Distinct mechanism of the long-B prokaryotic Argonaute-mediated nuclease activation in bacterial immunity

Distinct mechanism of the long-B prokaryotic Argonaute-mediated nuclease activation in bacterial immunity | 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 Research Article Distinct mechanism of the long-B prokaryotic Argonaute-mediated nuclease activation in bacterial immunity Min Liu, Qin Yu, Xiaoruo Zhang, Dujuan Shi, Qiong Xing, Fengtao Huang, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8163682/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Prokaryotic Argonautes (pAgos) are widely distributed and provide immunity against invading DNA. Based on their domain architecture, pAgos are classified into three major groups: long-A, long-B, and short pAgos. Among them, long-B pAgos remain the least understood subgroup. Here, we show that a long-B pAgo-nuclease system ( Ec BPAN) from Escherichia coli provides the first clear evidence of anti-phage activity in long-B pAgo systems. By combining structural determination, biochemical analyses, and in vivo phage-resistance assays, we elucidated the activation mechanism of Ec BPAN. We found that RNA-guided Ec Ago recognizes target DNA and subsequently recruits the autoinhibited dimer of its associated nuclease ( Ec bAgaN) to form an unprecedented 8:8 pAgo-nuclease complex with robust nonspecific DNase activity. The cryo-EM structure of the activated complex revealed a distinctive bowl-shaped architecture in which the C-terminal nuclease domains of Ec bAgaN form an active octamer, while the N-terminal domains engage four Ec Ago-guide RNA-target DNA ternary dimers in an interleaved manner, thereby relieving autoinhibition. Together, these findings provide the first mechanistic insights into a long-B pAgo defense system and reveal a mode of immune activation fundamentally distinct from those of long-A and short pAgo systems. long-B pAgo anti-phage activity EcBPAN system defense mechanism Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Argonaute proteins (Agos) are programmable enzymes that utilize nucleic acid guides for target recognition, and are widely distributed across all kingdoms of life [1] . Eukaryotic Agos (eAgos) initially characterized as the core constituents of RNA-induced silencing complex (RISC) and mediating RNA-relate cellular processes [2, 3] . In contrast, prokaryotic Agos (pAgos) serve as innate immune effectors that protect cells against invading plasmids and phages [4-6] . Compared with eAgos, pAgos exhibit more diversity in sequence and domain compositions. Based on domain architectures, pAgos can be classified into three distinct groups: long-A, long-B and short pAgos [7] . Long-A and long-B pAgos share significantly structural homology with eAgos and contain all four canonical domains: N, PAZ (PIWI–Argonaute–Zwille), MID (Middle) and PIWI (P-element Induced Wimpy Testis) domains, while short pAgos only consist of a MID domain and a catalytically inactive PIWI domain [7, 8] . Currently, the most extensively studied long-A pAgos utilize RNA or DNA guides to cleave target RNA or DNA [9-11] . Several long-A pAgos, such as Tt Ago from Thermus thermophilus and Cb Ago from Clostridium butyricum , have been shown to provide immunity by DNA-guided DNA interference [4, 12, 13] . Moreover, the structures of representative long-A pAgos have been determined [14, 15] . Approximately 60% of pAgos are classified as highly divergent short pAgos, which lack nuclease activity due to the absence of the DEDX catalytic tetrad in their PIWI domain [7, 8] . Recent studies have revealed that short pAgos from many bacteria constitute defense systems in association with effector proteins composed of an N-terminal enzymatic domain and a C-terminal APAZ (analog of PAZ) domain [7, 16, 17] . The APAZ domain is homologous to the N domain rather than PAZ domain of long pAgos. These characterized N-terminal enzymatic domains include the NADase domains TIR (Toll-interleukin-1 receptor) from SPARTA system and SIR2 (sirtuin 2) from SPARSA system, as well as nuclease domain DREN (DNA and RNA effector nuclease) from SPARDA system [17-21] . Unlike long-A pAgos, which directly recognize and cleave target nucleic acids, SPARTA and SPARSA systems activate their NADase domains upon target DNA recognition. This leads to the hydrolysis of cellular nicotinamide adenine dinucleotide (NAD⁺) and subsequently triggers cell death, thereby preventing viral infection and plasmids invasion [18, 22, 23] . For SPARDA system, target DNA binding activates associated nuclease, leading to degradation of cellular DNA and subsequent cell death or dormancy [24] . Structural and biochemical studies have provided a more in-depth understanding of the working mechanism for guide and target recognition and activation of short pAgos-associated systems [21, 24-26] . However, long-B pAgos have been studied much less extensively. Although their structural compositions are similar to that of long-A pAgos, long-B pAgos lack nuclease activity. Recent studies have shown that the gene neighborhood of long-B pAgos encode pAgo-associated proteins, which can be functionally connected with their corresponding long-B pAgos [7, 27] . These pAgo-associated proteins include nucleases from BPAN (long-B prokaryotic Argonaute nuclease) systems, SIR2-like proteins from BPAS (long-B prokaryotic Argonaute Sir2) systems and trans-membrane proteins from BPAM (long-B prokaryotic Argonaute trans-membrane) systems [27] . The study has shown that these systems can defend against invading plasmids but have not been demonstrated to provide detectable protection against phage infection. Moreover, the mechanisms of action of the long-B pAgos-associated systems remain elusive. In this study, we found that the Ec BPAN system from Escherichia coli ( E. coli ) provides strong protection against bacteriophage. We further discovered that RNA-guided Ec Ago recognizes target DNA and subsequently recruits Ec bAgaN to form an 8:8 complex with activated nuclease activity, which nonspecifically degrades double-stranded DNA (dsDNA). Our work provides the first mechanistic insights into a long-B pAgo-associated defense system. Results The anti-phage function and nuclease activation of Ec BPAN system Ec BPAN system consists of two proteins, Ec Ago and the nuclease Ec bAgaN (Fig. 1a). The previous study has demonstrated that the Ec BPAN system can act as a prokaryotic defense