: 79.3(cid:131)\nGood 2D classes 8,791 particles Helical rise: 8.92 (cid:1344)\nb c\n(cid:1828)A\n(cid:1828)E\nE192\n(cid:1828)B (cid:1828)C\nNo density observed for carbAND in active site\nFig. R1: CryoEM reconstruction of the SpbK filament in the presence of Carba-NAD+. (a)\nFlow-chart of the cryo-EM processing steps. (b) Gold-standard FSC curves of the final 3D\nreconstruction. (c) CryoEM map of active site region with fitted atomic model. The two\nTIR chains are highlighted in cyan and orange, respectively.\nComment: Second, the impact of YonE on the reaction mechanism of SPBK remains\nunclear to this reviewer. While the graph in The SEC profiles suggest that the two proteins\ninteract, yet the relevance of this interaction in the NAD+ consumption is unclear to this\nreviewer. The graph is figure 7a and 3e suggests that kinetics of the control SPBK is\ndieerent in two experiments at least in 16 hrs. Unless the mechanistic details of the\nreaction mechanism is clarified , the utility of Fig 8 remains inconsistent with the claims\nof this paper.\nResponse: We respectfully disagree with the reviewer’s assessment that Fig. 7a does not\ndemonstrate the relevance of the YonE:SpbK interaction in NAD+ consumption by SpbK.\nAt the 16-hour time point, over 80% of NAD+ is hydrolysed when SpbK is incubated with\nYonE, compared to less than 5% in its absence. This dieerence provides strong evidence\nfor a YonE-dependent enhancement of NAD+ consumption and is consistent with (Loyo\net al., 2025), which demonstrated that NAD+ levels in Bacillus subtilis cells drop\nsignificantly only when SpbK and YonE are co-expressed. To further demonstrate the\nimpact of YonE on the interaction, we have conducted additional kinetics experiments\nwith dieerent concentrations of YonE (0.2 µM, 1 µM and 2 µM) and updated Fig. 7a in the\nrevised manuscript accordingly (Fig. R7 on page 11 below).\nThe experiments in Fig. 3e and Fig. 7a of the manuscript are not directly comparable, as\nwe used dieerent concentrations of MBP-SpbK and substrate. For the TEV cleavage\nexperiments in Fig. 3e, we used 2 µM MBP-SpbK and 1 mM NAD+. For the YonE\nexperiments in Fig. 7a we used 0.2 µM MBP-SpbK and 500 µM NAD+. While MBP fusion\n3\nmarkedly reduces SpbK oligomerization, at 2 µM, we suspect the concentration is\nsueiciently high to allow some degree of SpbK self-association, which may account for\nthe low NADase activity. To make the results in Fig. 3e and Fig. 7a more directly\ncomparable, we have repeated the SpbK +/-TEV NADase experiments in Fig. 3e using 500\nµM NAD+ and two protein concentrations (0.2 and 2 uM) (Fig. R2 below).\nFig. R2: New Fig. 3e. Reaction progress curves of 0.2 μM MBP-SpbK +/-TEV protease + 500\nμM NAD+ (left panel) and 2 μM MBP-SpbK +/-TEV protease + 500 μM NAD+ (right panel).\nMBP-SpbK was incubated with TEV protease for 1 h before addition of NAD+.\nThe experiments were repeated two times with similar results.\nComment: While NMR has been used to monitor the enzymatic reaction of NAD\nconsumption, an orthogonal method might be required, since there can be contribution\nfrom NH signals of the protein side chain.\nResponse: We understand reviewer #1 is concerned about the potential interference\nfrom protein NH signals towards our NMR-based enzymatic assays. We ensure that this\nshould not be a concern as we have protein-only controls samples showing no such\ninterference (see Fig. R3 below). The NMR assay is also well established, with several\nrecent publications confirming its reliability (Bayless et al., 2025; Figley et al., 2021;\nHorsefield et al., 2019; Shi et al., 2022; Shi et al., 2024). Therefore, we believe that\nincorporating an additional NADase assay is unnecessary for this manuscript.