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Cyclic nucleotide-induced helical structure activates a TIR immune effector - Nature

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 pathway 1 , which originated in bacteria 2 . 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 biomolecules 3 . 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 bacteria 2 , 4 , 5 or during programmed nerve cell death 6 . 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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Main .
Cyclic nucleotide second messengers play a central role in prokaryotic antiviral defence by type III clustered regularly interspaced short palindromic repeats (CRISPR) 7 , 8 , cyclic nucleotide-based antiphage signalling systems (CBASS) 3 and the pyrimidine cyclase system for antiphage resistance (PYCSAR) 9 . These systems activate potent effector proteins that destroy key cellular components such as nucleic acids, cofactors or membranes to disrupt viral replication 3 , 10 . One example is the TIR domain, which functions as an enzyme that degrades NAD + to cause cell death in plants infected with pathogens 5 , 11 , antiphage immunity in the bacterial Thoeris 12 , 13 and TIR–STING (ref.? 2 ) systems, and programmed nerve cell death in metazoa 6 . TIR domain activation requires effector subunit assembly, but the molecular mechanisms are still not fully understood.
cA 3 activates TIR–SAVED to degrade NAD + .
Type III CRISPR and CBASS systems both use the SMODS-associated and fused to various effector domains (SAVED) cyclic nucleotide-binding sensor domain 14 , 15 . Here we focused on a type II-C CBASS from the Gram-positive bacterium Microbacterium ketosireducens 16 that has a nucleotide cyclase (CD-NTase), and TIR–SAVED and NucC effectors, along with a ubiquitin-like modification system of unknown function (Fig. 1a ). We designed synthetic genes for the expression of the CD-NTase and TIR–SAVED proteins in Escherichia coli and purified the recombinant proteins using cleavable amino-terminal His-tags and gel filtration (Supplementary Fig. 2 ). We investigated the activity of the cyclase by incubating the protein with a range of nucleotides, including [α- 32 P]ATP for visualization, and analysis by thin-layer chromatography. A radioactive product running at the position of a cyclic tri-adenylate (cA 3 ) standard was observed when ATP was present in the reaction (Fig. 1b ), and the addition of other nucleotides did not result in any observable change (Extended Data Fig. 1a ). The analysis of the reaction products by liquid chromatography confirmed that the cyclase uses ATP to produce cA 3 that co-elutes with a synthetic 3′,3′,3′-cA 3 standard (Fig. 1c ).
Fig. 1: M. ketosireducens CBASS generates a cA 3 second messenger to activate the TIR–SAVED NADase effector. a , The M. ketosireducens CBASS operon (RS81_contig000028). b , The cyclase (CD-NTase) uses ATP to produce cyclic oligoadenylate (cOA) products (lane 3), visualized after thin-layer chromatography. Lane 2 (C ? ), negative control without protein; lane 1 (C + ), positive control, cA 3 produced using the type III CRISPR complex of Vibrio metoecus 32 . Gel source data are available in Supplementary Fig. 1a . c , Analysis by high-performance liquid chromatography of the reaction products confirmed the cA 3 product. The original unprocessed figure is available in Supplementary Fig. 1b . d , The reaction used to analyse the NADase activity of TIR–SAVED. e , Cyclase products activate the NADase activity of TIR–SAVED. The cyclase was incubated with ATP for 2?h before adding εNAD + and TIR–SAVED?(blue). Controls were performed in the absence of?TIR–SAVED (green), cyclase (pink) or ATP (orange). a.u., arbitrary units. The data are means plotted with the standard deviation for triplicate experiments. f , The plasmid challenge assay. The type III CRISPR complex produces cA 3 on binding the target transcript, activating the NADase activity of TIR–SAVED. g , E. coli transformants after the plasmid immunity assay of M. tuberculosis Csm (type III CRISPR system) combined with TIR–SAVED. WT TIR–SAVED prevents the growth of transformants whereas the inactive E84Q variant does not. Other conditions, replicates and details of the constructs are shown in Extended Data Fig. 2 .
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To investigate the activity of the TIR–SAVED effector, we used etheno-NAD + (?NAD + ), an NAD analogue that emits a fluorescent signal on cleavage by TIR proteins 2 , 11 (Fig. 1d ). We screened a range of commercially available cyclic nucleotide molecules for the ability to activate the TIR–SAVED effector, observing that only cA 3 resulted in generation of a fluorescent signal (Extended Data Fig. 1b ). We proceeded to couple the cyclic nucleotide production by the cyclase with the NADase assay to follow the activation of TIR–SAVED. In the presence of ATP, the cyclase activated the TIR–SAVED effector to degrade NAD + (Fig. 1e ). Together these data demonstrate that the CD-NTase synthesizes a 3′,3′,3′-cA 3 product that can activate the NADase activity of TIR–SAVED.
The initial rate of NAD + cleavage increased linearly with cA 3 concentration up to a value of 0.5??M cA 3 , which corresponded with the concentration of TIR–SAVED in the assay, consistent with a 1:1 molar ratio of cA 3 to TIR–SAVED in the activated form of the effector (Extended Data Fig. 1c ). We next examined the Michaelis–Menten parameters of the NADase activity of TIR–SAVED, determining a Michaelis constant, K m , of 470??M and a catalytic efficiency, k cat / K m , in which k cat is the catalytic rate constant, of 2.08?×?10 3 ?M ?1 ?s ?1 (Extended Data Fig. 1d ). This K m value falls within the concentration range of NAD + found in mammalian cells and E. coli (200–640??M) 17 , 18 . Various bacterial TIR proteins have estimated K m values between 196 and 488??M (ref.? 4 ). In the Thoeris system of Bacillus cereus , the NADase enzyme ThsA activated by cyclic ADP ribose has a K m of 270??M for NAD and a k cat / K m of 2.1?×?10 3 ?M ?1 ?s ?1 (ref.? 13 ), in good agreement with our observations.