system against plasmids via abortive infection [27] . However, whether the Ec BPAN system can provide defense against phages remains to be explored. In this study, we tested the resistance of the Ec BPAN system against six E. coli phages, including T7, T4, T5, λ, lab02 and lab05 (Fig. 1b). We measured the efficiency of plaque formation (EOP) of these phages on E. coli cells expressing the Ec BPAN system. The results showed that the Ec BPAN system provided significant protection against phage lab02 (~10 000 fold), and decreased the plaque size formed by phage lab05, while showing no reduction in plaque formation for the other five phages (Supplementary information, Fig. S1). We further examined the anti-phage activity of Ec Ago and Ec bAgaN individually and found that neither protein alone exhibited detectable anti-phage activity, indicating that both components are required for the full defensive function of the Ec BPAN system (Fig. 1c). To further validate the cooperative function of these two proteins, Ec Ago and Ec bAgaN were expressed and purified for in vitro functional assays. Consistent with the previous findings [27] , efficient DNA cleavage was observed only when Ec Ago, Ec bAgaN, guide RNA and target DNA were all present in the reaction mixture (Fig. 1d), indicating that the interaction between Ec Ago and Ec bAgaN upon RNA-guided target recognition. Overall structure of the Ec BPAN complex bound to guide RNA and target DNA To further validate the interaction between Ec Ago and Ec bAgaN, we incubated Ec bAgaN with Ec Ago alone, Ec Ago loaded with guide RNA, Ec Ago bound to target DNA, or Ec Ago preloaded with both guide RNA and target DNA, followed by size-exclusion chromatography (SEC) analysis. The results showed that only Ec Ago preloaded with both guide RNA and target DNA was able to interact with Ec bAgaN to form a larger complex (Fig. 2a and Supplementary information, Fig. S2). The complex fraction was collected, and its structure was resolved by cryo-electron microscopy (cryo-EM) at a resolution of 3.27 Å (Fig. 2b, c and Supplementary information, Fig. S3 and Table S1). The well-defined cryo-EM density allowed us to build an atomic model with high confidence. The overall structure of Ec BPAN complex shows a distinct “bowl-shaped’’ architecture, composed of the Ec Ago-guide RNA-target DNA complex and Ec bAgaN in an 8:8 stoichiometric ratio. Within this arrangement, eight Ec Ago ternary complex molecules are arranged in a circular pattern to form the body of the bowl, while eight Ec bAgaN molecules are interconnected to constitute the bottom of the bowl. Interaction between Ec Ago and guide RNA-target DNA duplex The overall structure of Ec Ago is homologous to that of Rs Ago (Cα RMSD=2.173 Å) (Supplementary information, Fig. S4a). Based on the structure assembly, Ec Ago is divided into N domain (1-92), linker 1 (92-155), PAZ domain (155-218), linker 2 (218-308), MID domain (308-484) and PIWI domain (484-731) (Fig. 3a). Ec Ago adopts conserved domain architecture found in other Ago proteins [14] , however, its PAZ domain is significantly smaller than the canonical PAZ domain and comprises approximately 60 residues that adopts a hairpin-like structure (Supplementary information, Fig. S4b). The guide RNA-target DNA duplex is stably positioned between the N-PAZ and MID-PIWI lobes (Fig. 3b). Notably, the 5’-end of guide RNA remains unpaired with 3’-end of target DNA. The nucleotides at positions 20-21 of guide RNA strand, as well as the terminal nucleotide C21’ of target DNA are missing. Meanwhile, positions 2-19 of both strands engage in canonical Watson-Crick base pairing (Fig. 3c). The structure of Ec Ago bound guide RNA-target DNA complex reveals a network of intermolecular hydrogen-bond interactions between Ec Ago and guide RNA-target DNA duplex, particularly around 5’ end of the guide RNA and nucleotides at positions 2-13 of guide RNA-target DNA duplex (Fig. 3c). In contrast, there is almost no intermolecular interaction between nucleotides at positions 14-20 of guide RNA-target DNA duplex and Ec Ago. The 5’-phosphate group of guide strand is stably anchored within MID pocket and forms direct hydrogen bonds with conserved four-amino acids motif X-K-Q-K (Y426, K430, Q441, K469) and D442 from MID domain (Fig. 3d). Furthermore, the 5’-phosphate group of guide RNA coordinates to a Mg 2+ ion, which is simultaneously coordinated by the carboxylate group of C-terminal residue L731 of Ec Ago (Fig. 3d). To examine the role of the 5’-phosphate group of guide RNA in mediating interactions with MID domain, we performed an in vitro gnomic DNA degradation assay. Our results showed that the removal of the 5’-phosphate group from guide RNA significantly reduced DNA cleavage activity (Fig. 3e), suggesting that Ec Ago preferentially loads 5’- phosphorylated guide RNA for target DNA recognition. The nucleotides A1 of guide RNA and T1’ of target DNA are each inserted into their respective binding pockets (Fig. 3d, f). The nucleotide A1 of guide RNA strand is stabilized by stacking against Y426 in the MID domain, a conserved interaction observed among Argonaute proteins [28] . Alanine substitution of Y426 impaired the cleavage activity (Fig. 3g). Furthermore, the nucleotide A1 forms a hydrogen bond with side chain of residue N424 situated on the ‘‘specificity loops’’, a region known to discriminate different terminal nucleotides [9, 29] . To further probe the specificity of first RNA-DNA base pair recognized by Ec Ago, we assessed the cleavage activity using different RNA-DNA base pair combinations at this position. The results showed that substituting A·T with U·A reduced cleavage activity by approximately 30%, while replacing it with G·C or C·G led to a more pronounced reduction of about 60% in genomic DNA cleavage (Fig. 3e), suggesting that Ec Ago exhibits a bias toward A at the position 1 of guide RNA. The terminal nucleotide T1’ of the target DNA inserts into a hydrophilic pocket formed by E309, Y358, Y457, N460 and Y687, and forms polar contact with the side chain of N460 from the MID domain. Alanine substitution of N460 impaired the cleavage activity (Fig. 3g). To determine whether Ec Ago has a preference for thymine at the position 1’ of target DNA, we measured the genomic DNA cleavage activity after substituting T1’ with A’, or G’, or C’ in the target DNA. The results showed that the replacement of T1’ with any other nucleotides had no significant