\n*:1H NMR Peaks used for integration and quantification\n* *\n*\n2 μM SpbK+ 500 μM NAD+, 8 h\n* *\n2 μM SpbK+ 500 μM NAD+, 0.17 h\n2 μM SpbK, 8 h\n2 μM SpbK, 0.17 h\n(cid:3)(cid:2)(cid:1) (cid:3)(cid:2)(cid:7) (cid:3)(cid:2)(cid:6) (cid:3)(cid:2)(cid:5) (cid:4)(cid:2)(cid:3) (cid:4)(cid:2)(cid:1) (cid:4)(cid:2)(cid:7) (cid:4)(cid:2)(cid:6) (cid:4)(cid:2)(cid:5) (cid:1)(cid:2)(cid:3) (cid:1)(cid:2)(cid:1)\n(cid:8)(cid:5)(cid:9)(cid:10)(cid:11)(cid:11)(cid:12)(cid:13)\nReviewer 2:\nComment: The work in this paper is significant because it illustrates yet another\nmechanism in the arsenal of bacterial defense systems. The authors successfully\ndemonstrate that TIR-containing SpbK, when in the presence of the phage particle YonE,\nincreases the NADase activity required to defend against pathenogenic activity.\nResponse: We thank the reviewer for the positive feedback highlighting the significance\nof our work.\nComment: The structural mechanism proposed could benefit from a few more\nexperimental ideas: could cryoEM be performed on the stable dimer form of SpbK, and\nalso, SpbK in complex with YonE? Both experiments could further illustrate the\nmechanistic story and may be simple to do since purification was already performed of\neach.\nResponse: The SpbK TIR domain dimer has a molecular mass of only 30 kDa; therefore\nwe believe cryoEM would be extremely challenging if not impossible. The increased size\nof the fusion protein (monomer:~80 kDa; dimer ~160 kDa) is unlikely to help because\nMBP is connected to SpbK via a flexible linker and the linker between the N-terminal\ndomain and the TIR domain is also predicted to be flexible (based on AlphaFold modelling\nand lack of density for MBP and the N-terminal domain in our filament reconstruction).\nDue to this flexibility, the MBP and N-terminal domains are likely to adopt many dieerent\norientations relative to each other and the TIR domain. This variability will prevent\naccurate alignment and averaging, which are essential for high-resolution maps.\nWe agree with the reviewer that a cryoEM structure of a YonE:SpbK complex could be\ninformative. Unfortunately, it is not simple to do. We attempted to determine the cryoEM\nstructure of YonE in complex with both SpbKNtd (Fig. R4, below) and the SpbKNtd-SARM1SAM\nfusion protein (Fig. R5, below); however, we were unable to obtain high-quality 3D\nreconstructions.\nTo prepare the YonE:SpbKNtd complex for cryoEM, YonE (60 µM) was mixed with an excess\nof SpbKNtd (75 µM) and the mixture was subjected to analytical gel filtration,\nusing a Superdex 200 column. Elution peak 1 contained both YonE and SpbKNtd and was used for\ncryoEM analysis. During cryoEM processing, we encountered similar challenges as\nobserved with YonE alone, including preferred particle orientation and apparent flexibility\nin the stem and clip domains. Additionally, the gel filtration analysis revealed that only a\nsmall proportion of SpbKNtd forms a stable complex with YonE. The YonE:SpbKNtd peak\nlikely comprises a mixture of free YonE and YonE:SpbKNtd complexes, further\ncomplicating cryoEM data processing, particularly due to the limited range of particle\nviews. As with YonE alone, the reconstruction yielded interpretable density only for the\nwing and crown domains of YonE, providing no structural insight into the interaction\nbetween SpbKNtd and YonE.\n5\na b c\nPeak 1: Peak 2:\n9-11 mL 17-18 mL\nkDa\n72\n43\n34\n26\n17\nElution volume (mL)\nd\nC1, 4.3 (cid:1344) C12, 3.5 (cid:1344)\n2,359 movies (tilt angle: 0°)\n+\n2,299 movies (tilt angle: + 30°)\nCTF estimation\nBlob picking\n90°\nHomogenous\n1,560,000 particles Ab Initio, refinement\n3x classes\n35,914 particles\nClass 2D x3\nGood 2D classes 79,896 particles\nDensity for stem and clip domains are missing\nFig. R4: CryoEM reconstruction of the YonE:SpbKNtd complex. (a) Gel filtration analysis (b)\nSDS-PAGE analysis of gel filtration fractions containing the YonE:SpbKNtd complex (c)\nNegative-stain electron micrograph. (d) Flow-chart of the cryo-EM processing steps.\nAdditional 3D classification (not shown in this figure), did not identify any classes\ndisplaying density for the YonE:SpbKNtd complex.