Filamentation activates bacterial NLR-like antiviral protein

Filamentation activates bacterial NLR-like antiviral protein | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Filamentation activates bacterial NLR-like antiviral protein Jianting Zheng, Yiqun Wang, Yuqing Tian, Xu Yang, Feng Yu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5156926/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 11 Mar, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract Bacterial antiviral STANDs (Avs) are evolutionarily related to the nucleotide-binding leucine-rich repeat containing receptors (NLRs) widely distributed in immune systems across animals and plants. Ef Avs5, an Avs type 5 protein from Escherichia fergusonii , contains an N-terminal SIR2 effector domain, a nucleotide-binding oligomerization domain (NOD) and a C-terminal sensor domain, conferring protection against diverse phage invasions. Despite the established roles of SIR2 and STAND in prokaryotic and eukaryotic immunity, the mechanism underlying their collaboration remains unclear. Here we present cryo-EM structures of Ef Avs5 filaments, elucidating the mechanisms of dimerization, filamentation, filament clustering, ATP binding and NAD + hydrolysis, all of which are crucial for anti-phage defense. The SIR2 domains and NODs engage in the intra- and inter-dimer interaction to form an individual filament, while the outward C-terminal domains contribute to bundle formation. Filamentation potentially stabilizes the dimeric SIR2 configuration, thereby activating the NADase activity of Ef Avs5. Ef Avs5 is deficient in the ATPase activity, but elevated ATP concentrations can impede its NADase activity. Together, we uncover the filament assembly of Avs5 as a unique mechanism to switch enzyme activities and perform anti-phage defenses, emphasizing the conserved role of filamentation in immune signaling across diverse life forms. Biological sciences/Biochemistry/Structural biology Biological sciences/Microbiology Biological sciences/Biochemistry/Enzyme mechanisms Biological sciences/Immunology Sirtuin signal transduction ATPases with numerous domains bacterial defense system NAD+ hydrolysis filament formation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Key Points Ef Avs5 depletes NAD + for anti-phage defense. Ef Avs5 assembles as bundled filaments that can hydrolyze NAD + . The SIR2 domain and NOD collaborate to form an individual filament. The building block of the filament is Ef Avs5 dimer. The activity of the Ef Avs5 complex is regulated by ATP. Introduction Bacteria are in constant conflict with bacteriophages and develop diverse anti-phage defense systems of varying complexity 1 – 3 . The signal transduction ATPases with numerous domains (STAND) superfamily of P-loop NTPases, particularly the nucleotide-binding leucine-rich repeat containing receptors (NLRs) exemplified by animal inflammasomes and plant resistosomes 4 – 6 , recognizes pathogens and triggers downstream inflammatory responses or apoptotic processes through oligomerization, representing a conserved immune strategy in eukaryotes. Recently, this mechanism has also been discovered in the innate immunity of bacteria, known as antiviral STANDs (Avs) 3 . Avs proteins have a characteristic tripartite domain architecture: a central NTPase (alternatively called nucleotide-binding oligomerization domain, NOD); an extended C-terminal sensor with superstructure-forming repeats; and a variable N-terminal effector typically involved in cell death. Previous studies have revealed that Avs1-3 and Avs4 are activated by the phage terminases and portals, respectively, leading to the formation of tetramers that activate their N-terminal nucleases for antiviral defense 4 . Avs type 5 (Avs5) contains an N-terminal sirtuin (SIR2) effector and an unusual short C-terminal sensor domain, sometimes termed SIR2-STAND 3 . SIR2-mediated nicotinamide adenine dinucleotide hydrolase (NADase) activity represents a critical element in anti-phage immunity, triggering abortive infections via NAD + degradation and halting phage propagation, as exemplified by pAgo, Thoeris, DSR2, and the SIR2-HerA system 7 – 10 . In the Thoeris system, the signaling molecule 1''-3' gcADPR activates ThsA, which subsequently forms filaments and depletes NAD + , ultimately triggering cell death and thereby preventing the spread of phage infection 11 . Another defense system DSR2 tetramerizes to form a supramolecular complex that specifically recognizes phage tail tube proteins, and leads to cellular NAD + depletion 12 . Despite reports on SIR2 and STAND roles in prokaryotic and eukaryotic immunity, the collaborative mechanism between them and the mechanisms underlying Avs5 activation and assembly remain obscure. Here, we present a biochemical and structural analysis of Escherichia fergusonii Avs5 ( Ef Avs5), unveiling a unique mechanism of assembly-mediated activation by Ef Avs5. Ef Avs5 proteins form active filaments via SIR2 and NOD self-association, with sensor-mediated clustering enhancing their organization, allowing the NAD + depletion during phage infection. Moreover, the inactive NOD of Ef Avs5 requires ATP binding for its defensive role, whereas high ATP concentration impedes its NADase activity. Result 1. Ef Avs5 consumes NAD + to confer defense Avs5 system is widely distributed across Gram-negative phyla, especially Pseudomonadota (Fig. S1 ). To define the molecular anti-phage defense mechanism of Avs5, we investigated a single protein Avs5 defense system from E. fergusonii phage-inducible chromosomal islands (PICI), named Ef Avs5 (Fig. 1 A). Ef Avs5 is reported to defense against diverse Escherichia coli , Salmonella and Klebsiella pneumoniae phages 13 . We cultured E. coli cells expressing Ef Avs5 and found protection against T7 phages. The C terminal of Ef Avs5 is supposed to sense the phage invasion, while the SIR2 domain is suggested to be the effector depleting NAD + . Indeed, a single amino acid substitution at NADase active site (N141A) was sufficient to abolish phage defense by the Ef Avs5 system, confirming that the NADase activity is essential for defense. Similarly, a point mutation disrupting the NOD active site, K379A in walker A or D426A in walker B, as well as a deletion of the C-terminal sensor domain, also abolished defense (Fig. 1 A). Growth curves of E. coli cultures in the presence of Ef Avs5 kept growing when subjected to the low (0.2) or medium (2) multiplicity of infection (MOI) in contrast to the cells expressing N141A mutant or lacking Ef Avs5 (Fig. 1 B). We measured the survival rate of cells infected with T7 and found that Ef Avs5 increased cell survival after the first infection cycle (about 20 minutes) (Fig. 1 C). Further analysis showed that the phages amplified rapidly in the infected cells lacking Ef Avs5, but not in the presence of Ef Avs5 (Fig. 1 D). To investigate the effector’s activity during immunity, we detected the cellular NAD + content after infection with T7 phage. When subjected to the T7 phage infection, compared to cells lacking Ef Avs5, the concentration of NAD + in the Ef Avs5-expressing cells was declining rapidly after the first infection cycle, and began to rise in the second infection cycle (Fig. 1 E). These results suggest that Ef Avs5 consumes NAD + to effectively prevent phage amplification and leads to the population-level protection. 2. Architecture of Ef Avs5 filament assembly To biochemically characterize the Avs5 system, we purified Ef Avs5 protein overexpressed in E. coli BL21(DE3). The freshly isolated Ef Avs5 exists as soluble monomer (Fig. S2). Cryo-EM imaging of the monomer Ef Avs5 revealed small particles. We tried to resolve the structure but only obtained two-dimensional (2D) classes in poor resolution, possibly due to the flexibility (Fig. S3). However, the purified Ef Avs5 becomes visibly cloudy after stored at 4°C or -80°C for two weeks. Negative staining analysis of the suspension of Ef Avs5 confirmed that a significant fraction of the particles (> 50%) exhibited bundled 8-nm-wide fibrous assembly, and some of them extending up to ~ 400 nm in length (Fig. 2 A). Bundled fibrils can be connected parallelly or end-to-end and extend up to several micrometers. Since previous studies show that the activated SIR2 or Toll/interleukin-1 receptor (TIR) can assemble into helical filaments in Thoeris defense system, TIR-STING and TIR-SAVED effector, respectively 11 , 14 , 15 , we quantified the NADase activity of Ef Avs5. The freshly purified Ef Avs5 was deficient in the NADase activity, but the filamentous Ef Avs5 displayed robust NADase activity (Fig. 2 B). To gain more insight into the filament formation, we determined the cryo-electron microscopy (cryo-EM) structure of the active Ef Avs5. The final cryo-EM map was reconstructed using a total of 228,053 single particles and refined to a nominal resolution of 3.4 Å, with approximately 2.8 Å at the center of filament and approximately 6.8 Å at the peripheral sensor lobes (Fig. 2 C, Fig. S4 and Table S1 ). The model was built with eight Ef Avs5 molecules assembled into a helical filament, and extra densities suggest unlimited entries of both ends. The repeating building block of the filament is Ef Avs5 dimer with a C 2 symmetry arranged into a regular helical structure with a helical twist of 84.531° and a helical rise of 48.600 Å (Fig. 2 C). The filament is held together by 2 sets of inter-subunit interactions, one between subunit n and n + 3 and the other between subunit n + 1 and n + 2 (Fig. 2 D). The Ef Avs5 proteins fold into five domains: a catalytic SIR2 domain, a core NOD, which comprises the nucleotide-binding domain (NBD), helical domain (HD) and wing-helical domain (WHD) and the C-terminal sensor domain (Fig. 1 A and Fig. 2 E). The core of the helix is composed of the SIR2 and the NOD domain, whereas the C-terminal domain is pointing away and not involved in the helical contacts (Fig. 2 E). 3. The dimerization of Ef Avs5 In the Ef Avs5 filament, the individual dimeric unit adopts a C 2 symmetric conformation with a tight interface (2329.2 Å 2 of buried surface area) (Fig. 3 A). Within each Ef Avs5 dimer unit, abundant contacts are observed between the WHD of one subunit and the NBD of its adjacent subunit, encompassing salt bridges and hydrogen bonds, revealing an oligomerization pattern distinct from other characterized active STAND ATPases, which primarily oligomerize through NBD-NBD and WHD-WHD interactions (Fig. S5). The NBD of the n subunit interacts with the WHD of the n + 1 subunit, comprising the salt bridge of the K461(n)- E589(n + 1), K473(n)- E580(n + 1), and the hydrogen bonds of the T474(n)- Y583/R570(n + 1) (Fig. 3 B). Meanwhile, the WHD of the n subunit interacts with the NBD of the n + 1 subunit using slightly different residues, comprising the specific contacts of the E580(n)- H362(n + 1), and the hydrogen bonds of the Y583(n)- T474(n + 1). The dimeric interface is expanded by additional hydrogen bonds between its N-terminal SIR2 domains (Fig. 3 C). To probe the role of the dimer formation in Ef Avs5 activity, we generated mutants of the dimeric interface. The substitutions of Y241 and T262 in SIR2 domain, H362A and K461 in NOD abolished Ef Avs5 system antiviral defense, indicating that dimer formation is obligatory for Ef Avs5 anti-phage activity in vivo (Fig. 3 D). 