PLANT SCIENCE

NAD+ cleavage activity by animal andplant TIR domains in celldeath pathwaysShane Horsefield1*, Hayden Burdett1*, Xiaoxiao Zhang2,3*, Mohammad K. Manik1*,Yun Shi4*, Jian Chen2,3, Tiancong Qi5, Jonathan Gilley6,7, Jhih-Siang Lai1,Maxwell X. Rank1, Lachlan W. Casey1,8, Weixi Gu1, Daniel J. Ericsson9,Gabriel Foley1, Robert O. Hughes10, Todd Bosanac10, Mark von Itzstein4,John P. Rathjen3, Jeffrey D. Nanson1, Mikael Boden1, Ian B. Dry11, Simon J. Williams3,Brian J. Staskawicz5, Michael P. Coleman6,7, Thomas Ve1,4†,Peter N. Dodds2†, Bostjan Kobe1†

SARM1 (sterile alpha and TIR motif containing 1) is responsible for depletion ofnicotinamide adenine dinucleotide in its oxidized form (NAD+) during Walleriandegeneration associated with neuropathies. Plant nucleotide-binding leucine-richrepeat (NLR) immune receptors recognize pathogen effector proteins and triggerlocalized cell death to restrict pathogen infection. Both processes depend on closelyrelated Toll/interleukin-1 receptor (TIR) domains in these proteins, which, as weshow, feature self-association–dependent NAD+ cleavage activity associated withcell death signaling. We further show that SARM1 SAM (sterile alpha motif) domainsform an octamer essential for axon degeneration that contributes to TIR domainenzymatic activity. The crystal structures of ribose and NADP+ (the oxidized form ofnicotinamide adenine dinucleotide phosphate) complexes of SARM1 and plant NLRRUN1 TIR domains, respectively, reveal a conserved substrate binding site. NAD+

cleavage by TIR domains is therefore a conserved feature of animal and plant celldeath signaling pathways.

Toll/interleukin-1 receptor (TIR) domainsare usually found inmultidomain proteinsinvolved in innate immunity pathways inanimals and plants (1). Inmammals, TIR do-mains are located in the cytoplasmic regions

of Toll-like receptors (TLRs) and interleukin-1receptors (IL-1Rs) and in the cytosolic adaptorproteins involved in inflammatory signalingdownstream from these receptors (2). In these

molecules, TIR domains function as protein in-teractionmodules;molecular and structural char-acterizations of TIR domain signaling complexesin the TLR4 pathway suggest a nucleated assem-bly of open-ended complexes consistent with theSCAF (signaling by cooperative assembly forma-tion) mechanism prevalent in innate immunitypathways (3, 4). The protein SARM1 (sterile alphaand TIR motif containing 1) functions both asa TLR adaptor and as a key executor of axondegeneration (5–8). In plants, TIR domains aremost commonly found as the N-terminal signal-ing domains of cytoplasmic nucleotide-bindingleucine-rich repeat (NLR) resistance proteins,which directly or indirectly recognize effectorproteins from pathogens and initiate defenseresponses (9).Axon degeneration is a hallmark of many neu-

rological disorders (10), and understanding themolecular basis of SARM1-induced neuronal celldeath may offer therapeutic options. Axonal in-jury is associated with the breakdown of nicotin-amide adenine dinucleotide in its oxidized form(NAD+) (11), and SARM1 accelerates NAD+ de-pletion in nerves postinjury (8). SARM1 is amodular protein with several domains (Fig. 1A).Tandem sterile alpha motif (SAM) domains me-diate self-association of SARM1 and are requiredfor SARM1 function (12). Forced self-associationof SARM1TIRdomains induces axondegenerationin the absence of injury as a result of rapid NAD+

depletion (8), and the TIR domain has recentlybeen shown to have NAD+ cleavage activity (13).

Plant NLRs recognize pathogen effector pro-teins and trigger the hypersensitive response(HR), a process usually associated with local-ized cell death, to restrict pathogen infection.Isolated plant TIR domains can trigger cell deathwhen transiently expressed in planta, in the ab-sence of the corresponding pathogen effectorproteins (referred to as autoactivity), and thisactivity is dependent on two self-associationinterfaces (14–16). However, interacting partnersor a direct signaling pathway have not yet beendefined.In the current study, we determined the crys-

tal structure of the TIR domain of humanSARM1, and the structure revealed close sim-ilarity to plant NLR TIR domain structures. Inagreement, we show that like the SARM1 TIR do-main, the TIR domains from plant immune re-ceptors, including L6 (flax; Linum usitatissimum)and RUN1 (grapevine; Muscadinia rotundifolia)also have self-association–dependent NAD+-cleaving enzyme (NADase) activity. For SARM1,we show that the SAM domains contribute toNADase activity by assembling into an octa-mer. We demonstrate the mechanistic basis ofNAD+ cleavage through structural analysis. Col-lectively, this work suggests a conserved sig-naling mechanism involving nucleotide cleavagein cell death pathways and provides a basisfor rational drug design in the treatment ofaxonopathies.