\nTo prepare the YonE:S SpbKNtd-SARM1SAM complex for cryoEM, YonE (25 µM) was mixed\nwith SpbKNtd-SARM1SAM (25 µM) and the mixture was subjected to analytical gel filtration\nusing a Superdex 200 column. Elution peak 1 contained both YonE and YonE: SpbKNtd-\nSARM1SAM and was used for cryoEM analysis. To characterise the complex formed by YonE\nand the SpbKNtd-SARM1SAM fusion protein, we first collected negative stain EM images\nfrom gel filtration peak fractions. The initial portion of the peak appeared heterogeneous,\nwith numerous large aggregates. We suspect that fusion of SpbKNtd to the octameric\nSARM1SAM domain ring enables multivalent interactions with YonE dodecamers,\npromoting aggregation. The later portion of the gel filtration peak was more\nhomogeneous, predominantly containing ring-shaped particles consistent in size with\neither YonE dodecamers or SARM1SAM octamers. We focused on this fraction for cryoEM\nanalysis. Unfortunately, from a cryoEM dataset comprising ~4,400 movies, we were only\nable to obtain two single well-defined 'side-view' 2D classes containing ~14,000 particles\n(Fig. R5), which was insueicient for 3D reconstruction. We suspect that multivalent\ninteractions persist in the sample, complicating single-particle analysis. The side-view\n6\n)uAm(\n082A\n1\n2\nclasses revealed additional density beneath the stem/clip region of YonE, which we\nbelieve corresponds to the octameric SARM1SAM ring.\na\nSpbKNtd-SARMSAM + YonE\nYonE\nSpbKNtd-SARMSAM\nc d\nFig. R5: (a) Gel filtration analysis. (b) SDS-PAGE analysis of gel-filtration fractions. The\nchromatogram and gel in (a-b) are from Fig. 7a in manuscript. (c) Negative-stain electron\nmicrographs of the YonE:SpbKNtd-SARM1SAM gel filtration peak 1. Left and right panels\ncorrespond to the first and second halves of the gel filtration peak 1, respectively. (b)\nRepresentative 2D class averages from cryoEM processing of YonE:SpbKNtd-SARM1SAM in\nCryoSPARC.\nComment: Also, for the cryoEM reconstruction of YonE, I am curious to see the full\ncomputational processing workflow. Was any symmetry or 3D classification performed\nprior to the final reconstruction of this 12-mer density of YonE? If so, it would be ideal to\ninspect the C1 solution prior to any imposed symmetry, as well as any other resulting 3D\nclasses, to see if the missing base units can be found. This could be included in the\nsupplemental processing Fig., but it would lend to a better understanding or\ninterpretation of the missing density, which can potentially get averaged out if it does not\nfollow an imposed symmetry rule.\nResponse: We agree with the reviewer that the density corresponding to the stalk and clip\ndomain of YonE may be averaged out if it does not conform to the imposed C12 symmetry.\nIn addition to the workflow shown in Fig. S5, we also performed non-uniform refinement\nusing C1 symmetry (~4.6 Å resolution), as well as 3D classification. However, inspection\nof the resulting maps did not reveal any density for the missing regions (see Fig. R6\nbelow), indicating that the absence of density is unlikely to be a symmetry-related issue.\n7\n)uAm(\n082A\nb\nPeak 1 Peak 2\n2\nkDa\n72\n55\n1 43\n34\n26\n17\nYonEClip:SpbKNtd complex?\nSARM1SAM octamer?\nWe have updated Supplementary Fig. S5 to include the C1 symmetry processing step.\nThe 3D classification results were not included, as none of the resulting classes were\nused for further analysis.\na b Class 1 Class 2\n4,220 particles 1,511 particles\nNon-uniform refinement C1 symmetry\nClass 3\n2,563 particles Class 4\n1,148 particles\n90°\n1,30 C 8 l a p s a s r t 5 icles 2,05 C 9 l a p s a s r t 6 icles 1,47 C 1 l a p s a s r t 7 icles\nClass 8 Class 9 Class 10\n1,415 particles 5,512 particles 1,113 particles\nDensity for stem and clip domains are missing\nFig. R6: Additional cryoEM processing steps for YonE. a) Non-uniform refinement,\nC1\nsymmetry. b) 3D classification, C1 symmetry, 10 classes.\nComment: Overall the findings do support and elucidate SpbKs mechanism in the fight\nagainst SpB phage infection, and it adds to the expansive set of tools available to\nbacterial immune systems.