4. SIR2- and NOD-dependent filamentation The Ef Avs5 structure features both SIR2 and NOD engaging in intra- and inter-dimer interactions, resulting in two spirals: one dominated by SIR2, the other by NOD (Fig. 4 A, B and E). The SIR2 domain within Ef Avs5 system exhibits a typical two-domain fold (Fig. 4 D), resembling DSR2(PDB ID: 8WYB, rmsd of 1.160 Å for 74 aligned Cα atoms) and ThsA (PDB ID: 8BTO, rmsd of 1.247 Å for 84 aligned Cα atoms) 11 , 12 , except for a longer α3 creating a broader substrate channel (Fig. S6). The large domain (LD) has a Rossmann-fold core that is conserved among all the SIR2 proteins, while the small domain (SD) lacks the three-stranded zinc-binding motif compared to human Sirt5 16 . The interface between SIR2 dimers is non-parallel to the helical axis (Fig. 4 A), burying ∼1218.8 Å 2 of surface area (Fig. 4 B). SIR2 α4, α10 and α12 helices contribute to the intra-dimer interactions (Fig. 3 C), whereas the loop connecting β3 and β4 forms polar interactions and hydrogen bonds with the loop between β4 and α9 of the adjacent SIR2, thereby constituting the inter-dimer interactions and facilitating the assembly of Ef Avs5 filaments (Fig. 4 C). Interestingly, despite the similarity of the structure, the oligomerization mechanism of Ef Avs5-SIR2 is different from SIR2-HerA, ThsA and DSR2 (Fig. S7). Identical to the intra-dimer interaction, the inter-dimer interactions of NOD also mainly include the NBD-WHD contacts, but adopt a different mechanism (Fig. 4 E). The NBD interface of the n + 1 molecule is predominantly negatively charged (I303, E307, E404), complementing the positively charged interface of WHD domain of n + 2 molecule (K552, H622, K623) (Fig. 4 F and G). To probe the role of the filament formation in Ef Avs5 activity, we generated mutants D164A and R185A of the interface between n and n + 3 subunits, and double mutants I303A/E307A, E404A/K552A and H622A/K623A of the interface between n + 1 and n + 2 subunits. These substitutions abolished Ef Avs5 system antiviral defense, indicating that both SIR2-based and NOD-based filament formation is obligatory for Ef Avs5 anti-phage activity in vivo (Fig. 3 D). 5. NADase catalytic pocket As expected, modeling of NAD + within the active site in the cryo-EM map of Ef Avs5 was not feasible due to degradation by the active SIR2. Superimposing the SIR2 domain of Ef Avs5 onto both the active and inactive conformations of ThsA reveals a comparable active pocket, situated within the SIR2 dimer (Fig. 5 A and Fig. 2 D). Dimerization likely contributes to the stabilization of the active pocket. In the case of ThsA and DSR2, the position of the loop above the active pocket decides to block the NAD + access to the active site or enable NAD + binding, thus control the NADase activity of SIR2 11,17 . However, in the Ef Avs5, the longer α3 pull this loop far away the active pocket (Fig. 5 A and Fig. S6), indicating a different activation mechanism adopted by Ef Avs5. In the Ef Avs5-NAD + predicted model, the A41, T140, N141, Y142, N180, Y202, S233 in the pocket interact with NAD + , creating an ideal condition for the reaction (Fig. 5 B). The Y202 is also involved in SIR2-based dimerization. These residues are highly conserved in the bacterial SIR2 proteins, except the substitution of an Asn residue with His at residue 180 in Ef Avs5 (Fig. S8). 6. ATP inhibits the NADase activity of the Ef Avs5 system The NOD of Ef Avs5 is an AAA + ATPase, belongs to a novel STAND NTPase 3 family found in bacterial conflict systems and in metazoan TRADD-N associated counter-invader proteins 18 . The structural comparison in Dali server 19 suggested that Ef Avs5’s NOD is most similar to the plant NLR RPP1(PDB ID: 7CRC, rmsd of 1.160 Å for 56 aligned Cα atoms) and cell death protein 4 in Caenorhabditis elegans (PDB ID: 4M9S, rmsd of 1.239 Å for 45 aligned Cα atoms). Notably, we found that Ef Avs5 is an inactive ATPase either in the monomer or filament assembly (Fig. 5 C). Consistently, the cryo-EM map allows modelling of ATP molecules in each NOD active site (Fig. 5 D), although we did not add additional ATP during the sample preparation. In the structure, ATP molecules are buried in the active pocket between NBD and WHD (Fig. 5 E), with an adjacent magnesium ion coordinated by the canonical Walker A and B motif (Fig. 5 F). The K379 and S380 in Walker A form hydrogen bonds with the oxygens of the phosphate groups, determining the orientation of ATP. Meanwhile, the acidic D426 in Walker B binds to the Mg 2+ . The recognition of the γ-phosphate group of ATP in the ZAR1 resistosome and Apaf-1 apoptosome is facilitated by an arginine residue in the conserved 'TT/SR' motif, which is crucial for their activation and preserved as 'TTR' and R453 in Ef Avs5 20,21 . The I351 forms hydrogen bonds with the N6 atoms of the adenine ring (Fig. 5 F). The region spanning residues 300–366, sometimes described as the fish-specific NACHT-associated (FISNA) domain, resembles the active state of NLRP3 (PDB ID:8EJ4), triggering conformational alterations and oligomerization 22 . The accurate positioning of ATP is crucial for the functionality of the Ef Avs5 system, as evidenced by the phage plaque assays with WalkerA/B mutants (Fig. 1 A). The deficiency of ATP hydrolysis probably results from its WHD domain's unique conformations near the active site, particularly the hydrogen bonding between R537 and the ATP phosphate, contrasting with other active STAND ATPases (Fig. 5 F and Fig. S9). Based on the role of ATPase in sustaining Ef Avs5's activity in vivo , we investigated the potential impact of ATP on NAD + hydrolysis. Our findings revealed that low ATP concentrations (0.05 and 0.5 mM) had negligible effects, whereas 5 mM ATP significantly reduced Ef Avs5's NADase activity by 40% (Fig. S10). Notably, GTP and dGTP also effectively inhibited NADase activity, while other NTPs/dNTPs showed minimal effects, except for a slight inhibition by UTP (Fig. 5 G). These results indicate that specific NTP/dNTP and its concentration can inhibit Ef Avs5's NADase activities. 7. Filamentous cluster formation mediates Ef Avs5 anti-phage activity Ef Avs5 filaments are organized into higher-order structures, forming bundles or three-dimensional networks (Fig. 2 A). Although density maps of the filament bundle were not available, interactions between filaments were observed during 2D classification in cryo-EM analysis (Fig. 6 A), in which the Ef Avs5 exhibits an end-to-end linkage between dimers from adjacent filaments (Fig. 6 B). Notably, the C-terminal sensor possesses a predominantly positive charge, which complements the negatively charged interface of the N-terminal SIR2 domain residing in adjacent filaments (Fig. 6 C). These interactions would enable the Ef Avs5 filament to extend in four distinct directions, thereby promoting the formation of parallel bundles (Fig. 6 D). The C-terminus of Ef Avs5 does not participate in the formation of the individual filament but may play a crucial role in filament clustering. Deletion of the entire C-terminal sensor abolished Ef Avs5 activity (Fig. 1 A). Further analysis confirmed that deletion of C-terminal six positively charged amino acids similarly abolished Ef Avs5's resistance to phage invasion, as well as the mutations of D79A, E85A and K792A at interaction interfaces (Fig. 6 E). These results indicated that filament clustering may be essential for the anti-phage function of Ef Avs5. Discussion In this study, we determined the cryo-EM structures of the Ef Avs5 and found that Ef Avs5 assembles into bundled filaments for NADase activity and anti-phage defense. We propose the following model for Ef Avs5 (Fig. 6 F). Without phage infection, Ef Avs5 adopts an inactive, monomeric conformation in cells. Phage infection triggers Ef Avs5 to form filament bundles, in which SIR2 adopts a dimeric active state. This active state enables Ef Avs5 to rapidly degrade NAD + , leading to an abortive infection that prevents propagation of phage through a population of cells. The Ef Avs5 structure displays a filament assembly distinct from the characterized SIR2-containing defense systems and STAND proteins, underlining the universal yet diverse nature of filamentation as an antiviral mechanism across prokaryotic and eukaryotic immune systems. The Ef Avs5 filament is formed by Ef Avs5 dimers with a C2 symmetry arranged into a high-order complex, involving comprehensive SIR2-SIR2 and NBD-WHD interactions. In SIR2-APAZ/Ago, DSR2, SIR2-HerA and Thoeris system, SIR2 domains function as monomers, tetramers (dimer of dimers), dodecamers (hexamer of dimers) and filament (spiral of tetramers), respectively 7 – 10 . Compared with the SIR2-based filamentation in ThsA, the NOD and SIR2 domain work together to facilitate filament formation in Ef Avs5, highlighting their independent yet complementary roles in the assembly process. Moreover, to the best of our knowledge, Ef Avs5 represents the first STAND family protein reported to assemble into functional filaments. The activation of STAND family proteins typically involves oligomerization, which includes tetramers ( Se Avs3, Ec Avs4, and plant TIR-NLRs), pentamers (plant ZAR1), heptamers (animal apoptosome Apaf-1) or octamers (Drosophila dark apoptosome) 4 , 21 , 23 – 25 . Notably, Solanum lycopersicum NRC2 ( Sl NRC2) was recently found to form filaments at elevated concentrations, but adopt an inactive conformation to avoid self-activation. Distinct from Ef Avs5, the Sl NRC2 filament consists of three identical protofilaments, which contain copies of tetramers 26 . The activation observed in this study appears to be correlated with the accumulation of Ef Avs5 proteins, mirroring previous findings in plants where concentration-dependent hypersensitive response cell death was mediated by the plant NLR protein 27 , 28 . Considering its genomic localization on a PICI, intracellular expression of Ef Avs5 may be tightly regulated to avoid unnecessary activity. The phage signals activating Ef Avs5 remain unclear. We speculated that the activation of Ef Avs5 upon phage infection may involve binding of an as-yet-unidentified protein or compound; alternatively, it may be a response to changes in substrate concentration. Firstly, Ef Avs5 may interact with either phage-derived proteins, akin to the DSR2 system 9 , or host-derived proteins, such as that involved in the bNACHT25 system 29 , leading to the activation of filament formation and enzymatic activity. However, our attempt to identify potential activators through screening of escape phages was unsuccessful. Secondly, as suggested by the finding that the binding of the TIR substrates NAD + and ATP induces phase separation and activation of TIR in vitro 30 , one of the potential activation mechanisms of Ef Avs5 may be the substrate-based response. Above all, our structural and biochemical findings offer insights into the molecular mechanism of Avs5 bacterial immune system, highlighting the conservation of filamentation as a key regulatory mechanism governing antiviral defense across prokaryotes and metazoans. Limitations of the study Our findings elucidate the fundamental mechanisms underlying Ef Avs5 function, yet the activation signals associated with phage infection remain elusive and warrant further investigation. Apart from biochemical assays on mutants, our results lack direct evidence of Ef Avs5 forming fibrillar clusters in vivo . Additionally, we propose that ATP intricately modulates Ef Avs5 activity, but the underlying molecular mechanisms require detailed clarification. Methods Plasmid construction DNA fragments encoding Ef Avs5 from Escherichia fergusonii PICI EfCIRHB19-C05 (QML19490.1) were synthesized and inserted into the expressing plasmids pET28a with N-terminal His 6 -tag by Universe Gene Technology (Tianjin). The DNA fragments encoding Ef Avs5 was subcloned into the pBAD33 vector for phage infection assays. Plasmids carrying Ef Avs5 mutants were constructed using site-directed mutagenesis. Primers used are listed in Table S2. Protein expression and purification The vector pET28a- Ef Avs5 was transformed into E. coli BL21(DE3). Induction of expression was achieved by adding 0.3 mM isopropyl-β-D-thiogalactopyranoside (IPTG) and incubated for 16 h at 16°C and 220 rpm. The cells were then harvested and resuspended in buffer A (50 mM Tris, pH 8.0, 500 mM NaCl, 5 mM MgCl 2 , 10% glycerol, 1 mM β-mercaptoethanol and 5 mM imidazole). After purification by nickel-NTA, the eluate was further loaded onto a size exclusion chromatography column (SEC) (Superose 6, Cytiva) equilibrated with buffer B (20 mM Tris, pH 8.0, 150 mM NaCl, 5 mM MgCl 2 , 5% glycerol) and the purified proteins were stored at −80 °C. Plaque assays E. coli MG1655 possessing pBAD33- Ef Avs5, its mutants or an empty vector(pBAD33) were grown at 37℃ in LB medium. When the OD 600 reached 0.4, cells were induced with 0.5% L-arabinose for 1 hour. Then, 150 μL of the culture was mixed with 3 mL LB containing 0.8% low-melting agarose（with 0.5% L-arabinose added）, and the mixture was