effect on cleavage activity (Fig. 3e). Moreover, similar interaction patterns involving N460 were observed when the 3’-terminal nucleotide was A’, or G’, or C’ (Supplementary information, Fig. S5a), further supporting that Ec Ago lacks a strong base preference at the position 1’ of target DNA. Mismatches between guide and target strands have been demonstrated to impair the cleavage efficiency in several pAgos [20, 30, 31] . In this study, to evaluate the effect of mismatches on cleavage activity of the Ec BPAN system, we designed a series of target DNA, each containing a single mismatched nucleotide at position 2-21 relative to guide RNA (Supplementary information Table S2) and tested their cleavage reaction by Ec BPAN system. As shown in Supplementary information, Fig. S5b, single mismatches at positions 2-10, 12, and the 3’-end of the guide-target duplex exhibited minimal impact on cleavage efficiency. In contrast, single mismatches at positions 11, or 13-16 impaired the cleavage activity. Notably, the cleavage activity of Ec BPAN system is not affected by mismatches in the 5’-seed region (position 2-8) but is impaired by mismatches in the 3’ supplementary guide region (position 13-16). Positioning of putative catalytic pocket residues in Ec Ago The cleavage-competent prokaryotic Agos contain a catalytic pocket with a conserved DEDD tetrad in the PIWI domain. Ec Ago exhibits two substitutions in the DEDD tetrad (G492, E532, Q568, D700 as shown in Supplementary information, Fig. S6a) and Q568 forms a hydrogen bond with phosphate group of A10’ of target DNA (Fig. 3c). Ago-mediated cleavage requires insertion of the conserved “glutamate finger” into the catalytic pocket to form the functional DEDX tetrad [32] . In Tt Ago ( Thermus thermophilus ), a glutamate residue (E512) undergoes conformational transition from unplugged state in complex with guide strand to plugged-in state in complex with guide and target strands (Supplementary information, Fig. S6b, c), resulting in the cleavage of target [11] . The structure of guide RNA-target DNA-bound Ec Ago in the quaternary complex shows that E532, the glutamate residue corresponding to E512 ( Tt Ago) and E569 ( Rs Ago) (Supplementary information, Fig. S6), is located away from the catalytic pocket and adopts an unplugged conformation. This structural feature probably explains that Ec Ago complex with guide RNA and target DNA lacks of DNA cleavage activity (Fig. 2d). Structural basis of Ec bAgaN octamer Ec bAgaN is annotated as a member of the RecB-like protein family. Structural analysis reveals that Ec bAgaN comprises two distinct domains connected by a flexible, unstructured linker (Fig. 4a). Although Ec bAgaN shares low sequence identity with known structures, its C-terminal domain is highly identical to structurally characterized PD-(D/E)XK nucleases. The C-terminal domain exhibits a conserved α-β-α fold architecture, featuring a four-stranded mixed β-sheet sandwiched by two α-helices on both sides. The nuclease domain harbors a conserved D-EVK motif (D298, E309, V310 and K311) (Supplementary information, Fig. S7a), which is essential for the nuclease activity [27] . The previous study [27] and our SEC analysis both showed that Ec bAgaN forms a dimer in solution (Supplementary information, Fig. S2). Consistently, the Ec bAgaN dimeric state was observed in our structure. Within Ec bAgaN dimer, two copies stacked against each other by two interfaces: Interface I and Interface II, accompanied by cross-linking between the linker regions (Fig. 4a). At the Interface I, hydrogen bonds are formed between residues D14 and Y15’, as well as between N212 and Q189’ (residues marked with a prime [’] denote those from the opposing protomer). At the Interface II, a more extensive hydrogen-bonding network is observed. Specifically, Q259, H265, N268, D283 and Y271 form a hydrogen-bond interactions with W258’, E245’, T243’ R241’, and K240’. Alanine substitution of D14/Y15/Q189 impaired the cleavage activity. A multi-site alanine substitution mutant (designated M1: K240A, R241A, T243A, E245A, W258A, Q259A, H265A, N268A, Y271A and D283A) almost completely abolished the cleavage activity (Fig. 4b), highlighting the critical role of these interfaces in the Ec bAgaN dimer for the activation of Ec BPAN system. Furthermore, four Ec bAgaN dimer units further assemble into an octamer (Fig. 4c). Hydrogen-bond interactions are observed between the C-terminal regions of these dimers, involving the following residue pairs: I281 and K361, Q288 and Y340, Q292 and E358, Q357 and S382, N286 pair and S332 pair. Alanine substitution of S332/Y340, N286/Q288/Q292 and Q357/E358/K361 reduced the cleavage activity (Fig. 4b). Notably, the D14A/Y15A/Q189A and M1 mutations from the Ec bAgaN dimer interface and N286A/Q288A/Q292A mutation from the Ec bAgaN octamer interface disrupted the formation of the Ec BPAN 8:8 complex, resulting in smaller complexes compared to the wild-type Ec BPAN system (Fig. 4d). Taken together, these findings highlight the important role of Ec bAgaN- Ec bAgaN interactions in Ec BPAN 8:8 complex and demonstrate that the assembly of Ec BPAN 8:8 complex is indispensable for the activation of its nuclease activity. Interaction between Ec Ago and Ec bAgaN is essential for the formation of the Ec BPAN 8:8 complex Structural analysis uncovers that guide-directed target recognition by Ec Ago recruits the nuclease Ec bAgaN via a protruding loop (segments 81-90) from N-terminal domain of Ec bAgaN. This loop inserts into the interface between MID and PIWI domains of Ec Ago, forming an extensive hydrogen-bonding network (Fig. 5a). Specifically, residues A82, D86 and P89 of Ec bAgaN form hydrogen bonds with R552, S524 and Y318 of Ec Ago, respectively. In addition, hydrogen bonds also are observed between R77, S94, H134 of Ec bAgaN and N554, D559, R321 and F323 of Ec Ago. To validate the functional significance of this interface, we introduced alanine substitutions at these critical residues. These mutations lead to strong reduction in defense against invading DNA as assessed by bacteriophage infection assay (Fig. 5b). Consistently, the same mutations also reduced the genomic DNA cleavage activity in vitro (Fig. 5c). To investigate the role of interaction between Ec Ago and Ec bAgaN in the formation of the Ec BPAN 8:8 complex, we replaced this protruding loop of Ec bAgaN with a (GGGGS)₂ linker and subsequently performed in vitro genomic