\nResponse: We thank the reviewer for this positive assessment.\nComment: SpbK is a TIR domain containing protein from Bacillus, shown previously to be\nan enzyme with NAD+ depletion activity which presumably leads to cellular growth arrest\nin response to phage infection. The authors have elucidated the structure of higher order\nfilaments using cryo-EM and established structural similarity of these filaments to other\nknown TIR-domain containing proteins. In addition, the authors describe interactions\nbetween YonE (a portal protein from phage SPbeta) and SpbK. Based on separate\nstructural determinations of SpbK and YonE complemented with Alphafold predictions\nand mutagenesis experiments, the authors have determined residues/interfaces\n(namely, SpbK’s N terminal domain to YonE’s clip domain) which are key to interactions\nbetween SpbK and YonE.\nThe authors have presented us with a very thorough study that has layers of details which\nwill of interest to biochemists, structural biologists, phage biologists, and those studying\nimmune signaling. This represents one of the few research articles in recent memory that\nare explicit about the pitfalls of studying cyclic ADPR analogs and that provide clear and\nin-depth analysis of these molecules. The story is clear and the data and analysis are\noriginal and exciting. Minor comments mainly on the formatting of figures and\nsuggestions for one or two additional experiments to solidify alphafold predictions can\nbe found below.\nResponse: We thank the reviewer for the positive feedback and constructive suggestions.\nComment: First results subheading “an” NADase not “a”\nResponse: We thank the reviewer for the suggestion. The subheading has been updated\nin the revised manuscript.\nComment: For the double-peaks observed for N1-cADPR: Can the authors speculate\nmore on what component in lysate could be causing this phenomenon? Metals perhaps?\nCould the second peak which disappears following pre-purification be a less-stable\nisomer which simply does not survive the lyophilization procedure?\nResponse: Indeed, a metal cation is our leading hypothesis, because the phosphates\nmay bind it tightly, which could stabilise cADPR into dieerent conformational isomers.\nThe latter hypothesis is unlikely because we have lyophilized the sample and redissolved\nit (without a prior separation), and we still see both peaks; in other words, lyophilisation\nalone does not seem to make one of the peaks disappear.\nWe added a parenthetical statement about divalent metal cations to the revised\nmanuscript (page 6):\n“We hypothesised that either two cADPR variants were produced by the cell, or\ncomponents in the cell lysate (e.g., divalent metal cations) caused the N1-cADPR to\nadopt dieerent configurations with dieerent retention times.”\nthe authors should consider adding mutagenesis data to supplement the supposition.\nMutants of residues suspected to form the composite active site are especially\ninteresting (perhaps K224 stands out as the authors mention this residue specifically).\nThe composite active site has been observed across multiple bacterial, plant and\nanimal/human TIR NADase structures (Bhatt et al., 2024) and has already been\nextensively validated through site-directed mutagenesis in both bacterial (AbTir) and\nhuman (SARM1) TIR domains (Manik et al., 2022; Shi et al., 2022). Loyo and Grossman\ndemonstrated that E192, which interacts with the nicotinamide ribose, is essential for\nSpbK NADase activity (Loyo and Grossman, 2025). Importantly, our structural model\npredicts that D158 plays a key role in forming the composite active site, and we have\nshown that its mutation completely abolishes NADase activity. As such, we consider\nadditional mutagenesis data for the predicted SpbK:NAD+ interaction unlikely to yield\nsubstantial new insights.