Structure and NAD+ cleavage activity ofthe SARM1 TIR domain

The crystal structure of the TIR domain fromhuman SARM1 (hSARM1TIR; residues 560 to 700;1.8-Å resolution) (Fig. 1B and table S1) showsmoresimilarity to plant and TLR TIR domains [Dali(17)] (Fig. 1C and table S2) than to bacterialTIR domains, contrary to conclusions reachedfrom sequence analyses (18). These comparisonsalso reveal close similarities with enzymes suchas the bacterial N-glycosidase MilB, whichcleaves the nucleotide hydroxymethyl–cytidine5′-monophosphate (hydroxymethyl-CMP) (Fig. 1Dand table S2). The structure of hSARM1TIR con-tains a cleft with a bound glycerol molecule (glyc-erol was used as cryoprotectant). This region,consisting of residues from the bA strand, theAA and BB loops, and the aB and aC helices[the elements of the secondary structure arelabeled sequentially (1)], can be superimposedclosely with the catalytic sites of MilB as well ashuman CD38, including a catalytic glutamateresidue [residue 642 (E642) in SARM1, E103 inMilB, or E226 in CD38] (Fig. 1D and fig. S1).Structural evidence and the association of hSARM1with NAD+ depletion suggests hSARM1TIR maycleave NAD+. Based on molecular docking analy-ses (fig. S2), we show by nuclear magnetic reso-nance (NMR) and fluorescence-based assays (19)that hSARM1TIR cleaves NAD+ into nicotinamide(Nam) and ADP-ribose (ADPR) (Fig. 2, A and B,figs. S3, S4, and S5, and table S3). The Glu642→Ala(E642A) mutation in hSARM1TIR abolishes thisactivity, as do alanine mutations at the conservedactive site residues tyrosine 568 (Y568), arginine

RESEARCH

Horsefield et al., Science 365, 793–799 (2019) 23 August 2019 1 of 7

1School of Chemistry and Molecular Biosciences, Institutefor Molecular Bioscience and Australian InfectiousDiseases Research Centre, University of Queensland,Brisbane, QLD 4072, Australia. 2Agriculture and Food,Commonwealth Scientific and Industrial ResearchOrganisation, Canberra, ACT 2601, Australia. 3PlantSciences Division, Research School of Biology, TheAustralian National University, Canberra ACT 2601,Australia. 4Institute for Glycomics, Griffith University,Southport, QLD 4222, Australia. 5Department of Plant andMicrobial Biology, University of California Berkeley,Berkeley, CA 94720, USA. 6John van Geest Centre forBrain Repair, Department of Clinical Neurosciences,University of Cambridge, ED Adrian Building, Forvie Site,Robinson Way, Cambridge CB2 0PY, UK. 7BabrahamInstitute, Babraham, Cambridge CB22 3AT, UK. 8Centrefor Microscopy and Microanalysis, University ofQueensland, Brisbane, QLD 4072, Australia.9Macromolecular Crystallography (MX) Beamlines,Australian Synchrotron, Melbourne, VIC 3168, Australia.10Disarm Therapeutics, 400 Technology Square,Cambridge, MA 02139, USA. 11Agriculture and Food,Commonwealth Scientific and Industrial Research Organisation,Urrbrae, SA 5064, Australia.*These authors contributed equally to this work.†Corresponding author. Email: b.kobe@uq.edu.au (B.K.);t.ve@griffith.edu.au (T.V.); peter.dodds@csiro.au (P.N.D.)

on February 15, 2021

http://science.sciencem

ag.org/D

ownloaded from

569 (R569), and R569+R570 (RRAA) (Fig. 2A andfig. S4C). While this work was in progress, similarresults were reported by Essuman et al. (13).

Plant TIR domains cleave NAD+

Due to their involvement in cell death and struc-tural similarities with hSARM1TIR, we tested ifplant TIR domains can also cleaveNAD+. At highprotein concentrations, purified TIR domainsfrom the NLRs L6 and RUN1 were capable ofcleaving NAD+ into Nam and ADPR (Fig. 2C).The catalytic activities were lower than the ac-tivity of hSARM1TIR (Fig. 2D). Like hSARM1TIR,they were also able to cleave NADP+ but notFAD (fig. S6). Activity was not observed for thepurified TIR domains from theNLRsRPS4, SNC1,RPP1, RPV1, or ROQ1 (fig. S7). When Escherichiacoli lysates (rather than pure proteins) weretested in an enzyme-linked cycling NAD+ cleavageassay (20), NADase activity was observed alsofor RPS4TIR (fig. S8). Mutations of the residueequivalent to hSARM1TIR E642 in L6TIR andRUN1TIR (E135A and E100A, respectively) abol-ishedNAD+ cleavage activity (Fig. 2C and fig. S9A).

Structural basis of NAD+ cleavage byTIR domains

In NADases, a catalytic glutamate typically in-teracts with the C-2 and C-3 hydroxyl groups oftheNam ribose inNAD+. In the hSARM1TIR struc-ture, E642 forms hydrogen bonds with the C-2and C-3 hydroxyl groups of glycerol (Fig. 1D).Attempts to generate crystals of hSARM1TIR orits mutants bound to NAD+-related ligands re-vealed the structure of hSARM1TIR bound toribose (1.8-Å resolution) (fig. S10, A to E). TheC-2 and C-3 hydroxyl groups interact with theE642 carboxylate, with other hydroxyls inter-acting with Y568, R570, and aspartic acid resi-due 594 (D594). A chloride occupies the positionof the phosphate group attached to the C-5atom in the Nam ribose of NAD+. The structureof hSARM1TIR(G601P) (MES bound; 1.7-Å resolu-tion) (table S1) bound to a molecule of 2-(N-morpholino) ethanesulfonic acid (MES) (fig. S10,F toH) also shows interaction of E642, E599, andY568 with the heterocyclic ring and of R569 andR570 with the sulfonic acid group of MES, whichis located in a similar position to the chloride ionin the structure of an hSARM1TIR:ribose com-plex. MES has been found to mimic ligands inother nucleotide-binding proteins (21).Attempts to capture NAD+-related ligands

