Abstract
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.
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Data availability
The electron microscopy data deposition D_1292120121 has been assigned the following accession codes: PDB ID 7QQK and EMD-14122. Source data are provided with this paper. All other data are provided in the Supplementary Information.
Change history
11 January 2023
A Correction to this paper has been published: https://doi.org/10.1038/s41586-023-05701-9
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Acknowledgements
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 and acknowledge the European Synchrotron Radiation Facility for provision of synchrotron radiation facilities. 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.
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G.H. planned, carried out and analysed the results of the biochemical and structural analyses and drafted the manuscript; A.G. collected and analysed electron microscopy data; S. Graham cloned, expressed and purified the WT and variant proteins; H.R. carried out the preliminary biochemical analysis of the effector protein; S. Grüschow analysed the cyclic nucleotides; Q.B. carried out the SAXS; L.S. planned and analysed the EM analyses; M.F.W. conceptualized and oversaw the project, obtained funding and analysed data along with the other authors. All authors contributed to the drafting and revision of the manuscript.
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Extended data figures and tables
Extended Data Fig. 1 Cyclase and NADase activity.
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 Data Fig. 2 Plasmid immunity assay with TIR-SAVED activated by Type III CRISPR CSM.
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.
Extended Data Fig. 3 TIR-SAVED is a monomeric protein in diluted solution.
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.
Extended Data Fig. 4 Cryo-EM analysis of the TIR-SAVED filament.
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.
Extended Data Fig. 5 Surface representation of one TIR-SAVED subunit.
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.
Extended Data Fig. 6 Sequence alignment of TIR-SAVED proteins.
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.
Extended Data Fig. 7 Activity and oligomerisation of TIR mutants.
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.
Extended Data Fig. 8 cA3 binding properties of TIR-SAVED mutant R388E and W394A.
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.
Extended Data Fig. 9 Combination of TIR-SAVED variants.
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.
Supplementary information
Supplementary Information
This file contains Supplementary Figs. 1–6, legends for Supplementary Videos 1–7, and Supplementary Tables 1 and 2.
Supplementary Video 1
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.
Supplementary Video 2
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.
Supplementary Video 3
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.
Supplementary Video 4
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.
Supplementary Video 5
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.
Supplementary Video 6
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.
Supplementary Video 7
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.
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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
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DOI: https://doi.org/10.1038/s41586-022-05070-9
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