\nFurthermore, structural studies of both AbTIR and SARM1 bound to NAD mimetics have\nshown that the adenine ring forms key interactions with the protein backbone. Thus,\nmutations of side chains such as K224 may have limited impact on SpbK NADase activity.\nThis is exemplified by SARM1, where mutation of W662 (W662A), whose side chain stacks\nagainst the adenine ring, results in only a modest reduction in NADase activity (Shi et al.,\n2022).\nComment: Can the authors please comment here and in the main text why they chose to\nremove the first 45 amino acids of the YonE construct used for cryo-EM analysis? The\nstatement that the residues were truncated is made but no justification is provided.\nResponse: We removed the first 45 first amino acids of YonE in the expression construct\nas they are predicted to be disordered. We have included the following sentence in the\nmain text of revised manuscript:\nWe expressed and purified a truncated YonE construct lacking the first 45 residues\npredicted to be disordered (Jumper et al., 2021).\nComment: In experiments mixing YonE and SpbK WT/mutants, why were those\nconcentrations tested? (2uM and 0.2 uM, respectively)? If this is simply due to the\nexpected stoichiometry of the final complex, then the authors should amend the\nmanuscript with this detail for completeness and/or conduct additional experiment -\ntitration of [YonE] into SpbK.\nResponse: The ratio of 2 µM YonE to 0.2 µM MBP-SpbK was not selected based on\nstoichiometry, but rather on the significant enhancement in NADase activity observed\nrelative to MBP-SpbK alone. As suggested by the reviewer, we have conducted additional\nexperiments with dieerent concentrations of YonE (0.2 µM, 1 µM and 2 µM) and updated\nFig. 7a in revised manuscript accordingly (Fig. R7 below).\nThe initial NAD+ concentration was 500 μM. The experiments were performed two times\nwith similar results.\nComment: Can the authors share the SpbK NTD to YonE monomer to monomer alphafold\nprediction (possibly as a supplementary figure)? The statistics might look better and still\nprovide useful information regarding the binding interactions.\nResponse: We have included a figure below, showing the AlphaFold 3 models of the\nmonomeric SpbKNtd:YonEClip complex. The predicted ipTM scores are substantially lower\nthan those observed for the docameric complex (ipTM = 0.73), and there is no\nconsistency among the five predicted models (Fig. R8a). Importantly, none of these\nmodels recapitulate the interactions seen in the dodecameric complex (Fig. R8b). One\npossible explanation is that multiple interaction interfaces are required for stable\ncomplex formation. Specifically, in the model of the dodecameric complex, the a –a\n3 4\nloop of SpbKNtd docks onto the YonEClip:YonEClip interface, consistent with our\nmutagenesis data. The dodecameric complex model also suggests that\nSpbKNtd:SpbKNtd interactions are necessary for assembly, which is also supported by our\nmutagenesis data.\n11\na\nAF3 M1 AF3 M2 AF3 M3 AF3 M4 AF3 M5\nYonEclip YonEclip YonEclip YonEclip YonEclip\nSpbKNtd SpbKNtd\nSpbKNtd\nipTM = 0.12 ipTM = 0.05\nSpbKNtd SpbKNtd\npLDDT 90-100 70-90 50-70 0-50 ipTM = 0.09\nipTM = 0.15\nipTM = 0.16\nb AF3 M1 AF3 M2 AF3 M3 AF3 M4 AF3 M5\nYonEClip SpbKNtd YonEClip:SpbKNtd dodecamer model\nFig. R8: AlphaFold 3 models (M1-M5) of the monomeric YonE-SpbKNtd complex. Only the\nclip domain region of YonE (residues 270-225) was used for modelling. (a) Monomeric\nYonE-SpbKNtd complex models, colored by the confidence metric pLDDT (predicted local\ndieerence test). (b) Monomeric YonE-SpbKNtd complex models superimposed\nonto the dodecameric YonE-SpbKNtd complex model. The AlpaFold3 models were\nsuperimposed using the YonE clip domain.\nComment: Figures with SEC-MALS should clearly describe in the figure legends what is\nindicated by the orange trace observed in the graphs.