in crystals of RUN1TIR led to the structure ofthe NADP+ complex (Fig. 2, E and F, fig. S10I,and table S1). A molecule of bis-Tris [2,2-bis(hydroxymethyl)-2,2',2W-nitrilotriethanol], acomponent of the crystallization solution, is inthe catalytic site, while NADP+ interacts withthe periphery of the active site. The tryptophanat residue 96 (W96) forms a face-to-face aromaticstacking arrangement with the adenosine groupof NADP+, with the C-2′ phosphate of theadenosine ribose in NADP+ forming H-bondswith R34, G35, E36, and R39. The analogousC-5′ phosphate protrudes from the binding site,and the nicotinamide mononucleotide (NMN)

moiety has no interpretable electron density.The bis-Tris interacts with F33, G35, D60, andthe catalytic E100 site. Mutations to residuesin the proposed catalytic site affect effector-independent and effector-dependent HR in TIRdomains and full-length NLRs, respectively (tableS4). Conserved residue mutations R34A, S94A,W96A, and E100A in RUN1TIR also reduce NAD+

cleavage activity (fig. S7D). Double mutation oftwo arginine residues in the BB loop of RUN1TIR

(R64A+R65A) increases NAD+ cleavage activity(figs. S7D and S9, C and D). The bis-Tris inhibitsNADase activity (fig. S10J).

NADase activity dependence onSARM1TIR self-association

The disproportional increase of hSARM1TIR ac-tivity with concentration (fig. S4B) suggests that

self-associationmay be important for enzymaticactivity, consistent with known mechanisms ofTIR domain function (3, 15, 22). In agreementwith the SAMdomains facilitating self-associationof the TIR domains in hSARM1, we observed thatboth the SAMandTIRdomains ofCaenorhabditiselegans SARM1 (cSARM1tSAM-TIR) were requiredfor NADase activity (Fig. 2B). The lower NADaseactivity of cSARM1TIR compared to hSARM1TIR

is consistent with the delay in neuronal cell deathin C. elegans (23).The tandem SAM domains of human SARM1

(hSARM1tSAM; residues 409 to 561), producedin E. coli, were analyzed using size-exclusionchromatography (SEC) coupled withmultianglelight scattering (MALS) (Fig. 3A) and small-angle X-ray scattering (SAXS) (fig. S11). Thefindings suggested an octameric arrangement.

Horsefield et al., Science 365, 793–799 (2019) 23 August 2019 2 of 7

Fig. 1. hSARM1TIR crystal structure. (A) Schematic diagram of the SARM1 domain architecture.(B) Structure of hSARM1TIR [cartoon representation; catalytic E642 (orange) and glycerol (green) instick representation]. (C) Superposition of representative TIR domains from plant NLRs (L6; pink),TLR adaptors (MAL; yellow), TLRs (TLR2; orange) and bacterial proteins (TcpB; green) ontohSARM1TIR (blue). The glutamates equivalent to hSARM1TIR E642 are shown in stick representation.(D) Comparison of the catalytic pockets of hSARM1TIR and MilB. Cartoon representation of thecrystal structure of MilB (purple) bound to CMP (cyan) [Protein Data Bank (PDB) ID 4JEM]; below,rotated 180°, stick representation of residues in the catalytic pocket (green) coordinating with theligand (cyan), and hSARM1TIR structure (blue) bound to glycerol (magenta) with stick representationof residues in the catalytic pocket. (E) hSARM1TIR crystal packing. Three hSARM1TIR moleculesare shown in each of the antiparallel strands (colored orange and blue); each strand features a head-to-tail arrangement via the BB-loop (interacting with DE, bE, and aE regions) interface. Theassociation between strands is via the AE interface. (F) Superposition of one strand of the MALproto-filament (3) (yellow) onto the crystal packing arrangement of hSARM1TIR (orange and blue).

RESEARCH | RESEARCH ARTICLEon F

ebruary 15, 2021

http://science.sciencemag.org/

Dow

nloaded from

In the crystal structure of hSARM1tSAM (2.7-Åresolution; table S1), each of the two SAM do-mains (hSARM1SAM1 and hSARM1SAM2) adoptsa characteristic five–a-helix bundle (a1 to a5)separated by a 10–amino acid linker (residues477 to 486) (figs. S12 and S13 and table S5). Inthe asymmetric unit of the crystal, eight copiesof hSARM1tSAM form a ring (Fig. 3B). There arethree major protein interaction interfaces inthe octamer: an intramolecular hSARM1SAM1:hSARM1SAM2 interface (1034 Å2) and two inter-molecular interfaces between the hSARM1SAM1

domains (966 Å2) and between the hSARM1SAM2

domains (685 Å2) (fig. S14 and table S6). Typ-ically, SAM domains form open-ended polymericstructures (24), but the rigid association of thetwo SAM domains in hSARM1 results in ringformation (fig. S15).SARM1TIR behaves as a monomer in solution