poured into LB agar base layer in the 9 cm petri dish. Ten-fold dilutions of high-titers(>10 8 pfu/mL) of phage T7 were spotted onto the agar and incubated overnight at 37 ℃. The next day, plates were photographed with blue-white light reflection in the dark box. Phage-infection in liquid medium E. coli MG1655 possessing pBAD33- Ef Avs5, its mutant(N141A) or an empty vector(pBAD33) were grown until the OD 600 reached 0.4. These cultures were then induced with 0.5% L-arabinose for 1 hour, and diluted in LB containing 0.5% L-arabinose to an OD of ~0.1. Phage T7 was added to the culture at final MOI of 0.02, 0.2, 2, and 10. Subsequently, 200 μL of the diluted culture was placed into the wells of a 96-well plate. During shaking, the OD 600 was measured every 5 minutes at 37℃ for 6 hours. Cell survival assays Overnight cultures of E. coli MG1655, either containing the plasmid pBAD33- Ef Avs5 or an empty vector, were diluted 1:100 into fresh LB medium. These cultures were grown at 37°C with the addition of 0.5% L-arabinose until they reached an OD 600 of 0.4. Subsequently, the cells were harvested by centrifugation, washed with LB, and resuspended to an OD 600 of 0.2. To each sample, phage T7 was added at an MOI of 5, while control samples were left without phage addition. Following a 20-minute adsorption period, serial 10-fold dilutions of each sample were plated onto LB agar, and the plates were incubated overnight at 37°C. The cell survival rate was determined by comparing the CFU obtained from the samples with phage T7 addition to the CFU obtained from the control samples without phage, expressed as a percentage. Phage burst size measurements Overnight cultures of E. coli MG1655 harboring either the pBAD33- Ef Avs5 plasmid or an empty vector were diluted 1:100 in LB medium supplemented with 0.5% L-arabinose. These diluted cultures were then grown at 37°C until they reached an OD 600 of 0.4. Subsequently, T7 phages were introduced to the cultures at an MOI of 0.1, and the infection process was allowed to proceed at 37°C. To establish a baseline for the initial phage titer, an equal volume of phage was added to LB media and used as the reference for time 0 of infection. After infection for 20, 40, and 60 minutes at 37°C, which represent approximately one, two, and three cycles of T7 phage replication, respectively, 0.5 mL samples of the culture were withdrawn. These samples were centrifuged at 5000 rpm for 7 minutes and the supernatants were filtered through a 0.22 μm filter. The titer of the T7 phages present in the filtered supernatants was determined by a plaque assay using E. coli MG1655 as the host. NAD(H) degradation measurements NAD(H) concentrations were measured using the Innochem Coenzyme I NAD(H) Content Assay Kit (WST colorimetry), following the manufacturer's instructions. Briefly, overnight cultures were diluted 1:100 in 50 mL LB with 0.5% L-arabinose, grown to OD 600 of 0.4, and infected with T7 phage at an MOI of 2. At indicated times, 1 mL cultures were centrifuged, resuspended in acidic extract, sonicated, boiled, rapid cooled, and centrifuged. Supernatant was neutralized with alkaline extract. 50 μL of the neutralized supernatant was combined sequentially with 250 μL of Reagent 1, 75 μL of Reagent 2, 150 μL of Reagent 3, and 35 μL of Reagent 4 in the determination tube. After a 1-hour dark reaction, Reagent 5 was added to the mixture. In the control tube, Reagent 5 was added first, followed by the supernatant, maintaining identical steps thereafter. Absorbance was measured at 450 nm, and NAD(H) levels were calculated using a standard curve and normalized to the amount of NAD(H) present in an equivalent volume of sample with an OD 600 of 0.1 to allow for accurate comparisons between samples. Negative staining analysis The Ef Avs5 sample was directly applied to a glow-discharged 400-mesh Cu grid (Beijing ZhongJingKeYi Technology) for 30 s. After side blotting, the grid was immediately stained with 2% uranyl formate and then blotted again from the side. Staining was repeated twice with a 30 s incubation with uranyl formate in the final staining step. EM images were collected on a Talos F200C G2 at a nominal magnification of 52,000× and at a defocus of about 5 µm. Cryo-EM grid preparation and data acquisition 3.5 μL of the Ef Avs5 samples were added to freshly glow-discharged Quantifoil R1.2/1.3 Cu 300 mesh grids. In a Vitrobot (FEI, Inc.), grids were plunge-frozen in liquid ethane after being blotted for 2 s at 16°C with 100% chamber humidity. The grids were imaged using EPU on Titan Krios 300 kV microscopes with a K3 detector. Totally 4318 movies were collected under the defocus ranged from −0.8 to −1.6 μm, and the magnification was 81k in super-resolution mode. 32 frames per movie were collected with a total dose of 40 e – /Å 2 . Cryo-EM data processing and model building The cryo-EM data was processed using CryoSPARC suite v4.5.3 31 . After motion correction, patch CTF estimation and manual exposure curation, 1425 movies were selected. The first set of particles was picked using a filament tracer, and after two-dimensional (2D) classification, good templates were selected for template picking. A total of 1,392,237 particles were picked using a template picker and extracted (box size 360 pixels) from 1,425 accepted micrographs (Fig. S4). Two rounds of 2D classification were conducted and 364,551 particles were picked and used for ab-initio reconstruction (three classes). The largest 3D class (228,053 particles, 62.6%) was selected and refined by homogeneous refinement, non-uniform refinement and CTF refinement to give the final map (global resolution, 3.44 Å). Helical parameters (helical twist and helical rise) were determined by helical refinement job. The initial model of Ef Avs5 wes generated by AlphaFold 32 . The model of ATP was built in Coot 33 . The models of Ef Avs5 were fitted into the cryo-EM density maps using ChimeraX 34 . The model was refined in Coot and Phenix with secondary structure, rotamer and Ramachandran restraints 35 . The Map versus Model FSCs was generated by Phenix (Fig. S4). The statistics of cryo-EM data processing and refinement were listed in Table S1. In vitro NADase activity The Ef Avs5 NADase activity was evaluated through an εNAD + -based fluorescence assay, wherein the enzymatic cleavage of the nicotinamide glycosidic bond in εNAD + results in the formation of εADPR, which subsequently emits a fluorescent signal. In a 96-well black flat-bottom plate, 100 μL reaction mixtures were prepared, each containing 50 μM of εNAD + (Sigma catalogue No. N2630) and 0.5 μM Ef Avs5 proteins in reaction buffer (20 mM Tris, pH 8.0, 150 mM NaCl, and 5% glycerol, with and without the addition of nucleotides). A mixture containing only the reaction buffer and εNAD + was used as a control. The reactions were then loaded into a BioTek Synergy H1 microplate reader, and fluorescence intensities were recorded at 37°C every 20 seconds for 15 minutes, employing excitation and emission wavelengths of 310 nm and 410 nm, respectively. All experiments were conducted in triplicate to ensure reproducibility. ATPase activity ATPase activities were evaluated by using Malachite Green Phosphate Detection Kit (Beyotime). Each 200 μL reaction mixture consisted of 5μM Ef Avs5 proteins in a reaction buffer (20 mM Tris, pH 8.0, 150 mM NaCl, 5 mM MgCl 2 and 0.5 mM ATP). A mixture without the addition of Ef Avs5 proteins served as a control. Following a 30-minute incubation at 37°C, 70 μL of color reagent was added and the mixture was further incubated for another 30 minutes. Subsequently, the absorbance at 620 nm was measured using a multi-detection microplate reader (Tecan Spark). Phosphate levels were then calculated using a standard curve generated with phosphate standards. All experiments were conducted in triplicate to ensure reproducibility. Phylogenetic analysis of Avs5 systems The coding sequence of Avs5 was acquired from two sources: firstly, by utilizing the defense finder tool to scan through all bacterial and archaeal genomes housed in the IMG (Integrated Microbial Genomes & Microbiomes) database from November 2023, aiming to identify proteins that encode the Avs5 system 36 ; secondly, by leveraging the Foldseek search server to discover proteins (leveraging AlphaFold/UniProt50, ensuring a coverage exceeding 80%) that exhibited a high degree of structural similarity to Avs5 37 . To eliminate redundancy, MMseqs was employed with the specified parameters ‘–min-seq-id 0.90’ and ‘-c 0.8’, resulting in 263 filtered sequences 38 . These sequences were subsequently aligned using MAFFT, with the parameters set to '--maxiterate 1000 –globalpair' for optimal alignment 39 . Following alignment, trimAl was applied to refine the alignment by trimming unnecessary regions 40 . The phylogenetic tree was then constructed using IQ-TREE, incorporating the parameters '-nstop 500 -bb 1000 -m LG+F+I+G4' for enhanced accuracy and robustness 41 . Finally, iTOL was utilized for the visualization and annotation of the phylogenetic tree 42 . Declarations Acknowledgements This work was supported by National Natural Science Foundation of China (32370071, 32070040), National Key Research and Development Program of China (2020YFA0907900, 2019YFA0905400). We thank the staff members of the Electron Microscopy System at the National Facility for Protein Science in Shanghai (NFPS), Shanghai Advanced Research Institute, Chinese Academy of Sciences, China for providing technical support and assistance in data collection. We thank Jing Liu, Xinqiu Guo and Mengyu Yan at the Instrument Analysis Center (IAC) of Shanghai Jiao Tong University for providing assistance in data collection. Author contributions J.Z. and Y.W. designed the experiments and analyzed data. Y.W. and Y.T. performed biochemical assays. Y.W. and X.Y. conducted cryo-EM data collection. Y.W. conducted image processing, atomic model, building and refinement and structural analyses. Y.W. and Y.F performed bioinformatics analysis. Y.W. and J.Z. wrote the manuscript. Declaration of interests The authors declare no competing interests. Data availability The atomic coordinates have been deposited in the Protein Data Bank under accession codes 9JAP. The corresponding maps have been deposited in the Electron Microscopy Data Bank under the accession number EMD-61299. Original data for biochemical assays and uncropped gels were deposited to Figshare. 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Mol Biol Evol 32:268–274. https://doi.org:10.1093/molbev/msu300 Letunic I, Bork P (2021) Interactive Tree Of Life (iTOL) v5: an online tool for phylogenetic tree display and annotation. Nucleic Acids Res 49:W293–w296. https://doi.org:10.1093/nar/gkab301 Additional Declarations There is NO Competing Interest. Supplementary Files SupplementaryInformation20240926.pdf Cite Share Download PDF Status: Published Journal Publication published 11 Mar, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5156926","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":362339186,"identity":"3064a152-13bf-4a72-ab5e-7a594ccff286","order_by":0,"name":"Jianting Zheng","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5ElEQVRIiWNgGAWjYBACxgYGNhAtx8BwAEQzE6GlDaLFmIHhMJFagBrAWhIbIKqJ0MI8v/3Zg487atPnN54/JsFQYZ3YwH72ACGHpRvOPHM8d8OBw2wSDGfSExt48hIIaTkmzdt2LHcDA1ALY9vhxAYJHgMCWhjbpP+2HUuXbwBp+UeUFmY2aca2mgQGkMMYG4jSksYm2dt2wBDoF2OLhGPpxm08Ofi1GDYffybxs61OXn7GwYc3PtRYy/aznyGgpQFMAaNR4gADQwIDAySa8AF5CFXHwMDfQEjtKBgFo2AUjFQAAMyJRZZLvg/tAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0003-1250-3556","institution":"Shanghai Jiao Tong University","correspondingAuthor":true,"prefix":"","firstName":"Jianting","middleName":"","lastName":"Zheng","suffix":""},{"id":362339187,"identity":"4de8392c-7356-4590-8115-b37d1611d453","order_by":1,"name":"Yiqun Wang","email":"","orcid":"","institution":"Shanghai Jiao Tong University","correspondingAuthor":false,"prefix":"","firstName":"Yiqun","middleName":"","lastName":"Wang","suffix":""},{"id":362339188,"identity":"49875123-0b58-4440-bcda-e02818a40468","order_by":2,"name":"Yuqing Tian","email":"","orcid":"","institution":"Shanghai Jiao Tong University","correspondingAuthor":false,"prefix":"","firstName":"Yuqing","middleName":"","lastName":"Tian","suffix":""},{"id":362339189,"identity":"906ce96e-aa8e-4f89-95b5-31712485dd7a","order_by":3,"name":"Xu Yang","email":"","orcid":"https://orcid.org/0009-0000-4378-6232","institution":"Shanghai Jiao Tong University","correspondingAuthor":false,"prefix":"","firstName":"Xu","middleName":"","lastName":"Yang","suffix":""},{"id":362339190,"identity":"14649e75-aa29-44c2-a994-ca8958f7c6ec","order_by":4,"name":"Feng Yu","email":"","orcid":"","institution":"Shanghai Jiao Tong University","correspondingAuthor":false,"prefix":"","firstName":"Feng","middleName":"","lastName":"Yu","suffix":""}],"badges":[],"createdAt":"2024-09-26 08:21:26","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5156926/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5156926/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41467-025-57732-7","type":"published","date":"2025-03-11T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":66660511,"identity":"7038db48-c67a-4681-aee3-a00a7b922cf9","added_by":"auto","created_at":"2024-10-15 08:47:11","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":518120,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eEf\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eAvs5 consumes NAD\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e to confer defense.