\nResponse: We have added the following sentence to the legend of Fig. 3b, 5b and S4b\n“The blue line represents the refractive index trace, while the orange line represents the\naverage molecular mass distribution across the peak. “\nComment: Figure 5f and 5g- The wildtype spbK + yonE trace needs to be presented as\nwell for appropriate comparison and indeed it might be beneficial to combine both\npanels into one (which could look messier) or somehow redistribute the information to\nmake the meaning behind the figure clearer.\nResponse: In response to Reviewer #2's suggestion, we have added the wild-\ntype spbK traces and wild-type spbK + yonE from (Loyo and Grossman, 2025) to enable\nappropriate comparison (see Fig. R9 below). We believe that the inclusion of these\ncontrols improves the clarity of the data presented. However, we feel that combining the\nfigures would make them more cluttered and harder to interpret.\nTo improve clarity further, we expanded the description of these figures in the revised\nmanuscript:\n12\n100\n80\n60\n40\n20\n0\n1 1.4 1.8 2.2\nVolume (mL)\nd e\n“All of the mutants exhibited growth comparable to wild-type spbK (Fig. 5f), and\nconsistent with their loss of NADase activity, they did not induce growth arrest in the\npresence of YonE (Fig. 5g).”\nf g\nFig. R9: New Fig. 5f-g. (f) Growth curves of strains expressing various alleles of spbK. (g)\nGrowth curves of strains expressing both yonE and various alleles of spbK. 1 mM IPTG\nwas used to induce expression of yonE. IPTG was added directly after measuring OD600\nat time 0. Cells expressing yonE and spbK mutants do not exhibit the growth arrest\nphenotype observed when yonE is expressed with wild-type spbK. spbK alleles are\nexpressed under the native promoter of spbK. Data shown in f and g are from three\nbiological replicates. Error bars represent standard deviations. The wild-type spbK + yonE\nand wild-type spbK traces in (f-g) are from Loyo and Grossman, 2025 (Loyo and\nGrossman, 2025).\nComment: Figure 7c- Adding a legend on the figure would be useful and for consistency\namong other figure panels in this manuscript.\nResponse: We have added a legend to this figure in the revised manuscript.\nComment: Supplementary figure 1b- It might also be important and useful to show the\nNMR spectra for the individual bases as controls (in absence of enzyme).\nResponse: We understand reviewer #3’s concern about the absence of no-protein\ncontrol spectra. However, these bases do not show NMR signals in this anomeric region\n(5.4 to 6.4 ppm) and thus we believe it would be easier for readers to understand without\nno-protein controls. We have recently published a similar figure (Shi et al., 2024).\nComment: Explanation of what a portal protein is should likely come early on in the\nmanuscript rather than in the discussion. “Portal proteins such as YonE are required for\npro-capsid assembly and correct DNA packaging and are thus essential for phage\nproduction (41)”.\n13\n)aDk(\nthgiew\nraluceloM\nVolume (mL)\n)aDk(\nthgiew\nraluceloM\n200 100\n160 80\n120 60\n80 40\n40 20\n0 0\n1 1.4 1.8 2.2 1 1.4 1.8 2.2\nVolume (mL)\n)aDk(\nthgiew\nraluceloM\n100\n80\n60\n40\n20\n0\n1 1.4 1.8 2.2\nVolume (mL)\n)aDk(\nthgiew\nraluceloM\n200 nm 500 nm\n500 nm 500 nm\n500 nm\nstnuoC\nstnuoC\n0 100 200 300 400 500\nMass (kDa)\nstnuoC\n0 100 200 300 400 500 0 100 200 300 400 500\nMass (kDa)\nstnuoC\na D158R N191R c\n500 nm\nG194R P1 W197A\n500 nm\nb\nD158R N191R G194R P1 W197A\n0 100 200 300 400 500 0 100 200 300 400 500\nMass (kDa) Mass (kDa)\nResponse: We thank the reviewer for this suggestion. We have moved this sentence to\nthe introduction (page 4, paragraph 2) in the revised manuscript.\nReferences\nBayless, A.M., et al. (2025). The Arabidopsis TIRome informs the design of artificial TIR\n(Toll/interleukin-1 receptor) domain proteins. Proc Natl Acad Sci U S A 122, e2505893122.\nBhatt, A., Mishra, B.P., Gu, W., Sorbello, M., Xu, H., Ve, T., and Kobe, B. (2024). Structural\ncharacterization of TIR-domain signalosomes through a combination of structural\nbiology approaches. IUCrJ 11, 695-707.\nFigley, M.D., et al. (2021). SARM1 is a metabolic sensor activated by an increased\nNMN/NAD+ ratio to trigger axon degeneration. Neuron.