Activation of TIR–SAVED kills cells .
CBASS defence systems tend to be phage and host species specific, and the mechanism of activation in response to phage infection remains largely unknown 19 . As we could not analyse CBASS activity in the cognate host, we took advantage of the observation that TIR–SAVED is activated by cA 3 by coupling the effector with a type III CRISPR system from Mycobacterium tuberculosis , which generates a range of cyclic oligoadenylate species including cA 3 on detection of target RNA 20 . Here we replaced the cognate Csm6 effector with M. ketosireducens TIR–SAVED and programmed the CRISPR system with a guide RNA complementary to the tetracycline resistance gene tetR (Fig. 1f and Extended Data Fig. 2 ). When the active CRISPR system was present along with wild-type (WT) TIR–SAVED, transformation of a target plasmid containing the tetR gene resulted in no cell growth on plates containing tetracycline (Fig. 1g ). This phenotype required the production of cA 3 (Extended Data Fig. 2 ) and the NADase activity of TIR–SAVED, as the variant E84Q, which targets the active site (Fig. 1g ), relieved the effect 2 , 11 , 12 . This suggests that TIR–SAVED, activated by cA 3 , is responsible for cell death by NAD + hydrolysis.
Structure of the TIR–SAVED complex .
The structure of the TIR–SAVED monomer was predicted using Alphafold2 (ref.? 21 ) as implemented by the Colabfold server 22 (Supplementary Fig. 3 ). Size-exclusion chromatography combined with small-angle X-ray scattering indicated that TIR–SAVED tends to be monomeric in solution (Extended Data Fig. 3a,b; Supplementary Fig. 4 ). However, addition of cA 3 to the protein resulted in a?marked increase in global particle size as shown by dynamic light scattering (Fig. 2a ) and a marked shift in the elution volume on size-exclusion chromatography (Extended Data Fig. 3c ), suggesting the presence of high molecular weight complexes. These data strongly suggest that cA 3 binding induces multimerization of the TIR–SAVED protein, reminiscent of that observed for the SAVED-domain-containing Cap4 protein, in which head-to-tail multimers of two or three subunits were observed by electron microscopy 23 .
Fig. 2: Structure of the activated TIR–SAVED/cA 3 assembly. a , Dynamic light scattering shows that?wild-type TIR–SAVED increases in size on cA 3 binding.?The arrow highlights this change of average size. b , Left, a cryo-EM micrograph of TIR–SAVED in the presence of cA 3 . Scale bar,?50?nm. Right, TIR–SAVED/cA 3 filament density map. c , Cryo-EM density of the final processed tetramer in two orientations. d , The final atomic model of TIR–SAVED with cA 3 (space-fill) located between each of the four subunits (Protein Data Bank (PDB): 7QQK ). e , Analysis of TIR–SAVED/cA 3 /TIR–SAVED interaction interfaces based on the cryo-EM model. Key features and residues are highlighted. f , The interface of a TIR-domain dimer with NADP + modelled inside on the basis of structural alignment with Vitis rotundifolia RUN1 TIR domain (PDB: 6o0W ; ref.? 11 ). g , The NADase activity of D45A/L46A and Y115A variants compared to the WT (1.5??M TIR–SAVED incubated with 500??M ?NAD + in the presence of 27??M cA 3 ). The mean of triplicate experiments is shown with the standard deviation for the WT and the Y115A variant; the inactive D45A/L46A was assayed in duplicate.?Protein representations in panels c, d and f were created using BioRender.com.
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Analysis by cryogenic electron microscopy (cryo-EM) demonstrated that cA 3 drove the formation of ordered TIR–SAVED filaments (Fig. 2b ). The assembly is characterized by a right-handed superhelical solenoid with 22?nm diameter and a 14?nm pitch. Seventeen TIR–SAVED monomers are present in each turn of the filament (Fig. 2b ). Local resolution analysis performed with the CryoSPARC package highlighted a slightly anisotropic resolution, going from 2.5 to 5?? (Extended Data Fig. 4 ). Three-dimensional (3D) variability analysis of a structure including two turns of the solenoid highlighted a degree of flexibility over the filament (Supplementary Figs. 5 and 6 and Supplementary Videos? 1 – 6 ). The variability analysis shows a slight variation in the radius of the filaments, as well as the?pitch?of the structure; therefore, we chose to proceed with the cryo-EM reconstruction of one turn of the complex in isolation, and with the fitting of the four higher-resolution protomers, which provides information on protein/protein and protein/ligand interface.
To build a model of TIR–SAVED, we used four copies of the Alphafold2 output as a starting model for rigid body fitting using Chimera and the rigid-body-fitted tetramer was then refined as described in the Methods (Fig. 2c and Extended Data Figs. 4f and 5 ). Four densities that correspond to the characteristic shape and size of the cA 3 ligand 23 ??were fitted and refined (Fig. 2d ). The final atomic model confirmed the head-to-tail assembly in which the cA 3 was located in the binding pocket of the SAVED domain of subunit 1, interacting with conserved residues including W394 and K199. The binding site is completed by interactions with subunit 2, including?with the conserved residue R388 (Fig. 2e ), resulting in ‘sandwiching’ of cA 3 between the two SAVED domains (Supplementary Video? 7 ). The SAVED domain is a distant cousin of the cyclic oligoadenylate-sensing CARF domain associated with type III CRISPR systems 15 , and is found in 30% of CBASS operons 23 . SAVED domains fused to nucleases bind cyclic dinucleotides and trinucleotides, activating the associated nuclease domain for DNA degradation 23 , 24 . Our data suggest that head-to-tail stacking of SAVED domains, potentiated by cyclic nucleotide binding, is a defining feature of effector activation.