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Domain organization of the \u003cem\u003eEf\u003c/em\u003eAvs5 protein from \u003cem\u003eEscherichia fergusonii \u003c/em\u003eand efficiency of plating of T7 phages infecting \u003cem\u003eE. coli\u003c/em\u003e expressing \u003cem\u003eEf\u003c/em\u003eAvs5 or an empty vector. Data are representative images of n=3 biological replicates. Mutations used are indicated below the domain organization. NBD, nucleotide-binding domain; HD, helical domain; WHD, wing-helical domain; NOD, nucleotide-binding oligomerization domain. (\u003cstrong\u003eB\u003c/strong\u003e) Infection time courses for liquid cultures of \u003cem\u003eE. coli\u003c/em\u003e, with \u003cem\u003eEf\u003c/em\u003eAvs5, \u003cem\u003eEf\u003c/em\u003eAvs5 N141A or empty vector, infected at different multiplicities of infection (MOI) of phage T7. Data represent the mean of three replicates and shaded regions represent the standard error of the mean (SEM). (\u003cstrong\u003eC\u003c/strong\u003e) Survival of \u003cem\u003eE. coli\u003c/em\u003e cells expressing either the \u003cem\u003eEf\u003c/em\u003eAvs5 or empty vector as a control infected at an MOI of 5 with T7 for 20 minutes. Data represent the mean of three replicates and error bars represent the SEM. (\u003cstrong\u003eD\u003c/strong\u003e) Titer of T7 phage propagated on \u003cem\u003eE. coli\u003c/em\u003e cells expressing either the \u003cem\u003eEf\u003c/em\u003eAvs5 or empty vector as a control infected at an MOI of 0.1 with T7. Data represent the titer of T7 measured in PFU/mL after indicated time from initial infection, divided by the original phage titer prior to infection. Data represent the mean of three replicates and error bars represent the SEM. (\u003cstrong\u003eE\u003c/strong\u003e) Measurement of NAD\u003csup\u003e+\u003c/sup\u003e concentration in \u003cem\u003eE. coli\u003c/em\u003e expressing \u003cem\u003eEf\u003c/em\u003eAvs5 or empty vector as a control at the indicated time points after infection with phage T7 at an MOI of 2, normalized to an OD\u003csub\u003e600\u003c/sub\u003e of 0.1. The control group underwent near-complete lysis after 20 minutes of infection, precluding the quantification of NAD\u003csup\u003e+\u003c/sup\u003e content. Data represent the mean of two or three replicates and error bars represent the SEM.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-5156926/v1/c79485c3b63d2a5fbf339db2.png"},{"id":66660512,"identity":"48ef0eb4-be9f-481b-9598-ee8d9260c006","added_by":"auto","created_at":"2024-10-15 08:47:11","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":916637,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCryo-EM structure of the \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eEf\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eAvs5 filament.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Representative negative-stain micrograph images of \u003cem\u003eEf\u003c/em\u003eAvs5. The scale bar represents 100 nm. (\u003cstrong\u003eB\u003c/strong\u003e) \u003cem\u003eEf\u003c/em\u003eAvs5’s NADase activity is activated by filamentation. After the addition of ε-NAD\u003csup\u003e+\u003c/sup\u003e, total fluorescence was measured over time. Data represent the mean of three replicates and shaded regions represent the SEM. (\u003cstrong\u003eC\u003c/strong\u003e) Top and side views of the density map and the atomic model\u003cem\u003e \u003c/em\u003eof\u003cem\u003e Ef\u003c/em\u003eAvs5 filament. (\u003cstrong\u003eD\u003c/strong\u003e) Detailed view of \u003cem\u003eEf\u003c/em\u003eAvs5 filament to show the subunit interfaces. Two interfaces are indicated with boxes, and the NAD\u003csup\u003e+\u003c/sup\u003e active site is indicated with a grey arrow. (\u003cstrong\u003eE\u003c/strong\u003e) The core of \u003cem\u003eEf\u003c/em\u003eAvs5 filament is composed of the SIR2 domain and the NOD.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-5156926/v1/5f01f07ec75b87b4918d7d40.png"},{"id":66660516,"identity":"81d1a62b-7e34-4d83-885e-e3049072aefe","added_by":"auto","created_at":"2024-10-15 08:47:11","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":647038,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe mechanism of\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e Ef\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eAvs5 dimerization.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e) The structure of the \u003cem\u003eEf\u003c/em\u003eAvs5 dimer. (\u003cstrong\u003eB\u003c/strong\u003e) Details of the NOD-based dimeric interfaces. (\u003cstrong\u003eC\u003c/strong\u003e) Details of the SIR2-based dimeric interfaces. (\u003cstrong\u003eD\u003c/strong\u003e) Phage plaque assay of \u003cem\u003eE. coli\u003c/em\u003e expressing \u003cem\u003eEf\u003c/em\u003eAvs5 variants with mutations at the interfaces. The yellow background highlights the intra-dimer interactions, while the green background indicates the inter-dimer interactions. Data are representative images of n=3 biological replicates.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-5156926/v1/23f944cb39f0f5e94eb6be96.png"},{"id":66660517,"identity":"312f1698-4dc6-4054-9722-d4a3c1f39f71","added_by":"auto","created_at":"2024-10-15 08:47:11","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":798713,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSIR2- and NOD-dependent filamentation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e)Two spirals dominated by NOD (blue) and SIR2 domains (red), respectively. The C-terminal domains of \u003cem\u003eEf\u003c/em\u003eAvs5 are hided. The inter-dimer interactions are indicated by white boxes. (\u003cstrong\u003eB\u003c/strong\u003e) The intra-dimer and inter-dimer interface of SIR2 domain of the n+1, n, n+3 subunits. LD, large domain; SD, small domain. (\u003cstrong\u003eC\u003c/strong\u003e) Detailed inter-dimer interface of SIR2 domain. The n, n+3 subunits of \u003cem\u003eEf\u003c/em\u003eAvs5 was colored red and orange, respectively. (\u003cstrong\u003eD\u003c/strong\u003e) SIR2 topology diagrams of \u003cem\u003eEf\u003c/em\u003eAvs5. The small domain is shaded grey, and the large domain is shaded red. (\u003cstrong\u003eE\u003c/strong\u003e) The intra-dimer and inter-dimer interface of NOD domain of the n, n+1, n+2 subunits. (\u003cstrong\u003eF\u003c/strong\u003e) Detailed inter-dimer interface of NOD domain. The NBD of n+1 subunit, and the WHD of n+2 subunit of \u003cem\u003eEf\u003c/em\u003eAvs5 were colored dark and light blue, respectively. (\u003cstrong\u003eG\u003c/strong\u003e) The electrostatic potential inter-dimer interface of two adjacent NOD domains.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-5156926/v1/f91a05d610cb76d40defd52c.png"},{"id":66661869,"identity":"51336583-05d5-4b3a-935e-4c3447412f86","added_by":"auto","created_at":"2024-10-15 08:55:11","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":687277,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe NAD\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e hydrolyzing pocket and ATP binding pocket of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eEf\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eAvs5.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Overlay of SIR2 domain (red) of \u003cem\u003eEf\u003c/em\u003eAvs5 with the inactive (6LHX, light orange) and activated (light blue) \u003cem\u003eBc\u003c/em\u003eThsA SIR2 domains. NAD\u003csup\u003e+\u003c/sup\u003e from the activated ThsA (1ICI) is shown. The loops above the NAD\u003csup\u003e+\u003c/sup\u003e pocket of \u003cem\u003eEf\u003c/em\u003eAvs5 and ThsA SIR2 are indicated by arrows colored as in the color scheme. (\u003cstrong\u003eB\u003c/strong\u003e) Residues of \u003cem\u003eEf\u003c/em\u003eAvs5 interacting with NAD\u003csup\u003e+\u003c/sup\u003e molecule highlighted in sticks in the predicted \u003cem\u003eEf\u003c/em\u003eAvs5-NAD\u003csup\u003e+ \u003c/sup\u003ebinding\u003csup\u003e \u003c/sup\u003emodel. (\u003cstrong\u003eC\u003c/strong\u003e)ATPase activity assay of the monomer or the filament \u003cem\u003eEf\u003c/em\u003eAvs5. Data represent the mean of three replicates and error bars represent the SEM. (\u003cstrong\u003eD\u003c/strong\u003e) Cryo-EM map of ATP. (\u003cstrong\u003eE\u003c/strong\u003e) The binding pocket of ATP in the NOD dimer. (\u003cstrong\u003eF\u003c/strong\u003e) Residues of \u003cem\u003eEf\u003c/em\u003eAvs5 coordinating ATP molecule highlighted in sticks. The highly conserved Walker A motif and Walker B motif were indicated by arrows. (\u003cstrong\u003eG\u003c/strong\u003e) Effects of NTPs and dNTPs (5 mM) on NADase activity of the \u003cem\u003eEf\u003c/em\u003eAvs5. CK: no NTPs/dNTPs added. Data represent the mean of three replicates and error bars represent the SEM.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-5156926/v1/9b963ee47db8697ba01cf1b3.png"},{"id":66662431,"identity":"0ec5cb01-3eda-4902-a22d-2a027765e4c7","added_by":"auto","created_at":"2024-10-15 09:03:11","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1099300,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFilamentous cluster formation mediates \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eEf\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eAvs5 anti-phage activity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e) 2D class averages of the particle stacks, showing the bundled filament assembly. The scale bar represents 130 Å. (\u003cstrong\u003eB\u003c/strong\u003e)\u003cem\u003e \u003c/em\u003eInteracting \u003cem\u003eEf\u003c/em\u003eAvs5 dimers in adjacent filaments. (\u003cstrong\u003eC\u003c/strong\u003e) The electrostatic potential interface of C-terminal sensor and N-terminal SIR2 domain. (\u003cstrong\u003eD\u003c/strong\u003e) A schematic diagram of the filament cluster assembly of \u003cem\u003eEf\u003c/em\u003eAvs5. (\u003cstrong\u003eE\u003c/strong\u003e) Phage plaque assay of \u003cem\u003eE. coli\u003c/em\u003e expressing \u003cem\u003eEf\u003c/em\u003eAvs5 variants with mutations at the clustering interfaces. Data are representative images of n=3 biological replicates. (\u003cstrong\u003eF\u003c/strong\u003e) Proposed model of \u003cem\u003eEf\u003c/em\u003eAvs5 immune mechanism. Without phage infection, \u003cem\u003eEf\u003c/em\u003eAvs5 remains inactive as monomer. Phage infection activates \u003cem\u003eEf\u003c/em\u003eAvs5, forming filament bundles where SIR2 enable rapid NAD\u003csup\u003e+\u003c/sup\u003e degradation and aborting phage infection.