DNA cleavage activity assay and SEC analysis. The results confirmed that the substitution of this loop with (GGGGS)₂ linker impaired the cleavage activity and prevented the assembly of the Ec BPAN complex (Fig. 5c, d). Thus, our structural and biochemical data demonstrate that the specific Ec Ago- Ec bAgaN interaction, mediated by the Ec bAgaN loop, is a critical prerequisite for the formation of stable Ec BPAN 8:8 complex and the consequent activation of its nuclease function. Dimerization of the Ec Ago ternary complex upon Ec bAgaN recruitment is essential for Ec BPAN activation In the Ec BPAN 8:8 complex, eight Ec bAgaN molecules assemble into an octamer by forming four Ec bAgaN dimers, whereas the eight Ec Ago ternary complexes bound with guide RNA-target DNA duplex organize into four independent Ec Ago ternary dimers (Fig. 5e). Each Ec Ago molecule interacts specifically with one Ec bAgaN molecule. Interestingly, within each Ec Ago ternary dimer, one Ec Ago protomer binds to one protomer of an Ec bAgaN dimer, while the other Ec Ago protomer interacts with an adjacent Ec bAgaN dimer (Supplementary information, Fig. S7b). This cross-linking pattern connects the four Ec Ago dimers through the Ec bAgaN octamer, ultimately resulting in a globally stable Ec BPAN 8:8 complex. At the intermolecular interface of Ec Ago ternary dimer, the carboxylate group of D400 from the MID domain respectively forms hydrogen bonds with the side chains of R552 and imidazole ring of H726 from the PIWI domain of another Ec Ago molecule (Fig. 5f). The same interaction is also observed in the another MID-PIWI interface in the Ec Ago ternary dimer due to symmetry. Mutations of the interface residues impaired DNA cleavage activity (Fig. 5c), suggesting the dimerization of Ec Ago ternary complex is indispensable for activating the DNase activity of Ec BPAN system. Working model of the EcBPAN system Based on our findings and the previous study [27] , we propose a working model for Ec BPAN system (Fig. 6). Upon phage or plasmid invasion, Ec Ago binds to the transcripts derived from the invader and subsequently recognizes the complementary target DNA. This recognition induces a conformational change in Ec Ago, enabling the recruitment of an Ec bAgaN dimer and further assembly of an active Ec BPAN 8:8 complex with nuclease activity. The process involves target DNA recognition, Ec Ago dimerization, and Ec bAgaN octamerization. The activated Ec BPAN complex degrades the genome DNA, leading to host cell death and thereby conferring population-level immunity against the invading elements. Discussion Long-B pAgos are often associated with different types of effector proteins, including nuclease, NADase and transmembrane protein, and have been shown to confer bacterial immunity against invading plasmids [27] . Here, we characterized the anti-phages activity of bacterial long-B pAgo- Ec bAgaN nuclease ( Ec BPAN) system and determined its structure in complex with guide and target nucleic acids, thereby revealing the molecular mechanism underlying Ec BPAN system activation. Previous studies have primarily focused on elucidating the mechanisms of long-A and short pAgos. Long-A pAgos possess intrinsic nuclease activity and primarily function as single proteins against invading DNA. Structural studies have shown that most long-A pAgos act as monomers upon target recognition, while a few dimerize for activation [28] . In contrast, short pAgos lack catalytic activity and usually associate with APAZ-containing effector proteins to form heterodimers. Upon recognition of target nucleic acids, these heterodimers further oligomerize or assemble into filamentous structures to achieve activation [19, 21, 26, 33, 34] . Long-B pAgos share a similar overall domain architecture with long-A pAgos but are catalytically inactive. However, unlike the short pAgo systems, the associated effector proteins in long-B pAgo systems lack an APAZ domain and cannot directly form stable heterodimers with the pAgos. In this study, we uncovered the molecular mechanism of a long-B pAgo defense system for the first time. We found that upon target DNA recognition, long-B pAgo recruits its cognate nuclease dimer to assemble into a bowl-shaped 8:8 supramolecular complex, leading to activation of its nuclease activity. The action of the mechanism of this system is distinct from the mechanisms of both long-A and short pAgo systems and expands our understanding of the functional diversity of pAgo-mediated defense systems. Recently, a small subset of long-A pAgo systems has been found to contain an associated Cas4 family nuclease, which cooperates with the pAgo protein to defend against invading DNA [35] . In addition, the newly reported DdmDE defense system features a catalytically inactive pAgo-like protein, DdmE, that recognizes target DNA and recruits a helicase-nuclease, DdmD, to eliminate invading plasmids [36-38] . Although these recently discovered pAgo-based defense mechanisms differ from the long-B pAgo system characterized in this study, they collectively highlight the mechanistic diversity of pAgo-mediated strategies against foreign DNA elements. In our study, the long-B pAgo interacts with the N-terminal domain of its associated nuclease, a domain that is not found in other effector proteins of long-B pAgo systems, such as NADase and transmembrane protein. This is distinct from the APAZ domain found in diverse effector partners of short pAgo systems, suggesting that the activation and regulation of other long-B pAgo systems may differ from the one in this study. Future investigations into these long-B pAgo systems may bring new insights into pAgo-based defense mechanism. Ec bAgaN contains an N-terminal α-helical domain and a C-terminal PD-(D/E)XK nuclease domain. Under normal physiological conditions, Ec bAgaN exists as an autoinhibited dimer. Upon target DNA recognition by Ec Ago, the N-terminal domain of Ec bAgaN interacts with Ec Ago, which in turn promotes octamerization of its C-terminal nuclease domains and subsequent activation. Notably, no direct interaction is observed between the N-terminal and C-terminal domains in the active state. The N-terminal domain, which has no homologs in any structurally characterized proteins, appears to have specifically evolved within the long-B pAgo systems as a sophisticated molecular “switch” to regulate the activity of the C-terminal nuclease domain. Under normal