Generation of a composite active site .
TIR domains are ubiquitous, performing both protein scaffolding and enzymatic roles in different contexts across all three domains of life (reviewed in ref.? 4 ). As is the case for TIR–SAVED, the catalytically active TIR domains, which degrade NAD + to cause cell death, typically rely on multimerization linked to activation 2 , 4 , 5 , 11 , 25 , 26 , but the molecular mechanism for this activation is not well understood. In the filament assembly, TIR domains exhibit a conserved interaction interface involving the BB loop (Extended Data Fig. 6 ) of subunit 1, which is held in a configuration that exposes the active site, due to interaction with the DE loop from the adjacent subunit (Fig. 2f ). This BB-loop interface is also observed in TIR protofilament formation such as for the catalytically inactive TIR domain of the human MAL Toll-like receptor protein 27 and the active form of human SARM1 (ref.? 28 ). The BB loop is suspected, in other NAD + -consuming TIR proteins, to regulate the access to the active site 11 , 29 , 25 , 26 , and for the RUN1 TIR domain, it has recently been proposed that the DE loop of the adjacent subunit contributes to NAD binding 29 .
We therefore tested the hypothesis that a composite NADase active site is formed at the interface between two adjacent TIR domains in the TIR–SAVED filament (Fig. 2f ). We first investigated the BB loop, showing that a variant protein with the double alteration D45A/L46A completely lacked NADase activity (Fig. 2g ) without affecting cA 3 -dependent multimerization (Extended Data Fig. 7a ). We proceeded to explore the role of the putative NAD + -binding site by replacing the highly conserved residue Y115 in the DE loop, which is suitably positioned to interact with NAD + in the TIR–SAVED filament (Fig. 2f ). The Y115A variant had only 10% of the NADase activity of the WT enzyme, together with a threefold increase in K m for εNAD + , suggesting a direct role in the catalytic cycle rather than just substrate binding (Fig. 2g and Extended Data Fig. 7b,c ). This supports the model of a composite NADase active site that has also been proposed for the TIR-NLR RPP1 immune receptor 26 and SARM1 (ref.? 28 ), and is likely to be broadly relevant for catalytic TIR proteins.
Filamentation is essential for activation .
The composite active site of TIR–SAVED requires adjacent TIR domains to be brought together during the activation process. Nevertheless, it does not necessarily follow that the extended helical conformation of the effector observed here is relevant for function, as it could be an in vitro artefact of the assembly. To investigate this, three different site-directed variants were generated and assayed (Fig. 3a ). TIR–SAVED variant E (E84Q), which includes a substitution in the TIR active site, can multimerize but lacks NADase activity (Extended Data Fig. 8 ). Variant R (R388E), in which the charge of a conserved residue contributed by subunit 2 in the cA 3 interface is reversed (Fig. 2e ), retained the ability to bind cA 3 but was unable to multimerize and was catalytically inactive (Fig. 3b and Extended Data Fig. 8 ). Finally, we generated variant KW (K199E/W394A), targeting two conserved residues in the cA 3 -binding site of subunit 1 (Fig. 2e ). This variant no longer bound cA 3 and remained monomeric (Fig. 3b and Extended Data Fig. 8b ).
Fig. 3: TIR–SAVED oligomerization is required for NADase activity. a , Schematic diagram of the assembly of TIR–SAVED variants based on in vitro characterization. b , Electrophoretic mobility shift assay with radiolabelled cA 3 (alone, lane 1) incubated with TIR–SAVED WT and variants (at 0.22, 0.67 and 2.0??M). Protein/cA 3 complexes (monomers, dimers and filaments) were separated by native acrylamide gel and visualized by phosphor imaging. For gel source data, see Supplementary Fig. 1 . c , The?NADase activity of?a 0.25??M?concentration of the R388E variant?in the presence of increased variant?E84Q concentration (0, 0.125, 0.25, 0.5, 1.0, 2.0, 4.0 and 8.0??M), incubated with 500??M ?NAD + and 16.5??M cA 3 . As a positive control, 0.25??M WT TIR-SAVED?was incubated with 8.0??M E84Q (green bar, lane WT?+?E) in the same conditions. The lane R?+?KW corresponds to 0.25??M R388E incubated with 2.0??M K199E/W394A. The results of four experiments with the mean and standard deviation are shown. d , Colony-forming units (cfu) per millilitre of culture after the plasmid immunity assay with different combinations of TIR–SAVED variants. Consistent with the in vitro activities, the R?+?E filament was functionally active whereas the R?+?KW dimer provided no immunity. The data are the means for four biological replicates. NS, P ?>?0.05; P ? Source data
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Although each of these variants was inactive in isolation, they could be combined to generate further variations of the TIR–SAVED complex in a defined manner. First, we investigated the combination of the R and KW variants. Each has one WT and one compromised cA 3 -binding surface, and they can be combined along with cA 3 to generate a TIR–SAVED dimer with one composite active site (where the R and KW variants function as subunit 1 and 2, respectively, in Fig. 2e ). The dimeric composition was confirmed by analytical gel filtration and native polyacrylamide gel electrophoresis (Extended Data Fig. 9a,b ). Unexpectedly however, the dimer of TIR–SAVED showed no NADase activity on addition of cA 3 (Extended Data Fig. 9c ), despite the provision of both halves of the shared active site. We therefore combined the R variant with the E variant, a combination that can result in the generation of filaments of varied size, but with only one active site as the R variant can be extended only by successive addition of inactive E variant subunits (Fig. 3a ). By assaying this combination, we observed a recovery of NADase activity that increased progressively as the E variant was added. The initial activity continued to increase up to the highest ratio (1:32) of R/E studied, representing two turns of the helix on average—indicating that longer filaments of TIR–SAVED have higher levels of NADase activity even if there is only one active site in each filament (Fig. 3c and Extended Data Fig. 9d ). These observations highlight the requirement that catalytic TIR domains assemble into multimers, not just dimers, for activation.
