Cyclic nucleotide signalling is a key component of antiviral defence in all domains of life. Viral detection activates a nucleotide cyclase to generate a second messenger, resulting in activation of effector proteins. This is exemplified by the metazoan cGAS–STING innate immunity pathway1, which originated in bacteria2. These defence systems require a sensor domain to bind the cyclic nucleotide and are often coupled with an effector domain that, when activated, causes cell death by destroying essential biomolecules3. One example is the Toll/interleukin-1 receptor (TIR) domain, which degrades the essential cofactor NAD+ when activated in response to infection in plants and bacteria2,4,5 or during programmed nerve cell death6. Here we show that a bacterial antiviral defence system generates a cyclic tri-adenylate that binds to a TIR–SAVED effector, acting as the ‘glue’ to allow assembly of an extended superhelical solenoid structure. Adjacent TIR subunits interact to organize and complete a composite active site, allowing NAD+ degradation. Activation requires extended filament formation, both in vitro and in vivo. Our study highlights an example of large-scale molecular assembly controlled by cyclic nucleotides and reveals key details of the mechanism of TIR enzyme activation.
This is a preview of subscription content, access via your institution
Open Access articles citing this article.
Nature Open Access 20 July 2022
Subscribe to Nature+
Get immediate online access to the entire Nature family of 50+ journals
Subscribe to Journal
Get full journal access for 1 year
only $3.90 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Get time limited or full article access on ReadCube.
All prices are NET prices.
Cai, X., Chiu, Y. H. & Chen, Z. J. The cGAS-cGAMP-STING pathway of cytosolic DNA sensing and signaling. Mol. Cell 54, 289–296 (2014).
Morehouse, B. R. et al. STING cyclic dinucleotide sensing originated in bacteria. Nature 586, 429–433 (2020).
Millman, A., Melamed, S., Amitai, G. & Sorek, R. Diversity and classification of cyclic-oligonucleotide-based anti-phage signalling systems. Nat. Microbiol. 5, 1608–1615 (2020).
Essuman, K. et al. TIR domain proteins are an ancient family of NAD+-consuming enzymes. Curr. Biol. 28, 421–430 (2018).
Wan, L. et al. TIR domains of plant immune receptors are NAD+-cleaving enzymes that promote cell death. Science 365, 799–803 (2019).
Summers, D. W., Gibson, D. A., DiAntonio, A. & Milbrandt, J. SARM1-specific motifs in the TIR domain enable NAD+ loss and regulate injury-induced SARM1 activation. Proc. Natl Acad. Sci. USA 113, E6271–E6280 (2016).
Kazlauskiene, M., Kostiuk, G., Venclovas, C., Tamulaitis, G. & Siksnys, V. A cyclic oligonucleotide signaling pathway in type III CRISPR-Cas systems. Science 357, 605–609 (2017).
Niewoehner, O. et al. Type III CRISPR–Cas systems produce cyclic oligoadenylate second messengers. Nature 548, 543–548 (2017).
Tal, N. et al. Cyclic CMP and cyclic UMP mediate bacterial immunity against phages. Cell 184, 5728–5739 (2021).
Athukoralage, J. S. & White, M. F. Cyclic oligoadenylate signalling and regulation by ring nucleases during type III CRISPR defence. RNA 27, 855–867 (2021).
Horsefield, S. et al. NAD+ cleavage activity by animal and plant TIR domains in cell death pathways. Science 365, 793–799 (2019).
Ofir, G. et al. Antiviral activity of bacterial TIR domains via immune signalling molecules. Nature 600, 116–120 (2021).
Ka, D., Oh, H., Park, E., Kim, J. H. & Bae, E. Structural and functional evidence of bacterial antiphage protection by Thoeris defense system via NAD+ degradation. Nat. Commun. 11, 2816 (2020).
Burroughs, A. M., Zhang, D., Schaffer, D. E., Iyer, L. M. & Aravind, L. Comparative genomic analyses reveal a vast, novel network of nucleotide-centric systems in biological conflicts, immunity and signaling. Nucl. Acids Res. 43, 10633–10654 (2015).
Makarova, K. S. et al. Evolutionary and functional classification of the CARF domain superfamily, key sensors in prokaryotic antivirus defense. Nucl. Acids Res. 48, 8828–8847 (2020).