\nHorsefield, S., et al. (2019). NAD(+) cleavage activity by animal and plant TIR domains in\ncell death pathways. Science 365, 793-799.\nJumper, J., et al. (2021). Highly accurate protein structure prediction with AlphaFold.\nNature 596, 583-589.\nLoyo, C.L., and Grossman, A.D. (2025). A phage-encoded counter-defense inhibits an\nNAD-degrading anti-phage defense system. PLoS Genet 21, e1011551.\nManik, M.K., et al. (2022). Cyclic ADP ribose isomers: Production, chemical structures,\nand immune signaling. Science 377, eadc8969.\nShi, Y., et al. (2022). Structural basis of SARM1 activation, substrate recognition, and\ninhibition by small molecules. Mol Cell 82, 1643-1659 e1610.\nShi, Y., et al. (2024). Structural characterization of macro domain-containing Thoeris\nantiphage defense systems. Sci Adv 10, eadn3310.\nReviewer #1\nThe authors have performed experiments to full address my concerns/comments. The\nmanuscript can be accepted.\nResponse: We appreciate the reviewer’s recommendation for acceptance and are glad\nthe revisions addressed their concerns\nReviewer #2\nI am satisfied with the authors response to my first comments. Thank you for the edits\nand thorough response, the updated cryo-EM figures look great.\nResponse: We appreciate the reviewer’s positive feedback and are pleased that the\nrevisions and updated cryo-EM figures meet their expectations.\nReviewer #3\nThe authors have done more than expected during the interim and provide substantial\nnew data supporting their claims and corroborating prior results. An already strong\nmanuscript now feels even more robust. Might we suggest a few additional text edits\nstemming from the rebuttal discussion the authors have already expertly provided?\nResponse: We appreciate the reviewer’s positive assessment and provide our\nresponses to the suggested minor edits below.\nLikely the YonE:MBP-SbpK ratio that leads to maximal increase in NADase activity would\nbe the one that matches the stoichiometry observed in SEC (10:1). The new titration\ndata in Fig. R7 supports this idea and it could/should be stated more clearly in the text.\nResponse: We used diZerent SpbK variants in our experiments: the enzyme assays\nemployed the full-length protein fused to MBP, whereas size-exclusion chromatography\nutilised constructs lacking the TIR domain (either the N-terminal domain alone or fused\nto GST or SARM1SAM). MBP likely influences SpbK’s ability to self-associate (see Fig. 2 in\nthe manuscript). Additionally, initiation of TIR domain oligomerisation by YonE may\nstabilise the SpbK:YonE interaction and aZect stoichiometry. Given these complexities,\nwe prefer not to draw direct correlations between these experiments in the main text.\nThe authors should provide their rebuttal explanation for leaving out the no-protein\ncontrols in the NMR experiment in Supplementary Figure 1b in the main text, figure\nlegend, or methods for completeness.\nResponse: We have now included the reason 'for leaving out the no-protein controls in\nthe NMR experiment in the Supplementary Figure 1b legend: Spectra corresponding to\nno-protein samples are not shown as anomeric peaks for base-exchange products are\nevident by comparing with the control (bottom).\nIt would also round out the discussion about the stability of the ADPR molecules to\ninclude the authors rebuttal comments on this topic. Specifically regarding\nlyophilization.\nResponse: Addressing the impact of lyophilisation on N1-cADPR stability with suZicient\nrigor would require additional replicates and experimental work. We believe such eZort\nwould not add to the conclusions of this paper, and since the rebuttal will be published\nalongside the manuscript, we prefer to retain the lyophilisation discussion in the rebuttal\nrather than incorporating it into the main text.\nThe theory about metal ion impacts on observation of one or two ADPR species via LC-\nMS could be quickly tested for posterity. Add EDTA to see if it clears up the issue?