(based on SEC-MALS) (fig. S16A). Analysis ofcrystal packing of hSARM1TIR, to demonstratewhether SAM domain-mediated self-associationcontributes to NADase activity, revealed two

major self-association interfaces: an asymmetricBB loop–mediated one (796 Å2) analogous tothat observed in the MALTIR filament (3) and apseudosymmetric one (1452 Å2) similar to theAE interface in plant TIRs (mostly mediated bythe aA helices) (15, 22) (Fig. 1, E and F, figs. S17and S18, and table S7). Mutations in the BB loop(D594A, E596K, and G601P), aA helix (L579A),and EE loop (H685A) reduce the NADase ac-tivity of hSARM1TIR (Fig. 2A and fig. S4C), sug-gesting that both the BB-loop and AE interfacesobserved in the hSARM1TIR crystals are func-tionally important.In the crystal structure of the impaired BB-loop

mutant hSARM1TIR(G601P) (“ligand-free”; 2.1-Åresolution; table S1), the BB loop interaction ismodified whereas the AE interface is intact(fig. S19A). The BB loop folds over the catalyticcleft, with the lysine 597 residue (K597) insertedinto the active site and interacting with E642(via water molecules; no glycerol is present)(fig. S19B). This conformation may represent theinactive state of hSARM1TIR, presumably stabi-

lized in this protein by the BB loop mutation.In agreement, the K597E mutant of hSARM1is active in NAD+ depletion in axons (23). Thestructure of the inactive AE-interface mutanthSARM1TIR(H685A) (3.0-Å resolution) (table S1)reveals modified interactions between Y568,H685, and R570 in the AE interface (fig. S19C),yet the BB loop interface is intact. Moleculardynamics simulations of monomeric and oligo-meric hSARM1TIR reveal that the active site re-gion is less flexible in the oligomeric form (fig. S20and table S8), suggesting that self-associationmay stabilize the BB loop and the aB helix re-gion, reversing inhibition of the BB loop due tofolding of K597 into the catalytic cleft. Together,our results suggest that both the BB loop andAE interface interactions are required to stabi-lize the fully active enzyme conformation.

NADase activity dependence on plantTIR self-association

Self-association is important for the NADaseactivity of hSARM1TIR and cell death activities

Horsefield et al., Science 365, 793–799 (2019) 23 August 2019 3 of 7

Fig. 2. NADase activity of TIR domains. (A) NADase activity ofhSARM1TIR and its mutants, measured by the fluorescence assay usingeNAD. (B) NAD+ cleavage reaction time courses of human, Drosophila, andC. elegans SARM1 constructs, monitored by 1H NMR (298 K), using aprotein concentration of 20 mM and 1 mM NAD+. (C) NADase activityof RUN1TIR, RUN1TIR(E100A), L6TIR and L6TIR(E135A), measured by the

fluorescence assay using eNAD. (D) NAD+ cleavage reaction time coursesof RUN1TIR and L6TIR, monitored by 1H NMR (20°C), using an NAD+

concentration of 1 mM. (E) Structure of RUN1TIR in complex with NADP+ andbis-Tris (cartoon representation; ligands and selected residues are shownin a stick representation). (F) Comparison of the binding sites for CMP in MilB(PDB ID 4JEM) and NADP+/bis-Tris in RUN1TIR (stick representation).

RESEARCH | RESEARCH ARTICLEon F

ebruary 15, 2021

http://science.sciencemag.org/

Dow

nloaded from

of plant TIRs. In agreement, addition of Ni–nitrilotriacetic acid beads to 6xHis-tagged L6TIR

and RUN1TIR (figs. S7C and S9B) and macro-molecular crowding agents, including poly-ethylene glycol 400 (PEG400) and PEG3350(simulating a crowded environment inside cells),stimulated the NADase activities of L6TIR andRUN1TIR and led to measurable activities forSNC1TIR, RPP1TIR, ROQ1TIR, RPS4TIR, RPV1TIR,and ROQ1TIR (fig. S7, E and F).RUN1TIR behaves as a monomer in solution

(fig. S16B). In the crystals of RUN1TIR, however,both the AE (1400 Å2) and DE (1140 Å2) inter-faces common in plant TIR domains (15, 22) areobserved (fig. S21), comparable to the crystals ofArabidopsis SNC1TIR (15). Mutations in the AEand DE interfaces of L6 perturb self-associationof L6TIR in solution and its cell death signaling

activity in planta (15, 25). Mutation of the cat-alytic E100 still abrogates NAD+ cleavage activityin RUN1TIR(RRAA) (fig. S7D).

Axon degeneration requires SARM1 SAMdomain oligomerization

Based on the hSARM1tSAM crystal structure, wedesigned point mutations to prevent octamerformation. All the tested single-residue alaninemutations either resulted in insoluble pro-teins or showed no disruption of the oligomer(fig. S22). Therefore, we designed a mutant[hSARM1tSAM(5Mut)] with five hydrophobicinterface residues converted to arginines oraspartates (L442R, I461D, L514D, L531D, andV533D) (Fig. 3C and fig. S14, E to G). Thismutant protein is soluble and monomeric, basedon SEC-MALS (Fig. 3A), as is its mouse counter-

part mSARM1tSAM(5Mut) (fig. S16C). The equiv-alent mutations in cSARM1SAM-TIR abolish NADaseactivity (fig. S4D). We then compared the abil-ities of exogenously expressed full-length, wild-type mSARM1wt andmSARM15Mut to overcomethe delay to injury-induced (Wallerian) degen-eration of neurites in Sarm1−/− superior cervi-cal ganglion (SCG) neuron cultures. WhereasmSARM1wt restored rapid Wallerian degenera-tion, mSARM15Mut was expressed but essentiallynonfunctional (Fig. 3D and fig. S23). SAM-mediatedoligomerization therefore plays a pivotal role inthe axon degeneration activity of SARM1.