physiological conditions, this N-terminal domain likely maintains the nuclease in an inactive conformation by inhibiting octamer formation. The interaction between the N-terminal domain and Ec Ago upon target recognition releases this autoinhibition, allowing the C-terminal nuclease domain to assemble into an active octamer. Attempts to predict the dimeric structure of Ec bAgaN using AlphaFold3 were unsuccessful. Determination of the Ec bAgaN dimer structure in the future will be critical for elucidating how the N-terminal domain regulates the activation of the C-terminal nuclease domain. Materials and Methods Bacterial strains and phages E. coli strains DH5α and BL21 (DE3) were used for plasmid construction and protein expression, respectively. E. coli C600, which kindly provided by Professor Ruichao Li [39] , and E. coli MG1655 were used to assess the anti-phage activity of the Ec BPAN system. All bacterial strains were cultured in LB medium. Phages T4, T5, and T7 were kindly provided by Professor Bin Zhu [40] . Phage λ was kindly provided by Professor Lianrong Wang [41] . Two additional phages, lab02 and lab05, were isolated from a laboratory sink. Lab02 shares 98.9% nucleotide identity with E. coli phage TR2 (GenBank accession: OP251154), and lab05 shares 99.9% identity with E. coli phage phiD2-2 (GenBank accession: OR861627). Plasmid construction and mutagenesis The full-length Ec Ago and Ec bAgaN were respectively synthesized by GeneGreat Biological Engineering Co. Ltd. (Wuhan, China) and inserted into pET28a vector for protein expression. For site-directed mutagenesis, the plasmid fragments (Supplementary information, Table S3) were constructed by inverse polymerase chain reaction using the primers with mutant sites. All constructs were verified by DNA sequencing. Phage plaque assays The Ec BPAN operon was cloned into the pQE80L vector under the control of T5 promoter. For assays examining Ec BPAN activity against different phages, E. coli MG1655 strains carrying plasmids expressing Ec BPAN systems, or the empty vector control were grown overnight at 37℃ in LB medium supplemented with 100 μg/mL ampicillin. Phage resistance was assessed using a double-layer agar method, with 1.5% LB agar as the bottom layer and 0.5% LB agar containing bacterial cells as the top layer. Ec BPAN expression was induced with 0.01 mM IPTG. Serial 10-fold dilutions of phages were spotted onto the plates and incubated at 37℃ for the indicated times. For assays evaluating the anti-phage activity of Ec BPAN mutants against phage lab02, E. coli C600 strains were used to express Ec BPAN and its mutants upon induction with 0.01 mM IPTG. Serially diluted lab02 phage samples were spotted onto the plates and incubated at 37℃ for ~7 h. Protein expression and purification The recombinant plasmids used for the purification of Ec Ago and Ec bAgaN and their variants were transformed into E. coli BL21 (DE3) cells, plated onto agar plate with 50 μg/mL kanamycin. Cells were added into LB medium and grown to an optical density reached 0.6 at 600 nm, and then induced by adding 0.3 mM isopropyl- β -D-thiogalactopyranoside at 16 °C for 18 h. Cells were harvested by centrifugation (5000 rpm for 8 min at room temperature) and then resuspended in buffer A (25 mM Tris, 300 mM NaCl, pH 7.5, 20 mM imidazole). The lysate was clarified by centrifugation (12,000 rpm for 1 h at 4 °C) after disruption by a French press with 1000 bar (JuNeng Nano & Bio Technology Co. Ltd., Guangzhou, China). The His-tagged protein was loaded onto a Ni-NTA column pre-equilibrated in buffer A and eluted in buffer B (25 mM Tris, 300 mM NaCl, pH 7.5, 200 mM imidazole). Eluted protein was collected and concentrated in buffer C (25 mM Tris-HCl, 300 mM NaCl, pH 7.5). For Ec Ago, concentrated protein was loaded onto 5-mL HiTrap Heparin HP column (Cytiva) pre-equilibrated in buffer D (25 mM Tris-HCl, pH 7.5) and eluted with a linear gradient of buffer E (25 mM Tris-HCl, 1 M NaCl, pH 7.5). Eluted Ec Ago protein was pooled and concentrated in buffer C. The purity of Ec Ago and Ec bAgaN were confirmed by SDS-PAGE. In vitro activity assays The oligonucleotides sequences are listed in Supplementary information, Table S2. The genomic DNA were extracted from E. coli DH5a by Bacteria Genomic DNA Kit (Tsingke, Wuhan). Unless otherwise specified, 500 nM Ec Ago or its mutant was first incubated with 500 nM guide RNA at 37℃ for 15 min in buffer C with 5 mM MgCl 2 and 5 mM MnCl 2 . Then 500 nM target DNA was added and incubated at 37℃ for additional 15 min. Finally, ~300 ng genomic DNA and 50 nM Ec bAgaN or its mutant were added into the reaction mixtures, followed by incubation at 37℃ for 1 h. The samples were treated with 2 mg/mL proteinase K and stopped by mixing loading dye and analyzed by agarose gel electrophoresis. Ec BPAN complex assembly Guide and target nucleic acids were dissolved with buffer C. Ec Ago was first incubated with 21 nt long 5’-phosphorylated guide RNA in buffer C containing 5 mM MgCl 2 for 15 min, then 21 nt long target DNA was added and incubated for 15 min. Finally, the Ec bAgaN was supplemented into the reaction mixture and then incubated for 15 min. The molar ratio of Ec Ago, guide, target and Ec bAgaN was 1:1:1:1. All incubations were performed at room temperature. The acquired complex was applied to a size exclusion column (Superose 6 increase 10/300 GL, Cytiva) pre-equilibrated with 25 mM Tris-HCl, 150 mM NaCl, pH 7.5, 5 mM MgCl 2 and 1 mM DTT. The protein fractions at different elution time were characterized by SDS-PAGE and collected for subsequent structural analysis. Cryo-EM data acquisition and processing To prepare cryo-EM samples, 4 µL sample of Ec BPAN complex was applied onto glow-discharged holey carbon grids (Quantifoil Cu R1.2/1.3; 300 mesh). The grids were blotted for 5 seconds with force 2 in 100% humidity at 4 ℃, and then plunged into liquid ethane using a Vitrobot. Cryo-EM data collection was operated at 300 kV Titan Krios electron microscope equipped with a K3 summit electron direct detector. Micrographs were recorded in super-resolution mode at 105 000× magnification (0.425 Å/pixel). Each stack of 40 frames was exposed for 2.5 s with a defocus range from -1.0 µm to -2.2 μm, and the total dose of about 30 e − / Å 2 . 