\nResponse: We appreciate this suggestion and agree that metal ion interactions could be\nan interesting aspect to explore. However, implementing this test would require\ngenerating new lysates and additional LC-MS runs, which represents a considerable\namount of work. Since this experiment is not essential for the conclusions of this study,\nwe prefer to leave it for future investigations."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have performed experiments to full address my concerns/comments. The manuscript can be accepted.", "reviewer_2": "I am satisfied with the authors response to my first comments. Thank you for the edits and thorough response, the updated cryo-EM figures look great.", "reviewer_3": "The authors have done more than expected during the interim and provide substantial new data supporting their claims and corroborating prior results. An already strong manuscript now feels even more robust. Might we suggest a few additional text edits stemming from the rebuttal discussion the authors have already expertly provided?\nLikely the YonE:MBP-SbpK ratio that leads to maximal increase in NADase activity would be the one that matches the stoichiometry observed in SEC (10:1). The new titration data in Fig. R7 supports this idea and it could/should be stated more clearly in the text.\nThe authors should provide their rebuttal explanation for leaving out the no-protein controls in the NMR experiment in Supplementary Figure 1b in the main text, figure legend, or methods for completeness.\nIt would also round out the discussion about the stability of the ADPR molecules to include the authors rebuttal comments on this topic. Specifically regarding lyophilization.\nThe theory about metal ion impacts on observation of one or two ADPR species via LC-MS could be quickly tested for posterity. Add EDTA to see if it clears up the issue?", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "Response: We appreciate the reviewer’s recommendation for acceptance and are glad the revisions addressed their concerns\nResponse: We appreciate the reviewer’s positive feedback and are pleased that the revisions and updated cryo-EM figures meet their expectations.\nResponse: We appreciate the reviewer’s positive assessment and provide our responses to the suggested minor edits below.\nResponse: We used diZerent SpbK variants in our experiments: the enzyme assays employed the full-length protein fused to MBP, whereas size-exclusion chromatography utilised constructs lacking the TIR domain (either the N-terminal domain alone or fused to GST or SARM1SAM). MBP likely influences SpbK’s ability to self-associate (see Fig. 2 in the manuscript). Additionally, initiation of TIR domain oligomerisation by YonE may stabilise the SpbK:YonE interaction and aZect stoichiometry. Given these complexities, we prefer not to draw direct correlations between these experiments in the main text.\nResponse: We have now included the reason 'for leaving out the no-protein controls in the NMR experiment in the Supplementary Figure 1b legend: Spectra corresponding to no-protein samples are not shown as anomeric peaks for base-exchange products are evident by comparing with the control (bottom).\nResponse: Addressing the impact of lyophilisation on N1-cADPR stability with suZicient rigor would require additional replicates and experimental work. We believe such eZort would not add to the conclusions of this paper, and since the rebuttal will be published alongside the manuscript, we prefer to retain the lyophilisation discussion in the rebuttal rather than incorporating it into the main text.\nResponse: We appreciate this suggestion and agree that metal ion interactions could be an interesting aspect to explore. However, implementing this test would require generating new lysates and additional LC-MS runs, which represents a considerable amount of work. Since this experiment is not essential for the conclusions of this study, we prefer to leave it for future investigations."}]

Decision letter