Implications of NADase activity forplant immunity

The observed NADase activity of plant TIR do-mains may play a role in the cell death function

Horsefield et al., Science 365, 793–799 (2019) 23 August 2019 4 of 7

Fig. 3. Octameric structure of hSARM1tSAM is important for function.(A) Solution properties of hSARM1tSAM (wild type; red) and hSARM1tSAM(5Mut)

(blue), analyzed by SEC-MALS. Peaks indicate the traces from the refractiveindex (RI) detector during SEC; the lines under the peaks correspond tothe average molecular mass distributions across the peak.The averagemolecular mass of hSARM1tSAM is 169.8 kDa (± 0.5%), consistent with anoctamer (theoretical molecular mass 161.9 kDa). (B) Cartoon representationof the octameric ring assembly of hSARM1tSAM molecules. (C) Residuesmutated in hSARM1tSAM(5Mut). (D) Exogenously expressed mSARM1wt, butnotmSARM15Mut, restores the ability ofSarm1−/− neurites to degenerate aftercut. Constructs for mouse SARM1 variants were injected along with DsRed

vector (40 ng ml−1) to allow visualization of neurites of the injected neurons.The percentage of intact neurites at 24 hours after cut, relative to thosepresent at the time of transection, is plotted (left, individual values andmeans ± SEM are shown). Representative images of cut neurites at thetime of transection (0 hours) and at 24 hours are shown (right). mSARM15Mut

does not promote degeneration of cut Sarm1−/− neurites even at the highestconcentration used [n.s., not significant (P > 0.05); ***P < 0.001,separate one-way analyses of variance for each concentration withTukey’s multiple-comparison tests]. mSARM15Mut is expressed at theexpected size and at a higher level than mSARM1wt, both in injected SCGneurons and transfected HEK cells (fig. S25).

RESEARCH | RESEARCH ARTICLEon F

ebruary 15, 2021

http://science.sciencemag.org/

Dow

nloaded from

of plant NLRs, possibly by a mechanism similarto hSARM1. Mutation of the conserved cata-lytic glutamate in L6TIR-YFP, RUN1TIR-YFP,SNC1TIR-YFP, andRPS4TIR-YFP abrogated effector-independent HR detected by transient expressioninN. benthamiana leaves (Fig. 4A and fig. S24A).RUN1TIR(RRAA), which has increased NAD+ cleav-age activity, also showed increased HR of YFPandMyc fusion proteins (Fig. 4B and figs. S24Band S25).Expression inN. benthamianaof hSARM1tSAM-TIR,

but not hSARM1TIR or hSARM1tSAM, inducedcell death (Fig. 4C and fig. S24C). Disruption ofhSARM1tSAM-TIR oligomerization by introduc-tion of the fivemutations [hSARM1tSAM(5Mut)-TIR]abrogated cell death, indicating the need for SAM

domain–induced hSARM1TIR self-association inthe cell death process in planta. The E642A mu-tation in the hSARM1tSAM-TIR construct alsodisrupted cell death, implicating NAD+ cleavagein the process.Fusion of the oligomerizing hSARM1tSAM to

L6TIRd28-Myc, SNC1TIR-Myc, RPS4TIR-Myc, andRUN1TIR-Myc enhanced cell death induction ofthese proteins inN. benthamiana, while fusionof the nonoligomerizing hSARM1tSAM(5Mut) didnot (Fig. 4D and fig. S24D). Thus, like hSARM1TIR

neuronal degeneration activity, plant TIR-inducedHR requires both the catalytic glutamate and theability to self-associate.Plant TIR-containing NLR signaling requires

the downstream signaling component EDS1 (26).

However, hSARM1tSAM-TIR induced HRwhen itwas transiently expressed in N. benthaminaeds1-1 knockout mutant lines (27) (Fig. 4C andfig. S24C), implicating a mechanism differentto plant TIR-induced HR, or NADase activityinvolving a separate pathway distinct from theEDS1-mediated signaling pathway. By contrast,hSARM1tSAM fusions of plant TIR domains andRUN1TIR(RRAA) failed to induce HR in the eds1-1plants (Fig. 4, B and E, and fig. S26).Mutation of the conserved glutamate (E135A)

in the full-length L6 NLR also abolished itsability to induce cell death in the presence of theAvrL567 ligand (Fig. 4F and fig. S24E). Likewise,this mutation abolishes effector-independentsignaling by the constitutively active L6D541V

Horsefield et al., Science 365, 793–799 (2019) 23 August 2019 5 of 7

Fig. 4. TIR domainfunctions in plants.(A) Expression inN. benthamiana (N.b.) ofcatalytic glutamate mutantsof L6TIR, RUN1TIR, SNC1TIR,and RPS4TIR. (B) Expressionin N. benthamiana (left)and the eds1-1 mutant (right)of RUN1TIR-YFP andRUN1TIR(RRAA)-YFP, alone orcoexpressed with NdEDS1-YFP. (C) Left, Expressionin N. benthamiana ofhSARM1tSAM-TIR, hSARM1TIR,hSARM1tSAM(5Mut)-TIR,hSARM1tSAM-TIR(E642A),hSARM1TIR(E642A), andhSARM1tSAM. (Middle andright) Expression inN. benthamiana and theeds1-1 of hSARM1tSAM-TIR andhSARM1tSAM-TIR(E642A).(D) Expression inN. benthamiana of fusionproteins of TIR domainsfrom L6, SNC1, RPS4, andRUN1 to hSARM1tSAM

and hSARM1tSAM(5Mut).(E) Expression in inN. benthamiana (top) and theeds1-1 mutant (bottom)of TIR domains andTIR-hSARM1tSAM fusion pro-teins of RUN1 alone or coex-pressed with NbEDS1-YFP.(F) Expression of L6-YFP,L6MHV-YFP, L6E135A-YFP, andL6MHV-E135A-YFP inN. tabacum W38 (top) ortransgenic W38 expressingAvrL567 (bottom).