Takeuchi, M. & Hatano, K. Proposal of six new species in the genus Microbacterium and transfer of Flavobacterium marinotypicum ZoBell and Upham to the genus Microbacterium as Microbacterium maritypicum comb. nov. Int. J. Syst. Bacteriol. 48, 973–982 (1998).
Canto, C., Menzies, K. J. & Auwerx, J. NAD+ metabolism and the control of energy homeostasis: a balancing act between mitochondria and the nucleus. Cell Metab. 22, 31–53 (2015).
Zhou, Y. et al. Determining the extremes of the cellular NAD(H) level by using an Escherichia coli NAD+-auxotrophic mutant. Appl. Environ. Microbiol. 77, 6133–6140 (2011).
Lowey, B. & Kranzusch, P. J. CD-NTases and nucleotide second messenger signaling. Curr. Biol. 30, R1106–R1108 (2020).
Grüschow, S., Athukoralage, J. S., Graham, S., Hoogeboom, T. & White, M. F. Cyclic oligoadenylate signalling mediates Mycobacterium tuberculosis CRISPR defence. Nucl. Acids Res. 47, 9259–9270 (2019).
Jumper, J. et al. Highly accurate protein structure prediction with AlphaFold. Nature 596, 583–589 (2021).
Mirdita, M., Ovchinnikov, S. & Steinegger, M. ColabFold - making protein folding accessible to all. Nat. Methods 19, 679–682 (2022).
Lowey, B. et al. CBASS immunity uses CARF-related effectors to sense 3'-5'- and 2'-5'-linked cyclic oligonucleotide signals and protect bacteria from phage infection. Cell 182, 38–49 (2020).
Fatma, S., Chakravarti, A., Zeng, X. & Huang, R. H. Molecular mechanisms of the CdnG-Cap5 antiphage defense system employing 3',2'-cGAMP as the second messenger. Nature Commun. 12, 6381 (2021).
Martin, R. et al. Structure of the activated ROQ1 resistosome directly recognizing the pathogen effector XopQ. Science 370, eabd9993 (2020).
Ma, S. et al. Direct pathogen-induced assembly of an NLR immune receptor complex to form a holoenzyme. Science 370, eabe3069 (2020).
Ve, T. et al. Structural basis of TIR-domain-assembly formation in MAL- and MyD88-dependent TLR4 signaling. Nat. Struct. Mol. Biol. 24, 743–751 (2017).
Shi, Y. et al. Structural basis of SARM1 activation, substrate recognition, and inhibition by small molecules. Mol. Cell 82, 1643–1659 (2022).
Burdett, H., Hu, X., Rank, M. X., Maruta, N. & Kobe, B. Self-association configures the NAD+-binding site of plant NLR TIR domains. Preprint at bioRxiv https://doi.org/10.1101/2021.10.02.462850 (2021).
Vajjhala, P. R., Ve, T., Bentham, A., Stacey, K. J. & Kobe, B. The molecular mechanisms of signaling by cooperative assembly formation in innate immunity pathways. Mol. Immunol. 86, 23–37 (2017).
Morehouse, B. R. et al. Cryo-EM structure of an active bacterial TIR–STING filament complex. Nature https://doi.org/10.1038/s41586-022-04999-1 (2022).
Grüschow, S., Adamson, C. S. & White, M. F. Specificity and sensitivity of an RNA targeting type III CRISPR complex coupled with a NucC endonuclease effector. Nucl. Acids Res. 49, 13122–13134 (2021).
Rouillon, C., Athukoralage, J. S., Graham, S., Grüschow, S. & White, M. F. Investigation of the cyclic oligoadenylate signalling pathway of type III CRISPR systems. Methods Enzymol. 616, 191–218 (2019).
Whiteley, A. T. et al. Bacterial cGAS-like enzymes synthesize diverse nucleotide signals. Nature 567, 194–199 (2019).
Pernot, P. et al. Upgraded ESRF BM29 beamline for SAXS on macromolecules in solution. J. Synchrotron Radiat. 20, 660–664 (2013).