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-5156926/v1/7d99f0493c71473b83485366.png"},{"id":78331285,"identity":"e98bb9ad-265e-4660-852e-b96f996a2000","added_by":"auto","created_at":"2025-03-12 07:09:30","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5809778,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5156926/v1/4cb91563-f00e-4341-b8b3-b0f38c4b269e.pdf"},{"id":66660518,"identity":"56fcdb4f-78e6-4c3f-9541-c731378ce1dc","added_by":"auto","created_at":"2024-10-15 08:47:11","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2176950,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"SupplementaryInformation20240926.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5156926/v1/08999d55002bb92226149eb0.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Filamentation activates bacterial NLR-like antiviral protein","fulltext":[{"header":"Key Points","content":"\u003col\u003e\n \u003cli\u003e\u003cem\u003eEf\u003c/em\u003eAvs5 depletes NAD\u003csup\u003e+\u003c/sup\u003e for anti-phage defense.\u003c/li\u003e\n \u003cli\u003e\u003cem\u003eEf\u003c/em\u003eAvs5 assembles as bundled filaments that can hydrolyze NAD\u003csup\u003e+\u003c/sup\u003e\u003csub\u003e.\u003c/sub\u003e\u003c/li\u003e\n \u003cli\u003eThe SIR2 domain and NOD collaborate to form an individual filament.\u003c/li\u003e\n \u003cli\u003eThe building block of the filament is \u003cem\u003eEf\u003c/em\u003eAvs5 dimer.\u003c/li\u003e\n \u003cli\u003eThe activity of the \u003cem\u003eEf\u003c/em\u003eAvs5 complex is regulated by ATP.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Introduction","content":"\u003cp\u003eBacteria are in constant conflict with bacteriophages and develop diverse anti-phage defense systems of varying complexity\u003csup\u003e\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. The signal transduction ATPases with numerous domains (STAND) superfamily of P-loop NTPases, particularly the nucleotide-binding leucine-rich repeat containing receptors (NLRs) exemplified by animal inflammasomes and plant resistosomes\u003csup\u003e\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e, recognizes pathogens and triggers downstream inflammatory responses or apoptotic processes through oligomerization, representing a conserved immune strategy in eukaryotes. Recently, this mechanism has also been discovered in the innate immunity of bacteria, known as antiviral STANDs (Avs)\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Avs proteins have a characteristic tripartite domain architecture: a central NTPase (alternatively called nucleotide-binding oligomerization domain, NOD); an extended C-terminal sensor with superstructure-forming repeats; and a variable N-terminal effector typically involved in cell death. Previous studies have revealed that Avs1-3 and Avs4 are activated by the phage terminases and portals, respectively, leading to the formation of tetramers that activate their N-terminal nucleases for antiviral defense\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAvs type 5 (Avs5) contains an N-terminal sirtuin (SIR2) effector and an unusual short C-terminal sensor domain, sometimes termed SIR2-STAND\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. SIR2-mediated nicotinamide adenine dinucleotide hydrolase (NADase) activity represents a critical element in anti-phage immunity, triggering abortive infections via NAD\u003csup\u003e+\u003c/sup\u003e degradation and halting phage propagation, as exemplified by pAgo, Thoeris, DSR2, and the SIR2-HerA system\u003csup\u003e\u003cspan additionalcitationids=\"CR8 CR9\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. In the Thoeris system, the signaling molecule 1''-3' gcADPR activates ThsA, which subsequently forms filaments and depletes NAD\u003csup\u003e+\u003c/sup\u003e, ultimately triggering cell death and thereby preventing the spread of phage infection\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Another defense system DSR2 tetramerizes to form a supramolecular complex that specifically recognizes phage tail tube proteins, and leads to cellular NAD\u003csup\u003e+\u003c/sup\u003e depletion\u003csup\u003e12\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eDespite reports on SIR2 and STAND roles in prokaryotic and eukaryotic immunity, the collaborative mechanism between them and the mechanisms underlying Avs5 activation and assembly remain obscure. Here, we present a biochemical and structural analysis of \u003cem\u003eEscherichia fergusonii\u003c/em\u003e Avs5 (\u003cem\u003eEf\u003c/em\u003eAvs5), unveiling a unique mechanism of assembly-mediated activation by \u003cem\u003eEf\u003c/em\u003eAvs5. \u003cem\u003eEf\u003c/em\u003eAvs5 proteins form active filaments via SIR2 and NOD self-association, with sensor-mediated clustering enhancing their organization, allowing the NAD\u003csup\u003e+\u003c/sup\u003e depletion during phage infection. Moreover, the inactive NOD of \u003cem\u003eEf\u003c/em\u003eAvs5 requires ATP binding for its defensive role, whereas high ATP concentration impedes its NADase activity.\u003c/p\u003e "},{"header":"Result","content":"\u003cp\u003e1. \u003cb\u003eEf\u003c/b\u003e \u003cb\u003eAvs5 consumes NAD\u003c/b\u003e \u003csup\u003e \u003cb\u003e+\u003c/b\u003e \u003c/sup\u003e \u003cb\u003eto confer defense\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAvs5 system is widely distributed across Gram-negative phyla, especially Pseudomonadota (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). To define the molecular anti-phage defense mechanism of Avs5, we investigated a single protein Avs5 defense system from \u003cem\u003eE. fergusonii\u003c/em\u003e phage-inducible chromosomal islands (PICI), named \u003cem\u003eEf\u003c/em\u003eAvs5 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). \u003cem\u003eEf\u003c/em\u003eAvs5 is reported to defense against diverse \u003cem\u003eEscherichia coli\u003c/em\u003e, \u003cem\u003eSalmonella\u003c/em\u003e and \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e phages\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. We cultured \u003cem\u003eE. coli\u003c/em\u003e cells expressing \u003cem\u003eEf\u003c/em\u003eAvs5 and found protection against T7 phages. The C terminal of \u003cem\u003eEf\u003c/em\u003eAvs5 is supposed to sense the phage invasion, while the SIR2 domain is suggested to be the effector depleting NAD\u003csup\u003e+\u003c/sup\u003e. Indeed, a single amino acid substitution at NADase active site (N141A) was sufficient to abolish phage defense by the \u003cem\u003eEf\u003c/em\u003eAvs5 system, confirming that the NADase activity is essential for defense. Similarly, a point mutation disrupting the NOD active site, K379A in walker A or D426A in walker B, as well as a deletion of the C-terminal sensor domain, also abolished defense (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eGrowth curves of \u003cem\u003eE. coli\u003c/em\u003e cultures in the presence of \u003cem\u003eEf\u003c/em\u003eAvs5 kept growing when subjected to the low (0.2) or medium (2) multiplicity of infection (MOI) in contrast to the cells expressing N141A mutant or lacking \u003cem\u003eEf\u003c/em\u003eAvs5 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). We measured the survival rate of cells infected with T7 and found that \u003cem\u003eEf\u003c/em\u003eAvs5 increased cell survival after the first infection cycle (about 20 minutes) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Further analysis showed that the phages amplified rapidly in the infected cells lacking \u003cem\u003eEf\u003c/em\u003eAvs5, but not in the presence of \u003cem\u003eEf\u003c/em\u003eAvs5 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). To investigate the effector\u0026rsquo;s activity during immunity, we detected the cellular NAD\u003csup\u003e+\u003c/sup\u003e content after infection with T7 phage. When subjected to the T7 phage infection, compared to cells lacking \u003cem\u003eEf\u003c/em\u003eAvs5, the concentration of NAD\u003csup\u003e+\u003c/sup\u003e in the \u003cem\u003eEf\u003c/em\u003eAvs5-expressing cells was declining rapidly after the first infection cycle, and began to rise in the second infection cycle (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). These results suggest that \u003cem\u003eEf\u003c/em\u003eAvs5 consumes NAD\u003csup\u003e+\u003c/sup\u003e to effectively prevent phage amplification and leads to the population-level protection.\u003c/p\u003e \u003cp\u003e2. \u003cb\u003eArchitecture of\u003c/b\u003e \u003cb\u003eEf\u003c/b\u003e\u003cb\u003eAvs5 filament assembly\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo biochemically characterize the Avs5 system, we purified \u003cem\u003eEf\u003c/em\u003eAvs5 protein overexpressed in \u003cem\u003eE. coli\u003c/em\u003e BL21(DE3). The freshly isolated \u003cem\u003eEf\u003c/em\u003eAvs5 exists as soluble monomer (Fig. S2). Cryo-EM imaging of the monomer \u003cem\u003eEf\u003c/em\u003eAvs5 revealed small particles. We tried to resolve the structure but only obtained two-dimensional (2D) classes in poor resolution, possibly due to the flexibility (Fig. S3). However, the purified \u003cem\u003eEf\u003c/em\u003eAvs5 becomes visibly cloudy after stored at 4\u0026deg;C or -80\u0026deg;C for two weeks. Negative staining analysis of the suspension of \u003cem\u003eEf\u003c/em\u003eAvs5 confirmed that a significant fraction of the particles (\u0026gt;\u0026thinsp;50%) exhibited bundled 8-nm-wide fibrous assembly, and some of them extending up to ~\u0026thinsp;400 nm in length (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Bundled fibrils can be connected parallelly or end-to-end and extend up to several micrometers. Since previous studies show that the activated SIR2 or Toll/interleukin-1 receptor (TIR) can assemble into helical filaments in Thoeris defense system, TIR-STING and TIR-SAVED effector, respectively\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e, we quantified the NADase activity of \u003cem\u003eEf\u003c/em\u003eAvs5. The freshly purified \u003cem\u003eEf\u003c/em\u003eAvs5 was deficient in the NADase activity, but the filamentous \u003cem\u003eEf\u003c/em\u003eAvs5 displayed robust NADase activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo gain more insight into the filament formation, we determined the cryo-electron microscopy (cryo-EM) structure of the active \u003cem\u003eEf\u003c/em\u003eAvs5. The final cryo-EM map was reconstructed using a total of 228,053 single particles and refined to a nominal resolution of 3.4 \u0026Aring;, with approximately 2.8 \u0026Aring; at the center of filament and approximately 6.8 \u0026Aring; at the peripheral sensor lobes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, Fig. S4 and Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The model was built with eight \u003cem\u003eEf\u003c/em\u003eAvs5 molecules assembled into a helical filament, and extra densities suggest unlimited entries of both ends. The repeating building block of the filament is \u003cem\u003eEf\u003c/em\u003eAvs5 dimer with a C\u003csub\u003e2\u003c/sub\u003e symmetry arranged into a regular helical structure with a helical twist of 84.531\u0026deg; and a helical rise of 48.600 \u0026Aring; (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). The filament is held together by 2 sets of inter-subunit interactions, one between subunit n and n\u0026thinsp;+\u0026thinsp;3 and the other between subunit n\u0026thinsp;+\u0026thinsp;1 and n\u0026thinsp;+\u0026thinsp;2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). The \u003cem\u003eEf\u003c/em\u003eAvs5 proteins fold into five domains: a catalytic SIR2 domain, a core NOD, which comprises the nucleotide-binding domain (NBD), helical domain (HD) and wing-helical domain (WHD) and the C-terminal sensor domain (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA and Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). The core of the helix is composed of the SIR2 and the NOD domain, whereas the C-terminal domain is pointing away and not involved in the helical contacts (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003e3. \u003cb\u003eThe dimerization of\u003c/b\u003e \u003cb\u003eEf\u003c/b\u003e\u003cb\u003eAvs5\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIn the \u003cem\u003eEf\u003c/em\u003eAvs5 filament, the individual dimeric unit adopts a C\u003csub\u003e2\u003c/sub\u003e symmetric conformation with a tight interface (2329.2 \u0026Aring;\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e