RESEARCH | RESEARCH ARTICLEon F

ebruary 15, 2021

http://science.sciencemag.org/

Dow

nloaded from

variant (28). Consistent with the abrogated HRphenotypes observed for glutamate mutants, ourdata implicate TIR NADase activity in the celldeath process.

Bioinformatic analysis of TIR domains

To analyze the prevalence of NAD+ cleavageactivities among TIR domains and the relation-ships with enzymes such as MilB, we used arecently developed evolutionary model that isbased on secondary structure (29) to constructa phylogeny of proteins structurally similar tohSARM1TIR and plant TIR domains [fig. S27;for protein families with limited sequence sim-ilarities, such as the TIR domains, phylogeneticanalyses using sequence-based evolutionarymod-els are not reliable (30)]. This analysis suggeststhat TIR domains are part of a large superfamilyof enzymes that includes a number of structurallyrelated proteins (which are not usually associatedwith TIR domains) that bind nucleotides (e.g.,glycosyltransferases, nucleoside hydrolases, andflavodoxins) or carbohydrates (e.g., bacterial isom-erases) in the analogous region of the protein.

Outlook

We describe the structural basis of self-association–dependent NAD+ cleavage by TIRdomains from animal SARM1 and plant NLRs.TIR domains have a flavodoxin fold common tomany proteins with diverse functions, and ourbioinformatic analyses reveal that they are re-lated to a number of proteins with enzymaticactivities such as nucleotide and carbohydratehydrolysis, consistent with the NAD+ cleavageactivity observed for TIR domains. Some branchesmay have lost enzymatic activity but retainedscaffolding protein interaction functions. TLRand adaptor proteins show no NADase activities[fig. S7E; (13)]; although TLRs have glutamateresidues in the analogous region of the protein,these are not found in the same spatial loca-tions (Fig. 1). Some bacterial TIR domains havebeen shown to possess NAD+ cleavage activity(31), and the equivalent glutamate appears tobe conserved; however, the analogous regionsof the bacterial TIR domains with known struc-tures (1) differ from those in hSARM1TIR andplant TIR domains, with the Ca of the gluta-mate located >6 Å away (Fig. 1).In SARM1, the self-association of TIR domains

required for NADase activity is at least in partfacilitated by the octamer of SAM domains. TheARM domain (by interaction with the SAM andTIR domains) is suggested to hold hSARM1 in aninactive state (12); NMN has been proposed toremove this autoinhibition after axonal injury(32), allowing the TIR domains to self-associate,which activates the NADase activity and leadsto axon degeneration (fig. S28A). Full-lengthSARM1 has been reported to self-associate (8),suggesting it may be constitutively oligomeric.A structural model of activation may include

the following steps: Before activation, the inter-action of BB loop K597 with E642 prevents en-zymatic activity, and the N-terminal domain ofhSARM1 may stabilize this conformation. Upon

axon injury, SAM domain assembly facilitatesTIR domain association [analogous to animalNLRs causing caspase recruitment domain as-sociation (33)] through the BB loop interface,removing K597 from its inhibitory interaction.Further TIR domain association through the AEinterface facilitates the interactions of H685 andR570, leading to optimal configuration of the ac-tive site, NAD+ cleavage, and axon degeneration.In plant NLRs, we propose the nucleotide-

binding domains (NBDs) (34) facilitate self-association of TIR domains through AE andDE interfaces (15, 16). Downstream signalingby plant NLR TIR domains remains a mysterydespite substantial efforts by many researchgroups, with no direct signaling partners iden-tified. The observed NAD+ cleavage activityof TIR domains could represent the SCAF func-tion (22) responsible for the signaling event.Plant TIR domain cell death signaling is EDS1dependent (26). However, hSARM1tSAM-TIR in-duces cell death in eds1-1 N. benthamiana lines;hSARM1TIR hydrolyzes NAD+ much more rap-idly than the plant TIR domains examined andthus may cause necrosis through depletion ofNAD+, as suggested for axons. Plant TIRs maysignal cell death via a more controlled pathwaymediated by EDS1 (fig. S28B). Alternatively, theNAD+ hydrolysis catalyzed by plant TIRs maybe part of a pathway different from the EDS1-mediated signaling pathway.Consequently, the products of NAD+ hydroly-

sis may be involved in cell death signaling. CyclicADPR (cADPR), produced by hSARM1TIR (13)and CD38 (35), has been shown to stimulate Ca2+

influx as part of NLR-mediated HR through Ca2+

channels and is also involved in both abscisicacid and salicylic acid signaling pathways (36).NaADP is also involved in Ca2+ signaling inplants, although the enzymes responsible for itssynthesis are unknown (37). The NBDs couldplay a role by not only providing an oligomericplatform for TIR domain self-association butalso binding of the ADP group of NAD+ andremoving the cleaved ADPR group. Proteinsstructurally related to TIR domains have nucle-otide transfer activities (e.g., ADP ribosylation),and many ADP-ribosylases show low enzymaticactivity in the absence of the target of ribosyla-tion (38), which could explain the low NADaseactivities of plant TIR domains. The low NADaseactivities we observe may be due to poor self-association abilities of isolated plant TIR do-mains (supported by the stimulation of activitywith affinity beads or macromolecular crowd-ing reagents and in cell lysates, or NAD+ maynot be the preferred substrate [many NADasesshow high substrate promiscuity (39)].In summary, we show that NAD+ cleavage by

TIR domains is a conserved feature of animaland plant cell death signaling pathways, but thedifferences in structural organization of therelevant proteins and in cellular contexts resultin distinctive mechanisms. Our results providea foundation for future work on the role ofNADase activity in plant immunity and devel-opment of inhibitors of axon degeneration.