Although these observations confirmed the importance of filament assembly for TIR–SAVED activation in vitro, we wished to explore whether the same held true in vivo. To explore this, we created a second copy of the tir–saved gene to allow expression of combinations of variant proteins in our plasmid challenge assay. For the second copy of the gene, we used alternative codons to avoid any problems caused by recombination between two highly similar sequences in a plasmid. Using this experimental design, we tested whether the results obtained in vitro could be recapitulated in vivo (Fig. 3d ). The R variant alone afforded a modest degree (1 log) of protection in vivo, far lower than observed for the WT protein. A combination of the R and KW variant genes produced a null phenotype, confirming the lack of activity observed in vitro. However, when the R variant was combined with the inactive E variant, a?substantial level of effector activity was observed in the plasmid challenge assay, confirming the requirement for filament formation observed in vitro (Fig. 3c,d ). Thus, we conclude that extended filament formation is relevant and essential for the function of the TIR–SAVED effector.
Wider aspects of TIR domain activation .
Here we have explored a CBASS system with a TIR–SAVED effector that oligomerizes on activation by cA 3 . Oligomerization with ‘open symmetry’ (the ability to polymerize to infinity) seems to be a widespread property of prokaryotic innate immune effectors, encompassing the bacterial STING, SAVED and PYCSAR proteins 2 , 9 , 23 . We have demonstrated that this oligomerization is essential for TIR–SAVED function: first, as it probably allows access to the active site by formation of the BE interface, prising open the BB loop; second, as residues from the adjacent subunit participate in substrate binding and the catalytic cycle. Thus, the bacterial TIR-domain effectors seem to conform to the emerging paradigm of signalling by cooperative assembly formation proposed for the eukaryotic signalling complexes 30 . Notably, the bacterial TIR–SAVED filament exhibits the evolutionarily conserved head-to-tail arrangement in the TIR domain at the BE interface also found in eukaryotic TIR enzymes such as SARM1 (ref.? 28 ) and RPP1 (ref.? 29 ; Fig. 4 ), which is likely to be a prerequisite for TIR activation in all systems. However, our data demonstrate that assembly of this interface alone is not sufficient to result in an active state, as dimeric TIR–SAVED is inactive both in vitro and in vivo. Activation requires assembly of larger complexes, which may be essential to stabilize the TIR domain in an active conformation. In RPP1 and SARM1, TIR domains form tetrameric and octameric complexes with the AE interface formed by interactions between the αA and αE helices of TIR domains, perpendicular to the BE interface (Fig. 4 ). Here the TIR–SAVED assembly is unique as there is only a single filament of TIR domains, and it is notable that the SAVED domain occupies the space where the AE interface forms in the eukaryotic proteins (Fig. 4 ). Oligomerization-dependent activation of bacterial TIR effectors is likely to be the ancestral mechanism underlying the whole family of catalytic and non-catalytic TIR signalling complexes, exemplified by the accompanying study of the bacterial TIR–STING effector 31 . Our study reveals that activation is tightly controlled, requiring more than two TIR domains to be brought together. This suggests that the stabilization of the active form of TIR proteins requires extensive protein interactions, and possibly an alteration in dynamics, beyond the formation of the crucial BE interface—a property that may have evolved to avoid ‘accidental’ activation of TIR domains and autotoxicity.
Fig. 4: Multimerization of TIR–SAVED generates a conserved composite TIR active site. a , The TIR–SAVED assembly generated by cA 3 (PDB 7QQK)?promotes TIR self-association at the BE interface, composed of the BB loop and the DE loop. b , c , This asymmetric arrangement mediated by the BE interface is conserved in other eukaryotic TIR?enzyme assemblies such as the human SARM1 complex?(PDB 7NAK) 28 ( b ) and the plant NLR RPP1 receptor?(PDB 7DFV)? 29 ( c ), which are shown in the same orientation. The two-stranded TIR structure formed in the eukaryotic complexes involves the AE interface mediated by the αA and αE helices. In the TIR–SAVED filament, these two helices are oriented towards the SAVED domain.
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Methods .
Cloning and mutagenesis .