of buried surface area) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Within each \u003cem\u003eEf\u003c/em\u003eAvs5 dimer unit, abundant contacts are observed between the WHD of one subunit and the NBD of its adjacent subunit, encompassing salt bridges and hydrogen bonds, revealing an oligomerization pattern distinct from other characterized active STAND ATPases, which primarily oligomerize through NBD-NBD and WHD-WHD interactions (Fig. S5). The NBD of the n subunit interacts with the WHD of the n\u0026thinsp;+\u0026thinsp;1 subunit, comprising the salt bridge of the K461(n)- E589(n\u0026thinsp;+\u0026thinsp;1), K473(n)- E580(n\u0026thinsp;+\u0026thinsp;1), and the hydrogen bonds of the T474(n)- Y583/R570(n\u0026thinsp;+\u0026thinsp;1) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Meanwhile, the WHD of the n subunit interacts with the NBD of the n\u0026thinsp;+\u0026thinsp;1 subunit using slightly different residues, comprising the specific contacts of the E580(n)- H362(n\u0026thinsp;+\u0026thinsp;1), and the hydrogen bonds of the Y583(n)- T474(n\u0026thinsp;+\u0026thinsp;1). The dimeric interface is expanded by additional hydrogen bonds between its N-terminal SIR2 domains (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). To probe the role of the dimer formation in \u003cem\u003eEf\u003c/em\u003eAvs5 activity, we generated mutants of the dimeric interface. The substitutions of Y241 and T262 in SIR2 domain, H362A and K461 in NOD abolished \u003cem\u003eEf\u003c/em\u003eAvs5 system antiviral defense, indicating that dimer formation is obligatory for \u003cem\u003eEf\u003c/em\u003eAvs5 anti-phage activity \u003cem\u003ein vivo\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003e4. SIR2- and NOD-dependent filamentation\u003c/h3\u003e\n\u003cp\u003eThe \u003cem\u003eEf\u003c/em\u003eAvs5 structure features both SIR2 and NOD engaging in intra- and inter-dimer interactions, resulting in two spirals: one dominated by SIR2, the other by NOD (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, B and E). The SIR2 domain within \u003cem\u003eEf\u003c/em\u003eAvs5 system exhibits a typical two-domain fold (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD), resembling DSR2(PDB ID: 8WYB, rmsd of 1.160 \u0026Aring; for 74 aligned Cα atoms) and ThsA (PDB ID: 8BTO, rmsd of 1.247 \u0026Aring; for 84 aligned Cα atoms)\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e, except for a longer α3 creating a broader substrate channel (Fig. S6). The large domain (LD) has a Rossmann-fold core that is conserved among all the SIR2 proteins, while the small domain (SD) lacks the three-stranded zinc-binding motif compared to human Sirt5\u003csup\u003e16\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe interface between SIR2 dimers is non-parallel to the helical axis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA), burying \u0026sim;1218.8 \u0026Aring;\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e of surface area (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). SIR2 α4, α10 and α12 helices contribute to the intra-dimer interactions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC), whereas the loop connecting β3 and β4 forms polar interactions and hydrogen bonds with the loop between β4 and α9 of the adjacent SIR2, thereby constituting the inter-dimer interactions and facilitating the assembly of \u003cem\u003eEf\u003c/em\u003eAvs5 filaments (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Interestingly, despite the similarity of the structure, the oligomerization mechanism of \u003cem\u003eEf\u003c/em\u003eAvs5-SIR2 is different from SIR2-HerA, ThsA and DSR2 (Fig. S7). Identical to the intra-dimer interaction, the inter-dimer interactions of NOD also mainly include the NBD-WHD contacts, but adopt a different mechanism (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). The NBD interface of the n\u0026thinsp;+\u0026thinsp;1 molecule is predominantly negatively charged (I303, E307, E404), complementing the positively charged interface of WHD domain of n\u0026thinsp;+\u0026thinsp;2 molecule (K552, H622, K623) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF and G).\u003c/p\u003e \u003cp\u003eTo probe the role of the filament formation in \u003cem\u003eEf\u003c/em\u003eAvs5 activity, we generated mutants D164A and R185A of the interface between n and n\u0026thinsp;+\u0026thinsp;3 subunits, and double mutants I303A/E307A, E404A/K552A and H622A/K623A of the interface between n\u0026thinsp;+\u0026thinsp;1 and n\u0026thinsp;+\u0026thinsp;2 subunits. These substitutions abolished \u003cem\u003eEf\u003c/em\u003eAvs5 system antiviral defense, indicating that both SIR2-based and NOD-based filament formation is obligatory for \u003cem\u003eEf\u003c/em\u003eAvs5 anti-phage activity \u003cem\u003ein vivo\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD).\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e5. NADase catalytic pocket\u003c/h2\u003e \u003cp\u003eAs expected, modeling of NAD\u003csup\u003e+\u003c/sup\u003e within the active site in the cryo-EM map of \u003cem\u003eEf\u003c/em\u003eAvs5 was not feasible due to degradation by the active SIR2. Superimposing the SIR2 domain of \u003cem\u003eEf\u003c/em\u003eAvs5 onto both the active and inactive conformations of ThsA reveals a comparable active pocket, situated within the SIR2 dimer (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA and Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Dimerization likely contributes to the stabilization of the active pocket. In the case of ThsA and DSR2, the position of the loop above the active pocket decides to block the NAD\u003csup\u003e+\u003c/sup\u003e access to the active site or enable NAD\u003csup\u003e+\u003c/sup\u003e binding, thus control the NADase activity of SIR2\u003csup\u003e11,17\u003c/sup\u003e. However, in the \u003cem\u003eEf\u003c/em\u003eAvs5, the longer α3 pull this loop far away the active pocket (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA and Fig. S6), indicating a different activation mechanism adopted by \u003cem\u003eEf\u003c/em\u003eAvs5. In the \u003cem\u003eEf\u003c/em\u003eAvs5-NAD\u003csup\u003e+\u003c/sup\u003e predicted model, the A41, T140, N141, Y142, N180, Y202, S233 in the pocket interact with NAD\u003csup\u003e+\u003c/sup\u003e, creating an ideal condition for the reaction (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). The Y202 is also involved in SIR2-based dimerization. These residues are highly conserved in the bacterial SIR2 proteins, except the substitution of an Asn residue with His at residue 180 in \u003cem\u003eEf\u003c/em\u003eAvs5 (Fig. S8).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e6. \u003cb\u003eATP inhibits the NADase activity of the\u003c/b\u003e \u003cb\u003eEf\u003c/b\u003e\u003cb\u003eAvs5 system\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe NOD of \u003cem\u003eEf\u003c/em\u003eAvs5 is an AAA\u003csup\u003e+\u003c/sup\u003e ATPase, belongs to a novel STAND NTPase 3 family found in bacterial conflict systems and in metazoan TRADD-N associated counter-invader proteins\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. The structural comparison in Dali server\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e suggested that \u003cem\u003eEf\u003c/em\u003eAvs5\u0026rsquo;s NOD is most similar to the plant NLR RPP1(PDB ID: 7CRC, rmsd of 1.160 \u0026Aring; for 56 aligned Cα atoms) and cell death protein 4 in \u003cem\u003eCaenorhabditis elegans\u003c/em\u003e (PDB ID: 4M9S, rmsd of 1.239 \u0026Aring; for 45 aligned Cα atoms). Notably, we found that \u003cem\u003eEf\u003c/em\u003eAvs5 is an inactive ATPase either in the monomer or filament assembly (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Consistently, the cryo-EM map allows modelling of ATP molecules in each NOD active site (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD), although we did not add additional ATP during the sample preparation. In the structure, ATP molecules are buried in the active pocket between NBD and WHD (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE), with an adjacent magnesium ion coordinated by the canonical Walker A and B motif (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). The K379 and S380 in Walker A form hydrogen bonds with the oxygens of the phosphate groups, determining the orientation of ATP. Meanwhile, the acidic D426 in Walker B binds to the Mg\u003csup\u003e2+\u003c/sup\u003e. The recognition of the γ-phosphate group of ATP in the ZAR1 resistosome and Apaf-1 apoptosome is facilitated by an arginine residue in the conserved 'TT/SR' motif, which is crucial for their activation and preserved as 'TTR' and R453 in \u003cem\u003eEf\u003c/em\u003eAvs5\u003csup\u003e20,21\u003c/sup\u003e. The I351 forms hydrogen bonds with the N6 atoms of the adenine ring (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). The region spanning residues 300\u0026ndash;366, sometimes described as the fish-specific NACHT-associated (FISNA) domain, resembles the active state of NLRP3 (PDB ID:8EJ4), triggering conformational alterations and oligomerization\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. The accurate positioning of ATP is crucial for the functionality of the \u003cem\u003eEf\u003c/em\u003eAvs5 system, as evidenced by the phage plaque assays with WalkerA/B mutants (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The deficiency of ATP hydrolysis probably results from its WHD domain's unique conformations near the active site, particularly the hydrogen bonding between R537 and the ATP phosphate, contrasting with other active STAND ATPases (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF and Fig. S9).\u003c/p\u003e \u003cp\u003eBased on the role of ATPase in sustaining \u003cem\u003eEf\u003c/em\u003eAvs5's activity \u003cem\u003ein vivo\u003c/em\u003e, we investigated the potential impact of ATP on NAD\u003csup\u003e+\u003c/sup\u003e hydrolysis. Our findings revealed that low ATP concentrations (0.05 and 0.5 mM) had negligible effects, whereas 5 mM ATP significantly reduced \u003cem\u003eEf\u003c/em\u003eAvs5's NADase activity by 40% (Fig. S10). Notably, GTP and dGTP also effectively inhibited NADase activity, while other NTPs/dNTPs showed minimal effects, except for a slight inhibition by UTP (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG). These results indicate that specific NTP/dNTP and its concentration can inhibit \u003cem\u003eEf\u003c/em\u003eAvs5's NADase activities.\u003c/p\u003e \u003cp\u003e7. \u003cb\u003eFilamentous cluster formation mediates\u003c/b\u003e \u003cb\u003eEf\u003c/b\u003e\u003cb\u003eAvs5 anti-phage activity\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cem\u003eEf\u003c/em\u003eAvs5 filaments are organized into higher-order structures, forming bundles or three-dimensional networks (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Although density maps of the filament bundle were not available, interactions between filaments were observed during 2D classification in cryo-EM analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA), in which the \u003cem\u003eEf\u003c/em\u003eAvs5 exhibits an end-to-end linkage between dimers from adjacent filaments (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Notably, the C-terminal sensor possesses a predominantly positive charge, which complements the negatively charged interface of the N-terminal SIR2 domain residing in adjacent filaments (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). These interactions would enable the \u003cem\u003eEf\u003c/em\u003eAvs5 filament to extend in four distinct directions, thereby promoting the formation of parallel bundles (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). The C-terminus of \u003cem\u003eEf\u003c/em\u003eAvs5 does not participate in the formation of the individual filament but may play a crucial role in filament clustering. Deletion of the entire C-terminal sensor abolished \u003cem\u003eEf\u003c/em\u003eAvs5 activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Further analysis confirmed that deletion of C-terminal six positively charged amino acids similarly abolished \u003cem\u003eEf\u003c/em\u003eAvs5's resistance to phage invasion, as well as the mutations of D79A, E85A and K792A at interaction interfaces (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). These results indicated that filament clustering may be essential for the anti-phage function of \u003cem\u003eEf\u003c/em\u003eAvs5.