21 538 movies were collected in total in three individual times. All the cryo-EM movies were processed using CryoSPARC v4.2.1 [42] . The basic workflow was described in Supplementary information, Fig. S3. In brief, the collected movies were motion-corrected with Patch Motion Correction and then subjected to contrast transfer function (CTF) estimation with Patch CTF. Micrograph curation was applied to exclude frames with CTF fit >4 Å or ice contamination. Subsequent Particle picking was performed with Blob/Template-based particle peaking and followed by several rounds of Iterative 2D classification, ab initio reconstruction, and heterogeneous refinement. The final step was non-uniform refinement with imposed C2 symmetry. Model building and refinement Initial models generated by AlphaFold2 were rigid-body docked into cryo-EM maps using ChimeraX v1.6.1 [43] . Manual rebuilding in Coot v0.9.8 [44] resolved poorly fitted regions, followed by iterative real-space refinement in Phenix v1.20.1 [45] (global minimization, rotamer optimization). Validation included: MolProbity (Ramachandran outliers <0.5%; clash score 3); Map-model FSC (0.143 cutoff). Final structure was visualized in PyMOL v2.5.2 and ChimeraX. Refinement statistics are detailed in Supplementary information, Table S1 . Declarations Acknowledgements This work was supported by Project of Technological Innovation Plan in Hubei Province (2024BCA001); Natural Science Foundation of Wuhan City (2024040701010046). Author contributions M.L. and L.M. supervised the study. M.L. and Q.Y. prepared the samples and performed the activity assays. X.Z. and F.H. performed the phage plaque assays. M.L., Q.X. and D.S. collected and processed the cryo-EM data and built the atomic model. M.L. wrote the manuscript. L.M., F.H. and Q.X. reviewed and edited the manuscript. All authors analyzed the data. C ompeting interest s The authors claim no conflict of interest. References Swarts, D.C., et al. The evolutionary journey of Argonaute proteins. Nat. Struct. Mol. Biol 21 , 743-753 (2014). Ozata, D.M., Gainetdinov, I., Zoch, A., O'Carroll, D. & Zamore, P.D. PIWI-interacting RNAs: small RNAs with big functions. Nat. Rev. Genet. 20 , 89-108 (2019). Ding, S.W. RNA-based antiviral immunity. Nat. Rev. Immunol. 10 , 632-644 (2010). Jolly, S.M., et al. Thermus thermophilus Argonaute functions in the completion of DNA replication. Cell 182 , 1545-1559 (2020). Olina, A., et al. Genome-wide DNA sampling by Ago nuclease from the cyanobacterium Synechococcus elongatus . RNA Biol 17 , 677-688 (2020). Swarts, D.C., et al. DNA-guided DNA interference by a prokaryotic Argonaute. 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The nuclease-associated short prokaryotic Argonaute system nonspecifically degrades DNA upon activation by target recognition. Nucleic Acids Res. 52 , 844-855 (2024). Wang, F., Xu, H., Zhang, C., Xue, J. & Li, Z. Target DNA-induced filament formation and nuclease activation of SPARDA complex. Cell Res. (2025). Zaremba, M., et al. Short prokaryotic Argonautes provide defence against incoming mobile genetic elements through NAD(+) depletion. Nat. Microbiol. 7 , 1857-1869 (2022). Garb, J., et al. Multiple phage resistance systems inhibit infection via SIR2-dependent NAD(+) depletion. Nat. Microbiol. 7 , 1849-1856 (2022). Prostova, M., et al. DNA-targeting short Argonautes complex with effector proteins for collateral nuclease activity and bacterial population immunity. Nat. Microbiol. 9 , 1368-1381 (2024). Wang, C., Shen, Z., Yang, X.-Y. & Fu, T.-M. Structures and functions of short argonautes. RNA Biol. 21 , 883-889 (2024). Wang, X., et al. Structural insights into mechanisms of Argonaute protein-associated NADase activation in bacterial immunity. Cell Res. 33 , 699-711 (2023). Song, X., et al. Catalytically inactive long prokaryotic Argonaute systems employ distinct effectors to confer immunity via abortive infection. Nat. Commun. 14 , 6970 (2023). Wang, L., et al. Molecular mechanism for target recognition, dimerization, and activation of Pyrococcus furiosus Argonaute. Mol. Cell 84 , 675-686 (2024). Frank, F., Sonenberg, N. & Nagar, B. Structural basis for 5'-nucleotide base-specific recognition of guide RNA by human AGO2. Nature 465 , 818-822 (2010). Kuzmenko, A., Yudin, D., Ryazansky, S., Kulbachinskiy, A. & Aravin, A.A. Programmable DNA cleavage by Ago nucleases from mesophilic bacteria Clostridium butyricum and Limnothrix rosea . Nucleic Acids Res. 47 , 5822-5836 (2019). Sheng, G., et al. Structure/cleavage-based insights into helical perturbations at bulge sites within T. thermophilus Argonaute silencing complexes. Nucleic Acids Res. 45 , 9149-9163 (2017). Nakanishi, K., Weinberg, D.E., Bartel, D.P. & Patel, D.J. Structure of yeast Argonaute with guide RNA. Nature 486 , 368-374 (2012). Zhang, J.T., Wei, X.Y., Cui, N., Tian, R. & Jia, N. Target ssDNA activates the NADase activity of prokaryotic SPARTA immune system. Nat Chem Biol 20 , 503-511 (2024). Cui, N., et al. Tetramerization-dependent activation of the Sir2-associated short prokaryotic Argonaute immune system. Nat. Commun. 15 , (2024). Bobadilla Ugarte, P., et al. Cyanobacterial Argonautes and Cas4 family nucleases cooperate to interfere with invading DNA. Mol. Cell 85 , 1920-1937 (2025). Bravo, J.P.K., Ramos, D.A., Fregoso Ocampo, R., Ingram, C. & Taylor, D.W. Plasmid targeting and destruction by the DdmDE bacterial defence system. Nature 630 , 961-967 (2024). Loeff, L., et al. Molecular mechanism of plasmid elimination by the DdmDE defense system. Science 385 , 188-194 (2024). Yang, X.Y., Shen, Z., Wang, C., Nakanishi, K. & Fu, T.M. DdmDE eliminates plasmid invasion by DNA-guided DNA targeting. Cell 187 , 5253-5266 (2024). Liu, Z., et al. Adaptive evolution of plasmid and chromosome contributes to the fitness of a blaNDM-bearing cointegrate plasmid in Escherichia coli . Isme j 18 , (2024). Cheng, R., et al. Prokaryotic Gabija complex senses and executes nucleotide depletion and DNA cleavage for antiviral defense. Cell Host Microbe 31 , 1331-1344 (2023). Zou, X., et al. Systematic strategies for developing phage resistant Escherichia coli strains. Nat. Commun. 