Round, A. et al. BioSAXS Sample Changer: a robotic sample changer for rapid and reliable high-throughput X-ray solution scattering experiments. Acta Crystallogr. D 71, 67–75 (2015).
Costa, L. et al. Combined small angle X-ray solution scattering with atomic force microscopy for characterizing radiation damage on biological macromolecules. BMC Struct. Biol. 16, 18 (2016).
Hajizadeh, N. R., Franke, D., Jeffries, C. M. & Svergun, D. I. Consensus Bayesian assessment of protein molecular mass from solution X-ray scattering data. Sci. Rep. 8, 7204 (2018).
Manalastas-Cantos, K. et al. ATSAS 3.0: expanded functionality and new tools for small-angle scattering data analysis. J. Appl. Crystallogr. 54, 343–355 (2021).
Svergun, D., Barberato, C. & Koch, M. H. J. CRYSOL - a program to evaluate x-ray solution scattering of biological macromolecules from atomic coordinates. J. Appl. Crystallogr. 28, 768–773 (1995).
Mastronarde, D. N. Automated electron microscope tomography using robust prediction of specimen movements. J. Struct. Biol. 152, 36–51 (2005).
Punjani, A., Rubinstein, J. L., Fleet, D. J. & Brubaker, M. A. cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nat. Methods 14, 290–296 (2017).
Rubinstein, J. L. & Brubaker, M. A. Alignment of cryo-EM movies of individual particles by optimization of image translations. J. Struct. Biol. 192, 188–195 (2015).
Punjani, A. & Fleet, D. J. 3D variability analysis: resolving continuous flexibility and discrete heterogeneity from single particle cryo-EM. J. Struct. Biol. 213, 107702 (2021).
Casanal, A., Lohkamp, B. & Emsley, P. Current developments in Coot for macromolecular model building of electron cryo-microscopy and crystallographic data. Protein Sci. 29, 1069–1078 (2020).
Nicholls, R. A., Tykac, M., Kovalevskiy, O. & Murshudov, G. N. Current approaches for the fitting and refinement of atomic models into cryo-EM maps using CCP-EM. Acta Crystallogr. D 74, 492–505 (2018).
Liebschner, D. et al. Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix. Acta Crystallogr. D 75, 861–877 (2019).
This work was financed by the Biotechnology and Biological Sciences Research Council (references BB/S000313 and BB/T004789) and a European Research Council Advanced Grant (grant number 101018608) to M.F.W. We thank J. Athukoralage, T. Gloster and S. McQuarrie for discussions. We thank M. Tully for assistance in using beamline BM29. We acknowledge the Scottish Centre for Macromolecular Imaging, M. Clarke and J. Streetley for assistance with cryo-EM experiments and access to instrumentation, financed by the Medical Research Council (MC_PC_17135) and the Scottish Funding Council (H17007). This work used the platforms of the Grenoble Instruct-ERIC centre (Integrated Structural Biology Grenoble; UAR 3518 CNRS-CEA-UGA-EMBL) within the Grenoble Partnership for Structural Biology, supported by the French Infrastructure for Integrated Structural Biology (ANR-10-INBS-0005-02) and the Grenoble Alliance for Integrated Structural Cell Biology, financed within the University Grenoble Alpes graduate school (Ecoles Universitaires de Recherche) CBH-EUR-GS (ANR-17-EURE-0003). Components of Figs. 1 and 3 were created with BioRender.com.
The authors declare no competing interests.