The synthetic genes encoding M. ketosireducens CD-NTase?and TIR–SAVED were codon-optimized for expression in E. coli and purchased from Integrated DNA Technologies. Genes were cloned into the pEhisV5Tev vector 33 between the NcoI and BamHI restriction sites. The constructs were transformed into competent DH5α ( E. coli ) cells, and plasmids were extracted using GeneJET plasmid miniprep kit (Thermo Scientific) to verify sequence integrity by sequencing (Eurofins Genomics). Then plasmids were transformed into E. coli C43 (DE3) cells for protein expression. The TIR–SAVED variants E84Q, R388E, K199E/W394A, D45A/L46A and Y115A were generated on both constructs containing the tir–saved gene: the expression vector (pEhisV5spacerTev); and the vector used for the plasmid immunity assay (pRAT-Duet). Synthetic gene sequences and primers used for mutagenesis are listed in Supplementary Table 1 .
Protein expression and purification .
Recombinant CD-NTase and TIR–SAVED WT and variants were overexpressed in E. coli C43 (DE3) cells. After growing at 37?°C in lysogeny broth (LB) until the optical density at 600?nm reached 0.6–0.8, cultures were induced with 0.4??M final isopropyl-β- d -thiogalactoside concentration and incubated at 16?°C overnight. Cells were collected by centrifugation, and pellets were stored at ?80?°C. Cell pellets were lysed by sonication in buffer A (50?mM Tris-HCl, pH?7.5, 0.5?M NaCl, 10?mM imidazole, 10% glycerol) supplemented with 1?mg?ml ?1 final lysozyme concentration and protease inhibitors (cOmplete, EDTA-free protease inhibitor cocktail, Roche). Clarified lysates were loaded onto a 5-ml HiTrap FF column (GE Healthcare), pre-loaded with Ni 2+ and equilibrated with buffer B (50?mM Tris-HCl, pH?7.5, 0.5?M NaCl, 30?mM imidazole, 10% glycerol). Then His-tagged proteins were eluted along a linear gradient (50?mM to 500?mM imidazole). Recombinant His-tagged TEV protease was incubated with the eluted proteins to cleave the His-tag and the reaction was dialysed overnight at room temperature against buffer B. Dialysed samples were loaded again onto the Ni 2+ -loaded 5-ml HiTrap FF column (GE Healthcare) equilibrated with buffer B. The His-tagged cleaved proteins passed straight through the column and were then loaded onto a 26/60 Superdex 200 size-exclusion column (GE Healthcare) equilibrated with buffer C: 20?mM Tris-HCl, pH?7.5, 0.25?M NaCl, 10% glycerol. Proteins were concentrated with 30-kDa-cutoff Amicon centrifuge filters (Millipore), flash-frozen in liquid nitrogen and stored at ?80?°C. Purified protein was analysed by SDS–polyacrylamide gel electrophoresis (NuPage Bis-Tris 4–12%, Invitrogen) with Instant Blue staining (Expedeon). The final protein concentrations were determined by measuring absorbance at 280?nm using sequence-predicted extinction coefficients.
Cyclic nucleotide analysis by thin-layer chromatography .
Synthesis of cyclic nucleotides by the CD-NTase was analysed by using α- 32 P-labelled ATP mixed with ‘cold’ NTPs. The reactions were carried out at 37?°C for 2?h in cyclase buffer (50?mM CAPS, pH?9.4, 50?mM KCl, 10?mM MgCl 2 , 1?mM MnCl 2 , 1?mM dithiothreitol), as a high pH activates CD-NTases in vitro 34 . A 20??M concentration of CD-NTase was incubated with 50??M ATP and 30?nM [α- 32 P]ATP in a final volume of 20??l. Reactions were stopped by addition of phenol/chloroform and products were isolated by chloroform extraction. Then 1??l of the final volume was spotted on a silica gel thin-layer chromatography plate (Supelco Sigma-Aldrich). The plate was placed into a pre-warmed humidified chamber with running buffer composed of 30% H 2 O, 70% ethanol and 0.2?M ammonium bicarbonate, pH?9.2. Separated products were visualized by phosphor imaging. A control reaction run?with a characterized type III CRISPR system, VmeCMR 32 was performed with 1??M purified VmeCMR, 2??M target RNA which had been?incubated for 2?h at 37?°C in the reaction buffer (10?mM MgCl 2 , 10?mM Tris-HCl, pH?8, 50?mM NaCl).
Cyclic nucleotide analysis by liquid chromatography .
To analyse the nature of the cyclic nucleotide produced by the M. ketosireducens CD-NTase, the reaction was carried out with 50??M protein in cyclase buffer with 250??M ATP for 2?h at 37?°C. The reaction was diluted twofold with water and ultracentrifuged using spin filters with a molecular weight cutoff of 3?kDa (Pall). Liquid chromatography analysis was performed on the Dionex UltiMate 3000 system. Sample separation was carried out on a Kinetiex EVO C18 2.6??M (2.1?×?50?mm column, Phenomenex) with a 0–8% gradient of acetronitrile with 100?mM ammonium bicarbonate as the solvent. The flow rate was set at 300??l?min ?1 and the column compartment temperature was set at 40?°C. Data were collected at a wavelength of 250?nm, and a 20??M cA 3 commercial standard was used for comparison (Biolog).
NADase assay .
The NADase activity of M. ketosireducens TIR–SAVED was analysed by using ?NAD + as the substrate—when cleaved the ?ADP?ribose product could be detected by fluorescence. Reactions were prepared in a 30??l final volume with reaction buffer (50?mM Tris-HCl, pH?7.5, 50?mM KCl, 2.5?mM MgCl 2 ), 1??M cyclic trinucleotide (cA 3 ; Biolog), 0.5??M TIR–SAVEDand 0.5?mM ?NAD + (Sigma), or as stated in the figure. A master mix containing protein and activator was prepared on ice, and the substrate was added immediately before beginning analysis. Reaction samples were loaded into 96-well plates (Greiner 96 half-area) and fluorescence was measured continuously (cycle of 20?s) over 90?min using the FluoStar Omega (BMG Labtech) with an excitation filter at 300?nm and an emission filter at 410?nm. Reactions were carried out at 28?°C. A calibration curve was evaluated with the value obtained after 90?min of reaction with 10, 25, 75, 225, 500 and 675??M ?NAD + as the initial concentration.