13 , 4491 (2022). Punjani, A., Rubinstein, J.L., Fleet, D.J. & Brubaker, M.A. cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nat. methods 14 , 290-296 (2017). Pettersen, E.F., et al. UCSF ChimeraX: Structure visualization for researchers, educators, and developers. Protein Sci. 30 , 70-82 (2021). Emsley, P., Lohkamp, B., Scott, W.G. & Cowtan, K. Features and development of Coot. Acta Crystallogr D Biol Crystallogr 66 , 486-501 (2010). Afonine, P.V., et al. Real-space refinement in PHENIX for cryo-EM and crystallography. Acta Crystallogr D Biol Crystallogr 74 , 531-544 (2018). Additional Declarations The authors declare no competing interests. Supplementary Files Supplementaryinformation.docx Cite Share Download PDF Status: Posted 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-8163682","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":548113715,"identity":"5f6ef40b-0080-429d-a82f-94da767fd939","order_by":0,"name":"Min Liu","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Min","middleName":"","lastName":"Liu","suffix":""},{"id":548113716,"identity":"2f371d3b-9d21-4f29-b8f0-35284bab41f9","order_by":1,"name":"Qin 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system.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) Schematic representation of the BPAN system from \u003cem\u003eE. coli\u003c/em\u003e CAP29.\u003c/p\u003e\n\u003cp\u003e(b) Evaluation of the anti-phage activity of \u003cem\u003eEc\u003c/em\u003eBPAN system against various phages. A filled square indicates a significant reduction in EOP, and an “S” indicates smaller plaque formation.\u003c/p\u003e\n\u003cp\u003e(c) Both \u003cem\u003eEc\u003c/em\u003eAgo and \u003cem\u003eEc\u003c/em\u003ebAgaN are essential for the anti-phage activity of the \u003cem\u003eEc\u003c/em\u003eBPAN system. All experiments were performed in triplicate.\u003c/p\u003e\n\u003cp\u003e(d) The \u003cem\u003eEc\u003c/em\u003eBPAN system exhibits non-specific cleavage activity upon activation by RNA-guided target DNA recognition.\u003c/p\u003e","description":"","filename":"Fig.1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8163682/v1/0119b633a61d139b4555e3ac.jpg"},{"id":96433809,"identity":"c8e53095-ffd7-4a46-8d78-4509106a09fd","added_by":"auto","created_at":"2025-11-21 05:03:47","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":891640,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCryo-EM structure of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eEc\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eBPAN-guide RNA-target DNA complex\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) SEC and SDS-PAGE analyses of mixture of \u003cem\u003eEc\u003c/em\u003eAgo, guide, target and \u003cem\u003eEc\u003c/em\u003ebAgaN.\u003c/p\u003e\n\u003cp\u003e(b) Cryo-EM density map of \u003cem\u003eEc\u003c/em\u003eBPAN-guide RNA-target DNA complex.\u003c/p\u003e\n\u003cp\u003e(c) Cryo-EM structure of \u003cem\u003eEc\u003c/em\u003eBPAN-guide RNA-target DNA complex is shown in cartoon.\u003c/p\u003e","description":"","filename":"Fig.2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8163682/v1/17e35f06c1cdb1ef4e550858.jpg"},{"id":96454219,"identity":"19e8c864-0ea5-4481-a20f-078fe28af878","added_by":"auto","created_at":"2025-11-21 10:02:29","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":700003,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIntermolecular interactions between \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eEc\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eAgo and guide RNA-target DNA duplex\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) The domain architecture of \u003cem\u003eEc\u003c/em\u003eAgo. The N domain, linker 1, PAZ domain, linker 2, MID domain and PIWI domain are colored in pink, grey, cyan, salmon, orange and green, respectively.\u003c/p\u003e\n\u003cp\u003e(b) Cryo-EM structure of \u003cem\u003eEc\u003c/em\u003eAgo-guide RNA-target DNA complex is shown in surface (left) and carton (right) with color-coded domains and linkers.\u003c/p\u003e\n\u003cp\u003e(c) Schematic diagram of the interactions between \u003cem\u003eEc\u003c/em\u003eAgo and guide DNA-target DNA duplex. The nucleotides are numbered and the amino acids are colored as depicted in Fig 3a.\u003c/p\u003e\n\u003cp\u003e(d) Interaction between 5’-end of guide RNA and amino acids of \u003cem\u003eEc\u003c/em\u003eAgo. The guide RNA (red) is shown in sticks, with phosphorus atoms are colored in yellow.\u003c/p\u003e\n\u003cp\u003e(e) Analysis of cleavage activity by \u003cem\u003eEc\u003c/em\u003eBPAN complex with different guide RNA-target DNA duplexes. The different first RNA-DNA base pairs were shown. All experiments were performed in triplicate.\u003c/p\u003e\n\u003cp\u003e(f) Interaction between 3’-end of target DNA and amino acids of \u003cem\u003eEc\u003c/em\u003eAgo. The target DNA (blue) is shown in sticks, with phosphorus atoms are colored in yellow.\u003c/p\u003e\n\u003cp\u003e(g) Analysis of cleavage activity by \u003cem\u003eEc\u003c/em\u003eBPAN complex (wild-type)\u003cem\u003e \u003c/em\u003eand mutants (Y426 and N460 from \u003cem\u003eEc\u003c/em\u003eAgo). Cleavage activity assay was performed in triplicate, and the error bars represent the standard deviations. All experiments were performed in triplicate.\u003c/p\u003e","description":"","filename":"Fig.3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8163682/v1/c129dc31a4f5012a2681a9de.jpg"},{"id":96433817,"identity":"9b4eb335-7ec7-487c-b79f-9ac419ee31f5","added_by":"auto","created_at":"2025-11-21 05:03:47","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":870545,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe octamerization of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eEc\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003ebAgaN.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) Two \u003cem\u003eEc\u003c/em\u003ebAgaN molecules (\u003cem\u003eEc\u003c/em\u003ebAgaN.1 and \u003cem\u003eEc\u003c/em\u003ebAgaN.2) in \u003cem\u003eEc\u003c/em\u003eBPAN complex interacts with each other via their N-terminal and C-terminal domains. Close-up views of the interaction interfaces are shown.\u003c/p\u003e\n\u003cp\u003e(b) Analysis of cleavage activity by \u003cem\u003eEc\u003c/em\u003eBPAN complex(wild-type)\u003cem\u003e \u003c/em\u003eand \u003cem\u003eEc\u003c/em\u003ebAgaN mutants (D14A/Y15A/Q189A, M1, S332A/Y340A, N286A/Q288A/Q292A and Q357A/E358A/K361A). The results shown are the representative of three experiments, and the error bars represent the standard deviations.\u003c/p\u003e\n\u003cp\u003e(c) Cartoon representation of \u003cem\u003eEc\u003c/em\u003ebAgaN octamer is shown. Each \u003cem\u003eEc\u003c/em\u003ebAgaN molecule are depicted in different color. The interaction details of the \u003cem\u003eEc\u003c/em\u003ebAgaN octamer are presented.