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we determined the cryo-EM structures of the \u003cem\u003eEf\u003c/em\u003eAvs5 and found that \u003cem\u003eEf\u003c/em\u003eAvs5 assembles into bundled filaments for NADase activity and anti-phage defense. We propose the following model for \u003cem\u003eEf\u003c/em\u003eAvs5 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF). Without phage infection, \u003cem\u003eEf\u003c/em\u003eAvs5 adopts an inactive, monomeric conformation in cells. Phage infection triggers \u003cem\u003eEf\u003c/em\u003eAvs5 to form filament bundles, in which SIR2 adopts a dimeric active state. This active state enables \u003cem\u003eEf\u003c/em\u003eAvs5 to rapidly degrade NAD\u003csup\u003e+\u003c/sup\u003e, leading to an abortive infection that prevents propagation of phage through a population of cells.\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eEf\u003c/em\u003eAvs5 structure displays a filament assembly distinct from the characterized SIR2-containing defense systems and STAND proteins, underlining the universal yet diverse nature of filamentation as an antiviral mechanism across prokaryotic and eukaryotic immune systems. The \u003cem\u003eEf\u003c/em\u003eAvs5 filament is formed by \u003cem\u003eEf\u003c/em\u003eAvs5 dimers with a C2 symmetry arranged into a high-order complex, involving comprehensive SIR2-SIR2 and NBD-WHD interactions. In SIR2-APAZ/Ago, DSR2, SIR2-HerA and Thoeris system, SIR2 domains function as monomers, tetramers (dimer of dimers), dodecamers (hexamer of dimers) and filament (spiral of tetramers), respectively\u003csup\u003e\u003cspan additionalcitationids=\"CR8 CR9\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Compared with the SIR2-based filamentation in ThsA, the NOD and SIR2 domain work together to facilitate filament formation in \u003cem\u003eEf\u003c/em\u003eAvs5, highlighting their independent yet complementary roles in the assembly process. Moreover, to the best of our knowledge, \u003cem\u003eEf\u003c/em\u003eAvs5 represents the first STAND family protein reported to assemble into functional filaments. The activation of STAND family proteins typically involves oligomerization, which includes tetramers (\u003cem\u003eSe\u003c/em\u003eAvs3, \u003cem\u003eEc\u003c/em\u003eAvs4, and plant TIR-NLRs), pentamers (plant ZAR1), heptamers (animal apoptosome Apaf-1) or octamers (Drosophila dark apoptosome)\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Notably, \u003cem\u003eSolanum lycopersicum\u003c/em\u003e NRC2 (\u003cem\u003eSl\u003c/em\u003eNRC2) was recently found to form filaments at elevated concentrations, but adopt an inactive conformation to avoid self-activation. Distinct from \u003cem\u003eEf\u003c/em\u003eAvs5, the \u003cem\u003eSl\u003c/em\u003eNRC2 filament consists of three identical protofilaments, which contain copies of tetramers\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe activation observed in this study appears to be correlated with the accumulation of \u003cem\u003eEf\u003c/em\u003eAvs5 proteins, mirroring previous findings in plants where concentration-dependent hypersensitive response cell death was mediated by the plant NLR protein\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Considering its genomic localization on a PICI, intracellular expression of \u003cem\u003eEf\u003c/em\u003eAvs5 may be tightly regulated to avoid unnecessary activity. The phage signals activating \u003cem\u003eEf\u003c/em\u003eAvs5 remain unclear. We speculated that the activation of \u003cem\u003eEf\u003c/em\u003eAvs5 upon phage infection may involve binding of an as-yet-unidentified protein or compound; alternatively, it may be a response to changes in substrate concentration. Firstly, \u003cem\u003eEf\u003c/em\u003eAvs5 may interact with either phage-derived proteins, akin to the DSR2 system\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e, or host-derived proteins, such as that involved in the bNACHT25 system\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e, leading to the activation of filament formation and enzymatic activity. However, our attempt to identify potential activators through screening of escape phages was unsuccessful. Secondly, as suggested by the finding that the binding of the TIR substrates NAD\u003csup\u003e+\u003c/sup\u003e and ATP induces phase separation and activation of TIR \u003cem\u003ein vitro\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e, one of the potential activation mechanisms of \u003cem\u003eEf\u003c/em\u003eAvs5 may be the substrate-based response.\u003c/p\u003e \u003cp\u003eAbove all, our structural and biochemical findings offer insights into the molecular mechanism of Avs5 bacterial immune system, highlighting the conservation of filamentation as a key regulatory mechanism governing antiviral defense across prokaryotes and metazoans.\u003c/p\u003e\n\u003ch3\u003eLimitations of the study\u003c/h3\u003e\n\u003cp\u003eOur findings elucidate the fundamental mechanisms underlying \u003cem\u003eEf\u003c/em\u003eAvs5 function, yet the activation signals associated with phage infection remain elusive and warrant further investigation. Apart from biochemical assays on mutants, our results lack direct evidence of \u003cem\u003eEf\u003c/em\u003eAvs5 forming fibrillar clusters \u003cem\u003ein vivo\u003c/em\u003e. Additionally, we propose that ATP intricately modulates \u003cem\u003eEf\u003c/em\u003eAvs5 activity, but the underlying molecular mechanisms require detailed clarification.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003ePlasmid construction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDNA fragments encoding \u003cem\u003eEf\u003c/em\u003eAvs5 from \u003cem\u003eEscherichia fergusonii\u003c/em\u003e PICI EfCIRHB19-C05 (QML19490.1) were synthesized and inserted into the expressing plasmids pET28a with N-terminal His\u003csub\u003e6\u003c/sub\u003e-tag by Universe Gene Technology (Tianjin). The DNA fragments encoding \u003cem\u003eEf\u003c/em\u003eAvs5 was subcloned into the pBAD33 vector for phage infection assays. Plasmids carrying \u003cem\u003eEf\u003c/em\u003eAvs5 mutants were constructed using site-directed mutagenesis. Primers used are listed in Table S2.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProtein expression and purification\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe vector pET28a-\u003cem\u003eEf\u003c/em\u003eAvs5 was transformed into \u003cem\u003eE. coli\u003c/em\u003e BL21(DE3). Induction of expression was achieved by adding 0.3 mM isopropyl-\u0026beta;-D-thiogalactopyranoside (IPTG) and incubated for 16 h at 16\u0026deg;C and 220 rpm. The cells were then harvested and resuspended in buffer A (50 mM Tris, pH 8.0, 500 mM NaCl, 5 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 10% glycerol, 1 mM \u0026beta;-mercaptoethanol and 5 mM imidazole). After purification by nickel-NTA, the eluate was further loaded onto a size exclusion chromatography column (SEC) (Superose 6, Cytiva) equilibrated with buffer B (20 mM Tris, pH 8.0, 150 mM NaCl, 5 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 5% glycerol) and the purified proteins were stored at \u0026minus;80 \u0026deg;C.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePlaque assays\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eE. coli\u003c/em\u003e MG1655 possessing pBAD33-\u003cem\u003e\u0026nbsp;Ef\u003c/em\u003eAvs5, its mutants or an empty vector(pBAD33) were grown at 37℃ in LB medium. When the OD\u003csub\u003e600\u003c/sub\u003e reached 0.4, cells were induced with 0.5% L-arabinose for 1 hour. Then, 150 \u0026mu;L of the culture was mixed with 3 mL LB containing 0.8% low-melting agarose（with 0.5% L-arabinose added）, and the mixture was poured into LB agar base layer in the 9 cm petri dish. Ten-fold dilutions of high-titers(\u0026gt;10\u003csup\u003e8\u003c/sup\u003epfu/mL) of phage T7 were spotted onto the agar and incubated overnight at 37 ℃. The next day, plates were photographed with blue-white light reflection in the dark box.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhage-infection in liquid medium\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eE. coli\u003c/em\u003e MG1655 possessing pBAD33-\u003cem\u003e\u0026nbsp;Ef\u003c/em\u003eAvs5, its mutant(N141A) or an empty vector(pBAD33) were grown until the OD\u003csub\u003e600\u003c/sub\u003e reached 0.4. These cultures were then induced with 0.5% L-arabinose for 1 hour, and diluted in LB containing 0.5% L-arabinose to an OD of ~0.1. Phage T7 was added to the culture at final MOI of 0.02, 0.2, 2, and 10. Subsequently, 200 \u0026mu;L of the diluted culture was placed into the wells of a 96-well plate. During shaking, the OD\u003csub\u003e600\u003c/sub\u003e was measured every 5 minutes at 37℃ for 6 hours.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell survival assays\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOvernight cultures of \u003cem\u003eE. coli\u003c/em\u003e MG1655, either containing the plasmid pBAD33-\u003cem\u003eEf\u003c/em\u003eAvs5 or an empty vector, were diluted 1:100 into fresh LB medium. These cultures were grown at 37\u0026deg;C with the addition of 0.5% L-arabinose until they reached an OD\u003csub\u003e600\u003c/sub\u003e of 0.4. Subsequently, the cells were harvested by centrifugation, washed with LB, and resuspended to an OD\u003csub\u003e600\u003c/sub\u003e of 0.2. To each sample, phage T7 was added at an MOI of 5, while control samples were left without phage addition. Following a 20-minute adsorption period, serial 10-fold dilutions of each sample were plated onto LB agar, and the plates were incubated overnight at 37\u0026deg;C. The cell survival rate was determined by comparing the CFU obtained from the samples with phage T7 addition to the CFU obtained from the control samples without phage, expressed as a percentage.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhage burst size measurements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOvernight cultures of \u003cem\u003eE. coli\u003c/em\u003e MG1655 harboring either the pBAD33-\u003cem\u003eEf\u003c/em\u003eAvs5 plasmid or an empty vector were diluted 1:100 in LB medium supplemented with 0.5% L-arabinose. These diluted cultures were then grown at 37\u0026deg;C until they reached an OD\u003csub\u003e600\u003c/sub\u003e of 0.4. Subsequently, T7 phages were introduced to the cultures at an MOI of 0.1, and the infection process was allowed to proceed at 37\u0026deg;C. To establish a baseline for the initial phage titer, an equal volume of phage was added to LB media and used as the reference for time 0 of infection. After infection for 20, 40, and 60 minutes at 37\u0026deg;C, which represent approximately one, two, and three cycles of T7 phage replication, respectively, 0.5 mL samples of the culture were withdrawn. These samples were centrifuged at 5000 rpm for 7 minutes and the supernatants were filtered through a 0.22 \u0026mu;m filter. The titer of the T7 phages present in the filtered supernatants was determined by a plaque assay using \u003cem\u003eE. coli\u003c/em\u003e MG1655 as the host.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNAD(H) degradation measurements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNAD(H) concentrations were measured using the Innochem Coenzyme I\u0026nbsp;NAD(H) Content Assay Kit (WST colorimetry), following the manufacturer\u0026apos;s instructions. Briefly, overnight cultures were diluted 1:100 in 50 mL LB with 0.5% L-arabinose, grown to OD\u003csub\u003e600\u0026nbsp;\u003c/sub\u003eof 0.4, and infected with T7 phage at an MOI of 2. At indicated times, 1 mL cultures were centrifuged, resuspended in acidic extract, sonicated, boiled, rapid cooled, and centrifuged. Supernatant was neutralized with alkaline extract. 