Cyclase–NADase combined assays .
For the cyclase–NADase combined reaction, 5??M CD-NTase was incubated at 37?°C with 250??M ATP in a 25??l final volume containing 50?mM CAPS, pH?9.4, 50?mM KCl, 10?mM MgCl 2 , 1?mM MnCl 2 , 1?mM dithiothreitol. After 2?h, reactions were transferred into 96-well plates (Greiner 96 half-area) and supplemented with 0.5??M ?NAD + . Fluorescence measurements were performed as mentioned above, during 10?min before adding 0.5??M TIR–SAVED. Reactions were carried out at 37?°C for a further hour. Fluorescence data were plotted over time and analysed with GraphPad Prism.
Plasmid immunity assays .
To analyse the effect of TIR–SAVED NADase in vivo, we used the previously characterized 20 M. tuberculosis type III CRISPR system as an inducible producer of cA 3 when activated by the target RNA. Here we used the following plasmids to encode M. tuberculosis Csm1–5, Cas6 and a CRISPR array: pCsm1–5_ΔCsm6 ( M. tuberculosis Csm1–5 under the control of the T7 and lac promoters); and pCRISPR_TetR (CRISPR array with spacers targeting the tetracycline-resistance gene targeting and M. tuberculosis Cas6 under the control of the T7 promoter). Competent E. coli C43 (DE3) cells were co-transformed with two constructs: pCsm1–5_ΔCsm6 and pCRISPR_TetR. Plasmids were maintained by selection with 100??g?m l?1 ampicillin and 50??g?ml ?1 spectinomycin. The tir–saved gene was inserted into pRAT-Duet between NcoI and SalI (multiple cloning site 1) or KpnI and BglII (multiple cloning site 2). This plasmid contains the tetracycline resistant gene targeted by M. tuberculosis Csm. The plasmid immunity assay is based on the transformation of the target plasmid containing the gene encoding TIR–SAVED into recipient cells encoding M. tuberculosis Csm. Recipient cells were prepared and transformed with the target plasmid as described previously 20 . After the growth period in LB, cells were collected and resuspended in a LB volume adjusted to the same optical density at 600?nm (about 0.1). A total of 3??l of a 10-fold serial dilution was applied in duplicate to selective LB agar plates supplemented with 100??g?ml ?1 ampicillin, 50??g?ml ?1 spectinomycin, 25??g?ml ?1 tetracycline, 0.2% (w/v) d -lactose and 0.2% (w/v) l -arabinose. Plates were incubated overnight at 37?°C. This experiment was performed with two independent biological replicates using two technical replicates for each experiment. The variant Cas10(D630A) from M. tuberculosis Csm, which abolishes cyclase activity, was used as a control for no production of cyclic tri-adenylate. For colony counting, the same procedure was followed, except that 100??l of a 300??l growth volume was spread onto the selective LB agar plates. Two dilution factors were assayed for each condition and the experiments were performed in biological triplicates. Following incubation at 37?°C for 17–18?h, the resulting colonies were manually counted and the number of colony-forming units was reported per millilitre of culture volume. Data were statistically analysed by Prism8 (GraphPad) using non-pairing Brown–Forsythe and Welch analysis of variance tests. For multiple comparisons, the Dunnett T3 test was used.
Analytical gel filtration .
To analyse the oligomeric state of TIR–SAVED, 100??l protein (at least 100??M) was injected into a size-exclusion column (Superose 6 Increase 10/300?GL or Superose 12, GE Healthcare) equilibrated in 20?mM Tris-HCl, pH?8.0, 250?mM NaCl and 10% glycerol. In some experiments, 158??M cyclic tri-adenylate was added to the TIR–SAVED sample before centrifugation at 10,000 g for 10?min at 4?°C and loading onto the size-exclusion column. To analyse TIR–SAVED dimerization, 83??M R388E variant was first incubated with 500??M cA 3 and 127??M K199E/W394A variant and loaded into the Superose 12 size-exclusion column in similar conditions. The eluted fractions were then analysed by native polyacrylamide gel electrophoresis in a 4–16% Bis-Tris gel (Invitrogen).
Using similar gel filtration running conditions, standard proteins (number 1511901, BioRad) were eluted to calculate a calibration curve of the column. A 100??l volume of BSA (7.3?mg?ml ?1 ) was injected as an extra standard. The elution volume ( V e ) for each protein was determined on the basis of the elution profile. Then the K average was calculated as the ratio K average ?=?( V e ??? V 0 )/( V t ??? V 0 ), in which V 0 is the void volume (7.77?ml) and V t is the total volume (24?ml) of the column. The calibration curve corresponds to the plot of K average versus the molecular weight of each protein in log 10 . The trend line (logarithmic) was plotted with the following standards: y-globulin (158?kDa), BSA (66?kDa), ovalbumin (44?kDa) and myoglobin (17?kDa) to obtain the best R 2 value (0.997) corresponding to the range of the target protein.
Dynamic light scattering .