REFERENCES AND NOTES

1. T. Ve, S. J. Williams, B. Kobe, Apoptosis 20, 250–261(2015).

2. N. J. Gay, M. F. Symmons, M. Gangloff, C. E. Bryant,Nat. Rev. Immunol. 14, 546–558 (2014).

3. T. Ve et al., Nat. Struct. Mol. Biol. 24, 743–751 (2017).4. P. R. Vajjhala, T. Ve, A. Bentham, K. J. Stacey, B. Kobe,

Mol. Immunol. 86, 23–37 (2017).5. M. Carty et al., Nat. Immunol. 7, 1074–1081 (2006).6. J. M. Osterloh et al., Science 337, 481–484 (2012).7. Y. Kim et al., J. Exp. Med. 204, 2063–2074 (2007).8. J. Gerdts, E. J. Brace, Y. Sasaki, A. DiAntonio, J. Milbrandt,

Science 348, 453–457 (2015).9. P. N. Dodds, J. P. Rathjen, Nat. Rev. Genet. 11, 539–548

(2010).10. J. Gerdts, D. W. Summers, J. Milbrandt, A. DiAntonio,

Neuron 89, 449–460 (2016).11. J. Wang et al., J. Cell Biol. 170, 349–355 (2005).12. J. Gerdts, D. W. Summers, Y. Sasaki, A. DiAntonio, J. Milbrandt,

J. Neurosci. 33, 13569–13580 (2013).13. K. Essuman et al., Neuron 93, 1334–1343.e5 (2017).14. X. Zhang, P. N. Dodds, M. Bernoux, Annu. Rev. Phytopathol. 55,

205–229 (2017).15. X. Zhang et al., Proc. Natl. Acad. Sci. U.S.A. 114, E2046–E2052

(2017).16. M. T. Nishimura et al., Proc. Natl. Acad. Sci. U.S.A. 114,

E2053–E2062 (2017).17. L. Holm, L. M. Laakso, Nucleic Acids Res. 44, W351–W355

(2016).18. Q. Zhang, C. M. Zmasek, X. Cai, A. Godzik, Dev. Comp. Immunol.

35, 461–468 (2011).19. G. Pergolizzi, J. N. Butt, R. P. Bowater, G. K. Wagner,

Chem. Commun. 47, 12655–12657 (2011).20. Y. Gibon, F. Larher, Anal. Biochem. 251, 153–157

(1997).21. N. Czudnochowski, A. L. Wang, J. Finer-Moore, R. M. Stroud,

J. Mol. Biol. 425, 3875–3887 (2013).22. S. Nimma, T. Ve, S. J. Williams, B. Kobe, Curr. Opin. Struct.

Biol. 43, 122–130 (2017).23. D. W. Summers, D. A. Gibson, A. DiAntonio, J. Milbrandt,

Proc. Natl. Acad. Sci. U.S.A. 113, E6271–E6280 (2016).24. M. Bienz, Trends Biochem. Sci. 39, 487–495 (2014).25. M. Bernoux et al., Cell Host Microbe 9, 200–211

(2011).26. M. Wiermer, B. J. Feys, J. E. Parker, Curr. Opin. Plant Biol. 8,

383–389 (2005).27. A. Schultink, T. Qi, A. Lee, A. D. Steinbrenner, B. Staskawicz,

Plant J. 92, 787–795 (2017).28. M. Bernoux et al., Plant Cell 28, 146–159 (2016).29. J.-S. Lai, B. Rost, B. Kobe, M. Boden, bioRxiv 563452

[Preprint]. 28 February 2019; https://doi.org/10.1101/563452.

30. C. P. Ponting, R. R. Russell, Annu. Rev. Biophys. Biomol. Struct.31, 45–71 (2002).

31. K. Essuman et al., Curr. Biol. 28, 421–430.e4 (2018).32. Z. Y. Zhao et al., iScience 15, 452–466 (2019).33. M. Matyszewski et al., J. Biol. Chem. 293, 20240–20248

(2018).34. J. Wang et al., Science 364, eaav5870 (2019).35. Q. Liu et al., J. Biol. Chem. 284, 27637–27645 (2009).36. L. Hunt, F. Lerner, M. Ziegler, New Phytol. 163, 31–44