50 \u0026mu;L of the neutralized supernatant was combined sequentially with 250 \u0026mu;L of Reagent 1, 75 \u0026mu;L of Reagent 2, 150 \u0026mu;L of Reagent 3, and 35 \u0026mu;L of Reagent 4 in the determination tube. After a 1-hour dark reaction, Reagent 5 was added to the mixture. In the control tube, Reagent 5 was added first, followed by the supernatant, maintaining identical steps thereafter. Absorbance was measured at 450 nm, and NAD(H) levels were calculated using a standard curve and normalized to the amount of NAD(H) present in an equivalent volume of sample with an OD\u003csub\u003e600\u003c/sub\u003e of 0.1 to allow for accurate comparisons between samples.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNegative staining analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe \u003cem\u003eEf\u003c/em\u003eAvs5 sample was directly applied to a glow-discharged 400-mesh Cu grid (Beijing ZhongJingKeYi Technology) for 30 s. After side blotting, the grid was immediately stained with 2% uranyl formate and then blotted again from the side. Staining was repeated twice with a 30 s incubation with uranyl formate in the final staining step. EM images were collected on a Talos F200C G2 at a nominal magnification of 52,000\u0026times; and at a defocus of about 5 \u0026micro;m.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCryo-EM grid preparation and data acquisition\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e3.5 \u0026mu;L of the \u003cem\u003eEf\u003c/em\u003eAvs5 samples were added to freshly glow-discharged Quantifoil R1.2/1.3 Cu 300 mesh grids. In a Vitrobot (FEI, Inc.), grids were plunge-frozen in liquid ethane after being blotted for 2 s at 16\u0026deg;C with 100% chamber humidity. The grids were imaged using EPU on Titan Krios 300 kV microscopes with a K3 detector. Totally 4318 movies were collected under the defocus ranged from \u0026minus;0.8 to \u0026minus;1.6 \u0026mu;m, and the magnification was 81k in super-resolution mode. 32 frames per movie were collected with a total dose of 40 e\u003csup\u003e\u0026ndash;\u003c/sup\u003e/\u0026Aring;\u003csup\u003e2\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCryo-EM data processing and model building\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe cryo-EM data was processed using CryoSPARC suite v4.5.3\u003csup\u003e31\u003c/sup\u003e. After motion correction, patch CTF estimation and manual exposure curation, 1425 movies were selected. The first set of particles was picked using a filament tracer, and after two-dimensional (2D) classification, good templates were selected for template picking. A total of 1,392,237 particles were picked using a template picker and extracted (box size 360 pixels) from 1,425 accepted micrographs (Fig. S4). Two rounds of 2D classification were conducted and 364,551 particles were picked and used for ab-initio reconstruction (three classes). The largest 3D class (228,053 particles, 62.6%) was selected and refined by homogeneous refinement, non-uniform refinement and CTF refinement to give the final map (global resolution, 3.44 \u0026Aring;). Helical parameters (helical twist and helical rise) were determined by helical refinement job. The initial model of \u003cem\u003eEf\u003c/em\u003eAvs5 wes generated by AlphaFold\u003csup\u003e32\u003c/sup\u003e. The model of ATP was built in Coot\u003csup\u003e33\u003c/sup\u003e. The models of \u003cem\u003eEf\u003c/em\u003eAvs5 were fitted into the cryo-EM density maps using ChimeraX\u003csup\u003e34\u003c/sup\u003e. The model was refined in Coot and Phenix with secondary structure, rotamer and Ramachandran restraints\u003csup\u003e35\u003c/sup\u003e. The Map versus Model FSCs was generated by Phenix (Fig. S4). The statistics of cryo-EM data processing and refinement were listed in Table S1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eIn vitro\u003c/em\u003e NADase activity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe \u003cem\u003eEf\u003c/em\u003eAvs5 NADase activity was evaluated through an \u0026epsilon;NAD\u003csup\u003e+\u003c/sup\u003e-based fluorescence assay, wherein the enzymatic cleavage of the nicotinamide glycosidic bond in \u0026epsilon;NAD\u003csup\u003e+\u003c/sup\u003e results in the formation of \u0026epsilon;ADPR, which subsequently emits a fluorescent signal. In a 96-well black flat-bottom plate, 100 \u0026mu;L reaction mixtures were prepared, each containing 50 \u0026mu;M of \u0026epsilon;NAD\u003csup\u003e+\u003c/sup\u003e (Sigma catalogue No. N2630) and 0.5 \u0026mu;M \u003cem\u003eEf\u003c/em\u003eAvs5 proteins in reaction buffer (20 mM Tris, pH 8.0, 150 mM NaCl, and 5% glycerol, with and without the addition of nucleotides). A mixture containing only the reaction buffer and \u0026epsilon;NAD\u003csup\u003e+\u003c/sup\u003e was used as a control. The reactions were then loaded into a BioTek Synergy H1 microplate reader, and fluorescence intensities were recorded at 37\u0026deg;C every 20 seconds for 15 minutes, employing excitation and emission wavelengths of 310 nm and 410 nm, respectively. All experiments were conducted in triplicate to ensure reproducibility.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eATPase activity\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eATPase activities were evaluated by using Malachite Green Phosphate Detection Kit (Beyotime). Each 200 \u0026mu;L reaction mixture consisted of 5\u0026mu;M \u003cem\u003eEf\u003c/em\u003eAvs5 proteins in a reaction buffer (20\u0026thinsp;mM Tris, pH 8.0, 150 mM NaCl, 5\u0026thinsp;mM MgCl\u003csub\u003e2\u003c/sub\u003e and 0.5 mM ATP). A mixture without the addition of \u003cem\u003eEf\u003c/em\u003eAvs5 proteins served as a control. Following a 30-minute incubation at 37\u0026deg;C, 70 \u0026mu;L of color reagent was added and the mixture was further incubated for another 30 minutes. Subsequently, the absorbance at 620 nm was measured using a multi-detection microplate reader (Tecan Spark). Phosphate levels were then calculated using a standard curve generated with phosphate standards. All experiments were conducted in triplicate to ensure reproducibility.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhylogenetic analysis of Avs5 systems\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe coding sequence of Avs5 was acquired from two sources: firstly, by utilizing the defense finder tool to scan through all bacterial and archaeal genomes housed in the IMG (Integrated Microbial Genomes \u0026amp; Microbiomes) database from November 2023, aiming to identify proteins that encode the Avs5 system\u003csup\u003e36\u003c/sup\u003e; secondly, by leveraging the Foldseek search server to discover proteins (leveraging AlphaFold/UniProt50, ensuring a coverage exceeding 80%) that exhibited a high degree of structural similarity to Avs5\u003csup\u003e37\u003c/sup\u003e. To eliminate redundancy, MMseqs was employed with the specified parameters \u0026lsquo;\u0026ndash;min-seq-id 0.90\u0026rsquo; and \u0026lsquo;-c 0.8\u0026rsquo;, resulting in 263 filtered sequences\u003csup\u003e38\u003c/sup\u003e. These sequences were subsequently aligned using MAFFT, with the parameters set to \u0026apos;--maxiterate 1000 \u0026ndash;globalpair\u0026apos; for optimal alignment\u003csup\u003e39\u003c/sup\u003e. Following alignment, trimAl was applied to refine the alignment by trimming unnecessary regions\u003csup\u003e40\u003c/sup\u003e. The phylogenetic tree was then constructed using IQ-TREE, incorporating the parameters \u0026apos;-nstop 500 -bb 1000 -m LG+F+I+G4\u0026apos; for enhanced accuracy and robustness\u003csup\u003e41\u003c/sup\u003e. Finally, iTOL was utilized for the visualization and annotation of the phylogenetic tree\u003csup\u003e42\u003c/sup\u003e.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by National Natural Science Foundation of China (32370071, 32070040), National Key Research and Development Program of China (2020YFA0907900, 2019YFA0905400).\u003c/p\u003e\n\u003cp\u003eWe thank the staff members of the Electron Microscopy System at the National Facility for Protein Science in Shanghai (NFPS), Shanghai Advanced Research Institute, Chinese Academy of Sciences, China for providing technical support and assistance in data collection. We thank Jing Liu, Xinqiu Guo and Mengyu Yan at the Instrument Analysis Center (IAC) of Shanghai Jiao Tong University for providing assistance in data collection.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJ.Z. and Y.W. designed the experiments and analyzed data.\u003c/p\u003e\n\u003cp\u003eY.W. and Y.T. performed biochemical assays.\u003c/p\u003e\n\u003cp\u003eY.W. and X.Y. conducted cryo-EM data collection.\u003c/p\u003e\n\u003cp\u003eY.W. conducted image processing, atomic model, building and refinement and structural analyses.\u003c/p\u003e\n\u003cp\u003eY.W. and Y.F performed bioinformatics analysis.\u003c/p\u003e\n\u003cp\u003eY.W. and J.Z. wrote the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe atomic coordinates have been deposited in the Protein Data Bank under accession codes 9JAP. The corresponding maps have been deposited in the Electron Microscopy Data Bank under the accession number EMD-61299. Original data for biochemical assays and uncropped gels were deposited to Figshare.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMillman A et al (2022) An expanded arsenal of immune systems that protect bacteria from phages. Cell Host Microbe 30:1556\u0026ndash;1569e1555. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1016/j.chom.2022.09.017\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1016/j.chom.2022.09.017\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVassallo CN, Doering CR, Littlehale ML, Teodoro GIC, Laub MT (2022) A functional selection reveals previously undetected anti-phage defence systems in the E. coli pangenome. 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Despite the established roles of SIR2 and STAND in prokaryotic and eukaryotic immunity, the mechanism underlying their collaboration remains unclear. Here we present cryo-EM structures of \u003cem\u003eEf\u003c/em\u003eAvs5 filaments, elucidating the mechanisms of dimerization, filamentation, filament clustering, ATP binding and NAD\u003csup\u003e+\u003c/sup\u003e hydrolysis, all of which are crucial for anti-phage defense. The SIR2 domains and NODs engage in the intra- and inter-dimer interaction to form an individual filament, while the outward C-terminal domains contribute to bundle formation. Filamentation potentially stabilizes the dimeric SIR2 configuration, thereby activating the NADase activity of \u003cem\u003eEf\u003c/em\u003eAvs5. \u003cem\u003eEf\u003c/em\u003eAvs5 is deficient in the ATPase activity, but elevated ATP concentrations can impede its NADase activity. Together, we uncover the filament assembly of Avs5 as a unique mechanism to switch enzyme activities and perform anti-phage defenses, emphasizing the conserved role of filamentation in immune signaling across diverse life forms.\u003c/p\u003e","manuscriptTitle":"Filamentation activates bacterial NLR-like antiviral protein","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-10-15 08:47:06","doi":"10.21203/rs.3.rs-5156926/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"77db332c-3681-4d75-888c-9aef2ce91395","owner":[],"postedDate":"October 15th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":38551595,"name":"Biological sciences/Biochemistry/Structural biology"},{"id":38551596,"name":"Biological sciences/Microbiology"},{"id":38551597,"name":"Biological sciences/Biochemistry/Enzyme mechanisms"},{"id":38551598,"name":"Biological sciences/Immunology"}],"tags":[],"updatedAt":"2025-03-12T07:09:23+00:00","versionOfRecord":{"articleIdentity":"rs-5156926","link":"https://doi.org/10.1038/s41467-025-57732-7","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2025-03-11 04:00:00","publishedOnDateReadable":"March 11th, 2025"},"versionCreatedAt":"2024-10-15 08:47:06","video":"","vorDoi":"10.1038/s41467-025-57732-7","vorDoiUrl":"https://doi.org/10.1038/s41467-025-57732-7","workflowStages":[]},"version":"v1","identity":"rs-5156926","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5156926","identity":"rs-5156926","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}