Dynamic light scattering measurements were performed with the Zetasizer Nano S90 (Malvern) instrument. In the protein dilution buffer (20?mM Tris-HCl, pH?7.5, 250?mM NaCl, 10% glycerol), 21??M TIR–SAVED was prepared with 32??M cyclic nucleotide (cyclic tri-adenylate or others) when stated in the figure. After centrifugation at 12,000 g for 10?min at 4?°C and filtration with 0.22??m filters, 12??l of sample was loaded into the quartz cuvette (ZMV1012). The measurements were carried out at 25?°C with 3 measures of 13 runs. The curves of WT TIR–SAVED are the mean of three technical replicates and two independent experiments.
Small-angle X-ray scattering .
Small-angle X-ray scattering (SAXS) datasets were recorded at the European Synchrotron Radiation Facility (Grenoble, France) on the BioSAXS beamline BM29 (ref.? 35 ) using a 2D Pilatus detector. Data were collected at room temperature (20?°C) using a standard set up (automated sample mounting to a capillary by a robot) 36 . A 100??l volume of TIR–SAVED (10.9?mg?ml ?1 ) was injected into the Superose 12 column 10/300?GL (GE Healthcare) in 20?mM Tris-HCl, pH?8.0, 250?mM NaCl and 10% glycerol with a flow rate of 0.4?ml?min ?1 .
Sample scattering curves were obtained after subtraction of the averaged buffer signals using standard protocols with PRIMUS 37 . The values for the radius of gyration, R g , and the forward scattering intensity, I (0), were extracted using the Guinier approximation. The molecular weight range was estimated by Bayesian inference 38 implemented in the ATSAS suite 3.0.5 (ref.? 39 ). The theoretical SAXS curve of the TIR–SAVED structure predicted by Alphafold2 was back-calculated and fitted with the experimental SAXS datasets with the program CRYSOL 40 . SAXS parameters for data collection and analysis are reported in Supplementary Table 2 .
Electrophoretic mobility shift assays .
Radiolabelled cA3, [α- 32 P]cA 3 , was prepared with the type III CRISPR complex, VmeCMR, as described in the section entitled Cyclic nucleotide analysis by thin-layer chromatography. The reaction product was incubated with a threefold dilution range of TIR–SAVED (0.22, 0.67 and 2.0??M) for 15?min at 25?°C in a final 15??l volume. The reaction buffer was the same as that for NADase activity: 50?mM Tris-HCl, pH?7.5, 50?mM KCl, 2.5?mM MgCl 2 and 25?mM NaCl from the protein dilution buffer. Ficoll was added to the samples to give a final concentration of 4%, and samples were then loaded into a native 6% acrylamide gel (acrylamide/bis-acrylamide 29:1). TIR–SAVED/cA 3 complexes were separated by electrophoresis into 1× TBE buffer for 1?h at 200?V and visualized by phosphor imaging.
Thermal shift assays .
A 2??M concentration of TIR–SAVED WT or variants were incubated with a range of cA 3 concentrations (0, 0.4, 2 and 10??M) in the following buffer: 20?mM Tris-HCl, pH?7.5, 250?mM NaCl, 10% glycerol supplemented with 5× SYPRO Orange Fluorescent Dye (BioRad). A temperature gradient was applied from 25 to 95?°C with 1?°C increments, and fluorescence was measured in a Stratagene MX3005. The curves are the mean of two independent experiments with technical triplicates.
Transmission electron microscopy .
Samples for negative-stain electron microscopy analysis were prepared by diluting purified TIR–SAVED protein alone, or with an equimolar ratio of cyclic trinucleotide as indicated, to a concentration of 1?mg?ml ?1 in buffer (20?mM Tris-HCl pH?8.0, 250?mM NaCl, 10% glycerol).
Electron microscopy images of negatively stained TIR–SAVED were collected using a JEOL 1200?transmission electron microscope operating at 120?keV and equipped with a Gatan Orius CCD (charge-coupled device) camera at a nominal magnification of ×100,000, and a pixel size of 9.6??. A 4?μl volume of the diluted sample was applied onto a glow-discharged 400-mesh copper grid (Agar Scientific) coated with a layer of continuous carbon, followed by a 1-min absorption step and side blotting to remove bulk solution. The grid was immediately stained with 2% uranyl acetate at pH?7 and then blotted from the side and air-dried before imaging.
Cryo-EM grids were prepared using an FEI Vitrobot Mark IV (Thermo Fisher) at 4?°C and 95% humidity. A 3??l volume of TIR–SAVED complex was applied to holey carbon grids (Quantifoil Cu R1.2/1.3, 300 mesh), glow-discharged for 45?s at a current of 45?mA in an EMITECH K100X glow discharger. The grids were then blotted with filter paper once to remove any excess sample, and plunge-frozen in liquid ethane. All cryo-EM data presented here were collected on a JEOL CRYO ARM 300 microscope, equipped with a DE-64 direct detector at the Scottish Centre for Macromolecular Imaging, Glasgow, UK. A total of 3,907 videos were collected in accurate hole centring mode using SerialEM 3.8 (ref.? 41 ). The CryoSPARC 3.3.1 software 42 was used for motion correction, CTF estimation and manual exposure correction, as well as for the selection of the 2,319 videos used in the analysis. CryoSPARC 3.3.1 was also used for the whole single-particle reconstruction workflow, from manual particle picking to classification to generate templates for autopicking and subsequent 2D classification and 3D processing, including per-particle motion correction 43 , sharpening and 3D variability analysis 44 , obtaining a structure with an overall resolution of 3.8??. The final reconstruction was obtained from 596,378 particles selected from classes representing both circular and elongated particles at a sampling rate of 0.997?? per pixel and had an overall resolution of 3.8??, as calculated by Fourier shell correlation at 0.143 cutoff during post-processing. The Alphafold2 model was fitted to the map using the Chimera software, taking into account?the handedness. The inverted handedness was the only compatible solution and was therefore chosen for further modelling and refinement using the software packages Coot 45 , Refmac-Servalcat as implemented in the CCP-EM suite 46 and PHENIX 47 . The cryo-EM data collection, refinement and validation statistics are summarized in Extended Data Table 1 .