Peer review information
Nature thanks Martin Jinek and Andrew Lovering for their contribution to the peer review of this work. Peer reviewer reports are available.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data figures and tables
a, Cyclase products separated by thin layer chromatography. Radiolabelled ATP was mixed with 50 µM “cold” NTP (ATP, GTP, CTP, TTP, or UTP) and incubated with 20 µM cyclase for 2 h at 37 °C. As controls, cyclic oligoadenylate produced by the Type III CRISPR complex VmeCMR21 (C+) and the reaction without protein (C−). b, Cyclic nucleotide screening for TIR-SAVED NADase activity. 0.5 µM TIR-SAVED was incubated without (apo condition) or with 5 µM cyclic: AMP, di-AMP, tri-AMP, tetra-AMP, hexa-AMP, GMP, di-GMP, AMP-GMP, di-AMP-GMP or ADP ribose. The fluorescence intensity (a.u.) at 90 min of reaction was plotted for each cyclic oligoadenylate. c, Initial rate of TIR NADase activity depends on cA3 concentration. 0.5 µM TIR-SAVED was incubated with 0, 0.1, 0.3, 0.5, 1.0, 2.5 µM cA3 and 500 µM ɛNAD+. d, Enzymatic characterization of TIR-SAVED NADase activity. Initial rate is plotted against a range of ɛNAD+ concentration. Experimental data were fitted using the Michaelis-Menten equation. e, TIR NADase activity is proportional to TIR-SAVED concentration. 0, 0.1, 0.2, 0.4, 0.5, 0.8, 1.0 µM TIR-SAVED were incubated with 1 µM cA3 and 2 mM ɛNAD+. Data are the means of triplicate experiments with standard deviation (b, c, d, e).
Extended dataset from Fig. 1g. a, Schematic of the plasmid and competent cells used for the transformation. On the left, tetracycline resistant plasmid used for to transform the recipient cell. The tir-saved gene was cloned into the multiple cloning site 1 of the plasmid 2 and 3. In the plasmid 3, the catalytic residue E84 was mutated to prevent NADase activity. Recipient cell A and B both expressed the MtbCsm targeting the tetracycline resistant plasmid. In B, MtbCsm Cas10 was mutated (D630A) to prevent cOA production and is used as a control. In recipient cell C, the tetracycline resistant plasmid is not recognized as a target by MtbCsm. b, Cloning strategy of TIR-SAVED variants into the pRAT-Duet plasmid (left panel). To co-express two TIR-SAVED variants, one version was cloned into the multiple cloning site 1 (MSC-1) under the pBAD promotor while the R variant was cloned into the multiple cloning site 2 (MSC-2) under the T7 promoter. c, Transformed colonies after incubation overnight on induced plate. The different recipient cell/plasmid combination are annotated as “A.1” (recipient cell A transformed by plasmid 1). Results from two independent experiments with technical duplicates.
a, SEC-SAXS profile. Rg (radiation gyrus) was calculated based on Guinier approximation (see Material & Method section) and plotted against eluted volume. In red, the fractions used to estimate the global Rg and the molecular weight range of the protein. The theoretical MW value of the recombinant TIR-SAVED is 47.3 kDa. b, AF2 model fitted with the SEC-SAXS experimental dataset selected (red fractions in a.). c, Elution profile of TIR-SAVED after size exclusion chromatography in absence (blue) or presence (orange) of cA3 for a molar ratio of 1:1.5 (protein:cA3). Additional analysis of TIR-SAVED molecular weight by analytical gel filtration are shown in Supplemental Data Fig. 4.
a, Cryo-EM micrograph with single particle picked. b, Extract of single particle selected. c, 2D classes from both top and side views particles. d, 3D model of the filament. In blue, the extracted map used for refinement. e, Final refined 3D map coloured by local resolution (blue 2.5 A to red, 5 Å). f, Atomic model fitted into the density map for four TIR-SAVED/cA3 subunits.
a, position of the cA3 molecule bound to the SAVED protein. b, Representative map densities. Example map densities that allowed construction of the atomic model. The labels refer to the chain identities and residue numbers. The regions part of TIR-SAVED main features (BB loop, DE loop and cA3 binding pocket) are highlighted.
Secondary structure features are displayed for the TIR domain to match the conserved features of protein TIR family. Conserved residues are shaded. Mutated residues used in biochemical experiments are highlighted.
a, Dynamic Light Scattering analysis confirms the oligomerisation of Y115A and D45AL46A in presence of 1:1.5 protein:cA3 molar ratio. Experiment in technical triplicates for each condition. b, NADase activity comparison of the Y115A and D45AL46A mutants with the WT for a 3-fold serial dilution in protein concentrations (0.16, 0.5, 1.5, 4.5, 13.5 µM). 27 µM cA3 was used to activate TIR-SAVED incubated with 500 µM ɛNAD+ substrate. c, Enzymatic properties of Y115A mutant. Based on a NAD range concentration experiment, the initial rate of fluorescent ADP ribose production was calculated and fitted following a Michaelis-Menten model. In these experiments, as used previously for the WT, 0.5 µM Y115A was mixed in presence of 1 µM cA3 to hydrolyse 25, 75, 225, 500, 1000, 1500, 2000 and 3000 µM ɛNAD+. The right panel compares the final Michaelis-Menten parameters of Y115A with the WT protein. Data are the means of three experiments with Standard deviation indicated.