(2004).37. B. Gakière et al., Crit. Rev. Plant Sci. 2018, 1–49

(2018).38. N. J. Oppenheimer, A. J. Handlon, in The Enzymes,

D. S. Sigman, Ed. (Academic Press, 1992), pp. 453–505.39. H. C. Lee, Mol. Med. 12, 317–323 (2006).

ACKNOWLEDGMENTS

We acknowledge the use of the University of Queensland RemoteOperation Crystallization and X-Ray Diffraction (UQ-ROCX)Facility and the macromolecular crystallography (MX) andsmall/wide-angle X-ray scattering (SAXS/WAXS) beamlines at theAustralian Synchrotron, Victoria, Australia. MD simulations wereperformed on the High Performance Computing cluster “Gowonda”at Griffith University. We thank V. Masic and N. Deerain fortechnical contributions. Funding: The work was supported by theNational Health and Medical Research Council (NHMRC grants1107804 and 1160570 to B.K. and T.V.. 1071659 to B.K., and1108859 to T.V.), and the Australian Research Council (ARC grantsDP160102244 and DP190102526 to B.K. and P.N.D.). B.K. was anNHMRC Principal Research Fellow (1110971) and ARC LaureateFellow (FL180100109). T.V. received ARC DECRA (DE170100783)funding and S.J.W. received ARC DECRA DE160100893 funding.J.C. received a Chinese Scholarship Council (CSC) postgraduate

Horsefield et al., Science 365, 793–799 (2019) 23 August 2019 6 of 7

RESEARCH | RESEARCH ARTICLEon F

ebruary 15, 2021

http://science.sciencemag.org/

Dow

nloaded from

scholarship. Y.S. was a Griffith University postdoctoral fellow. J.G.was supported by the UK Medical Research Council and M.P.C. wassupported by the John and Lucille van Geest Foundation. M.K.M.was supported by the Australian Government Research TrainingProgram (RTP). Author contributions: S.H., H.B., X.Z., M.K.M.,Y.S., J.G., R.O.H., T.B., S.J.W., T.V., P.D., and B.K. designed theresearch; S.H., H.B., X.Z., M.K.M., Y.S., J.G., J.C., L.W.C., T.Q.,J.S.L., W.G., M.X.R., D.J.E., G.F., R.O.H., T.B., and T.V. performed theresearch; S.H., H.B., X.Z., M.K.M., Y.S., J.G., L.W.C., R.O.H., T.B., J.S.L.,

M.X.R., D.J.E., G.F., M.v.I., J.P.R., J.D.N., M.B., I.B.D., B.J.S., S.J.W.,M.P.C., T.V., P.N.D., and B.K. analyzed the data; S.H., H.B., X.Z.,M.K.M., Y.S., T.V., and B.K. wrote the paper; all authors edited andcontributed to writing. Competing interests: B.K. is a consultant forDisarm Therapeutics. B.K. and S.H. receive research funding fromDisarm Therapeutics. Data and materials availability: Coordinatesand structure factors for all crystal structures determined in thisstudy have been deposited in the Protein Data Bank with IDs 6O0S,6O0T, 6O0R, 6O0Q, 6O0U, 6O1B, 6O0V, and 6O0W.

SUPPLEMENTARY MATERIALS

science.sciencemag.org/content/365/6455/793/suppl/DC1Materials and MethodsFigs. S1 to S28Table S1 to S8References (40–88)

28 February 2019; accepted 23 July 201910.1126/science.aax1911

Horsefield et al., Science 365, 793–799 (2019) 23 August 2019 7 of 7

RESEARCH | RESEARCH ARTICLEon F

ebruary 15, 2021

http://science.sciencemag.org/

Dow

nloaded from

cleavage activity by animal and plant TIR domains in cell death pathways+NAD

Staskawicz, Michael P. Coleman, Thomas Ve, Peter N. Dodds and Bostjan KobeBosanac, Mark von Itzstein, John P. Rathjen, Jeffrey D. Nanson, Mikael Boden, Ian B. Dry, Simon J. Williams, Brian J.Jhih-Siang Lai, Maxwell X. Rank, Lachlan W. Casey, Weixi Gu, Daniel J. Ericsson, Gabriel Foley, Robert O. Hughes, Todd Shane Horsefield, Hayden Burdett, Xiaoxiao Zhang, Mohammad K. Manik, Yun Shi, Jian Chen, Tiancong Qi, Jonathan Gilley,

DOI: 10.1126/science.aax1911 (6455), 793-799.365Science 

, this issue p. 793, p. 799Science depletion during Wallerian degeneration of neurons.+signaling links mammalian TIR-containing proteins to NAD

) as part of their cell-death signaling in response to pathogens. Similar+nicotinamide adenine dinucleotide (NAD report that these TIR domains cleave the metabolic cofactoret al. and Wan et al.domains. In two papers, Horsefield

leucine-rich repeat immune receptors responsible for this hypersensitive response carry Toll/interleukin-1 receptor (TIR) One way that plants respond to pathogen infection is by sacrificing the infected cells. The nucleotide-binding

NAD depletion as pathogen response

ARTICLE TOOLS http://science.sciencemag.org/content/365/6455/793

MATERIALSSUPPLEMENTARY http://science.sciencemag.org/content/suppl/2019/08/21/365.6455.793.DC1

REFERENCES

http://science.sciencemag.org/content/365/6455/793#BIBLThis article cites 87 articles, 20 of which you can access for free

PERMISSIONS http://www.sciencemag.org/help/reprints-and-permissions

Terms of ServiceUse of this article is subject to the

is a registered trademark of AAAS.ScienceScience, 1200 New York Avenue NW, Washington, DC 20005. The title (print ISSN 0036-8075; online ISSN 1095-9203) is published by the American Association for the Advancement ofScience

Science. No claim to original U.S. Government WorksCopyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of

on February 15, 2021

http://science.sciencem

ag.org/D

ownloaded from