Reporting summary .
Further information on research design is available in the? Nature Research Reporting Summary linked to this article.
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 .
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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. 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 .
Author information .
Author notes Quentin Bertrand
Present address: Laboratory of Biomolecular Research, Biology and Chemistry Division, Paul Scherrer Institute, Villigen, Switzerland
Authors and Affiliations .
School of Biology, University of St Andrews, St Andrews, UK
Ga?lle Hogrel,?Shirley Graham,?Hannah Rickman,?Sabine Grüschow?&?Malcolm F. White
Institute of Molecular, Cell and Systems Biology, University of Glasgow, Glasgow, UK
Abbie Guild?&?Laura Spagnolo
Centre for Regenerative Medicine, Institute for Regeneration and Repair, The University of Edinburgh, Edinburgh, UK
Abbie Guild
Université Grenoble Alpes, CNRS, CEA, IBS, Grenoble, France
Quentin Bertrand
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Contributions .
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.
Corresponding authors .
Correspondence to Laura Spagnolo or Malcolm F. White .
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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??M “cold” NTP (ATP, GTP, CTP, TTP, or UTP) and incubated with 20??M cyclase for 2?h at 37?°C. As controls, cyclic oligoadenylate produced by the Type III CRISPR complex VmeCMR 21 (C + ) and the reaction without protein (C ? ). b , Cyclic nucleotide screening for TIR-SAVED NADase activity. 0.5??M TIR-SAVED was incubated without (apo condition) or with 5??M 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 cA 3 concentration. 0.5??M TIR-SAVED was incubated with 0, 0.1, 0.3, 0.5, 1.0, 2.5??M cA 3 and 500??M ?NAD + . d , Enzymatic characterization of TIR-SAVED NADase activity. Initial rate is plotted against a range of ?NAD + 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??M TIR-SAVED were incubated with 1??M cA 3 and 2?mM ?NAD + . Data are the means of triplicate experiments with standard deviation (b, c, d, e).
Source data
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 cA 3 for a molar ratio of 1:1.5 (protein:cA 3 ). Additional analysis of TIR-SAVED molecular weight by analytical gel filtration are shown in Supplemental Data Fig. 4 .
Source data
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??). f , Atomic model fitted into the density map for four TIR-SAVED/cA 3 subunits.
Extended Data Fig. 5 Surface representation of one TIR-SAVED subunit. .
a , position of the cA 3 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 cA 3 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:cA 3 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??M). 27??M cA 3 was used to activate TIR-SAVED incubated with 500??M ?NAD + 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??M Y115A was mixed in presence of 1??M cA 3 to hydrolyse 25, 75, 225, 500, 1000, 1500, 2000 and 3000??M ?NAD + . 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.
Source data
Extended Data Fig. 8 cA 3 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:cA 3 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??M) was incubated with 27??M cA 3 and 500??M ?-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??M) was incubated with increased cA 3 concentration (from 0.03 to 13.5??M) and 500??M ?-NAD + . Data are means from duplicate experiments. e , Comparison of the thermal denaturation profile of TIR-SAVED in presence of three concentrations of cA 3 indicated as protein:cA 3 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.
Source data
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 cA 3 . b , Analysis of the eluted fraction from (a) by native PAGE. As controls, separated TIR-SAVED were loaded in absence or presence of cA 3 . c , The combination of R+KW is catalytically dead. The R variant (0.25??M) was incubated with increased KW concentration (from 0.125 to 2??M) in presence of 4.5??M cA 3 and 500??M ?-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??M) was incubated with 8??M E variant in presence of 16.5??M cA 3 . Created with BioRender.com.
Source data
Extended Data Table 1 Cryo-EM data collection, refi nement and validation statistics Full size table
Supplementary information .
Supplementary Information .
This file contains Supplementary Figs. 1–6, legends for Supplementary Videos 1–7, and Supplementary Tables 1 and 2.
Reporting Summary .
Peer Review File .
Supplementary Video 1 .
Dynamic of the two tiers TIR–SAVED/cA 3 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/cA 3 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/cA 3 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/cA 3 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/cA 3 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/cA 3 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 .
cA 3 binding by two TIR–SAVED subunits. The cA 3 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.
Source data .
Source Data Fig. 1 .
Source Data Fig. 2 .
Source Data Fig. 3 .
Source Data Extended Data Fig. 1 .
Source Data Extended Data Fig. 3 .
Source Data Extended Data Fig. 7 .
Source Data Extended Data Fig. 8 .
Source Data Extended Data Fig. 9 .
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Hogrel, G., Guild, A., Graham, S. et al. Cyclic nucleotide-induced helical structure activates a TIR immune effector. Nature (2022). https://doi.org/10.1038/s41586-022-05070-9
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Received : 12 January 2022
Accepted : 01 July 2022
Published : 10 August 2022
DOI : https://doi.org/10.1038/s41586-022-05070-9
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