a, Schematic of TIR-SAVED mutations. b, Dynamic Light Scattering profile of TIR-SAVED variants in presence of 1:1.5 protein:cA3 molar ratio. c, NADase activity of E84Q and R388E compared to the WT TIR-SAVED enzyme. A range of protein concentration (0, 0.16, 0.5, 1.5, 4, 13.5 µM) was incubated with 27 µM cA3 and 500 µM ɛ-NAD+ for 60 min. Data are means plotted with standard deviations for triplicate experiments. d, The K199E/W394A variant is catalytically dead. The protein (1.5 µM) was incubated with increased cA3 concentration (from 0.03 to 13.5 µM) and 500 µM ɛ-NAD+. Data are means from duplicate experiments. e, Comparison of the thermal denaturation profile of TIR-SAVED in presence of three concentrations of cA3 indicated as protein:cA3 molar ratio of 1:0.2, 1:1, 1:5. The melting temperature Tm for the condition 1:0 and 1:5 was plotted for each protein (right panel). Experiments were done in triplicates with three measures for each temperature.
a, Purification by gel filtration of the dimeric TIR-SAVED variant in comparison to the wild-type apo protein. The R388E variant (R) was incubated with K199E/W394A (KW) in presence of cA3. b, Analysis of the eluted fraction from (a) by native PAGE. As controls, separated TIR-SAVED were loaded in absence or presence of cA3. c, The combination of R+KW is catalytically dead. The R variant (0.25 µM) was incubated with increased KW concentration (from 0.125 to 2 µM) in presence of 4.5 µM cA3 and 500 µM ɛ-NAD+. d, Kinetic analysis of subunit mixing experiments over 60 min. The WT+E and R+E combinations yielded similar activation kinetics. At later time points, weak activation was observed for the KW+E combination, whilst the E and KW controls showed no activity. In each mixing experiment, the indicated variant (0.25 µM) was incubated with 8 µM E variant in presence of 16.5 µM cA3. Created with BioRender.com.
This file contains Supplementary Figs. 1–6, legends for Supplementary Videos 1–7, and Supplementary Tables 1 and 2.
Dynamic of the two tiers TIR–SAVED/cA3 filament, eigenvector 1. Video associated with the Supplementary Fig. 5. A prominent bending of the long spring-shaped assembly was observed along the long axis.
Dynamic of the two tiers TIR–SAVED/cA3 filament, eigenvector 2. Video associated with the Supplementary Fig. 5. A prominent bending of the long spring-shaped assembly was observed along the long axis.
Dynamic of the two tiers TIR–SAVED/cA3 filament, eigenvector 3. Video associated with the Supplementary Fig. 5. A prominent bending of the long spring-shaped assembly was observed along the long axis.
Dynamic of the one tier TIR-SAVED/cA3 filament, eigenvector 1. Video associated with the Supplementary Fig. 6. A slight change in the pitch of one round of the spring-shaped assembly was observed along the long axis.
Dynamic of the one tier TIR–SAVED/cA3 filament, eigenvector 2. Video associated with the Supplementary Fig. 6. A slight change in the pitch of one round of the spring-shaped assembly was observed along the long axis.
Dynamic of the one tier TIR–SAVED/cA3 filament, eigenvector 3. Videos associated with the Supplementary Fig. 6. A slight change in the pitch of one round of the spring-shaped assembly was observed along the long axis.
cA3 binding by two TIR–SAVED subunits. The cA3 is located into the binding pocket of TIR–SAVED 1 (pink) closed by the tail side of TIR–SAVED 2 (green). Video associated with Fig. 2e.
About this article
Cite this article
Hogrel, G., Guild, A., Graham, S. et al. Cyclic nucleotide-induced helical structure activates a TIR immune effector. Nature 608, 808–812 (2022). https://doi.org/10.1038/s41586-022-05070-9