\u003c/p\u003e\n\u003cp\u003e(d) SEC analysis of \u003cem\u003eEc\u003c/em\u003eBPAN complex and \u003cem\u003eEc\u003c/em\u003ebAgaN mutants (D14A/Y15A/Q189A, M1 and N286A/Q288A/Q292A).\u003c/p\u003e","description":"","filename":"Fig.4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8163682/v1/14435f9ea4ae8ccc82d0aed6.jpg"},{"id":96433812,"identity":"c5ff3946-a946-4904-b618-1bd12fc76404","added_by":"auto","created_at":"2025-11-21 05:03:47","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":820939,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInteractions between \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eEc\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eAgo\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e–Ec\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003ebAgaN\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eand \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eEc\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eAgo–\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eEc\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eAgo.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) \u003cem\u003eEc\u003c/em\u003eAgo interacts with \u003cem\u003eEc\u003c/em\u003ebAgaN (magenta) via MID and PIWI domain. The amino acids of \u003cem\u003eEc\u003c/em\u003eAgo are colored based on Fig 3a.The protruding loop of \u003cem\u003eEc\u003c/em\u003ebAgaN are outlined with a black box. Close-up view of the interface of \u003cem\u003eEc\u003c/em\u003eAgo and \u003cem\u003eEc\u003c/em\u003ebAgaN is shown. Key interacting residues are shown in sticks.\u003c/p\u003e\n\u003cp\u003e(b) Plaque assay to evaluate the effect of key residue mutations on \u003cem\u003eE. coli\u003c/em\u003e’s defense against lab02 bacteriophage invasion. The results shown are the representative of three experiments.\u003c/p\u003e\n\u003cp\u003e(c) Analysis of cleavage activity by \u003cem\u003eEc\u003c/em\u003eBPAN complex (wild-type)\u003cem\u003e \u003c/em\u003eand its mutants (mutation of interact residues between \u003cem\u003eEc\u003c/em\u003eAgo and \u003cem\u003eEc\u003c/em\u003ebAgaN). The results shown are the representative of three experiments, and the error bars represent the standard deviations.\u003c/p\u003e\n\u003cp\u003e(d) SEC analysis of \u003cem\u003eEc\u003c/em\u003eBPAN complex (wild-type) and (GGGGS)₂ linker mutation.\u003c/p\u003e\n\u003cp\u003e(e) \u003cem\u003eEc\u003c/em\u003eBPAN complex contains four \u003cem\u003eEc\u003c/em\u003eAgo ternary dimer units shown in cartoon.\u003c/p\u003e\n\u003cp\u003e(f) Two Ago molecules interact with each other via the MID and PIWI domains. Detailed interaction interface was outlined with a black box. The same color scheme as in Fig 3A is adopted. Close-up view of the interface of \u003cem\u003eEc\u003c/em\u003eAgo and \u003cem\u003eEc\u003c/em\u003ebAgaN is shown. Key interacting residues are shown in sticks.\u003c/p\u003e","description":"","filename":"Fig.5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8163682/v1/278057deea1f45345d3b9a56.jpg"},{"id":96454275,"identity":"7b44712d-4a86-438e-b173-163fbb3c6949","added_by":"auto","created_at":"2025-11-21 10:02:32","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":570051,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProposed working model of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eEc\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eBPAN complex.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eEc\u003c/em\u003eBPAN complex senses the invasion of bacteriophage and plasmid, forms \u003cem\u003eEc\u003c/em\u003eBPAN 8:8 complex and activates its nuclease activity against genomic DNA to cause cell death. Guide RNA and target DNA are respectively colored in red and blue. The cartoon of \u003cem\u003eEc\u003c/em\u003eAgo is drawn in the same view angle of Fig 3a. The \u003cem\u003eEc\u003c/em\u003ebAgaN dimer is colored in magenta and yellow.\u003c/p\u003e","description":"","filename":"Fig.6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8163682/v1/1cd423a9e5b1e5dbc30d8b6b.jpg"},{"id":96456981,"identity":"07f7acbb-6edd-459e-a1f0-d0ebd05409d5","added_by":"auto","created_at":"2025-11-21 10:08:51","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5325927,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8163682/v1/21cd9a41-bf22-4b0b-ad15-ad8d4575e78f.pdf"},{"id":96433819,"identity":"43bae6d6-b0bd-49e0-a149-4f97176c0f20","added_by":"auto","created_at":"2025-11-21 05:03:48","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":16814772,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryinformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-8163682/v1/a640af7d4a895de75d2cd09d.docx"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eDistinct mechanism of the long-B prokaryotic Argonaute-mediated nuclease activation in bacterial immunity\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eArgonaute proteins (Agos) are programmable enzymes that utilize nucleic acid guides for target recognition, and are widely distributed across all kingdoms of life\u0026nbsp;\u003csup\u003e[1]\u003c/sup\u003e. Eukaryotic Agos (eAgos) initially characterized as the core constituents of RNA-induced silencing complex (RISC) and mediating RNA-relate cellular processes\u0026nbsp;\u003csup\u003e[2, 3]\u003c/sup\u003e. In contrast, prokaryotic Agos (pAgos) serve as innate immune effectors that protect cells against invading plasmids and phages\u0026nbsp;\u003csup\u003e[4-6]\u003c/sup\u003e. Compared with eAgos, pAgos exhibit more diversity in sequence and domain compositions. Based on domain architectures, pAgos can be classified into three distinct groups: long-A, long-B and short pAgos\u0026nbsp;\u003csup\u003e[7]\u003c/sup\u003e. Long-A and long-B pAgos share significantly structural homology with eAgos and contain all four canonical domains: N, PAZ (PIWI\u0026ndash;Argonaute\u0026ndash;Zwille), MID (Middle) and PIWI (P-element Induced Wimpy Testis) domains, while short pAgos only consist of a MID domain and a catalytically inactive PIWI domain \u003csup\u003e[7, 8]\u003c/sup\u003e. Currently, the most extensively studied long-A pAgos utilize RNA or DNA guides to cleave target RNA or DNA\u0026nbsp;\u003csup\u003e[9-11]\u003c/sup\u003e. Several long-A pAgos, such as \u003cem\u003eTt\u003c/em\u003eAgo from \u003cem\u003eThermus thermophilus\u003c/em\u003e and \u003cem\u003eCb\u003c/em\u003eAgo from \u003cem\u003eClostridium butyricum\u003c/em\u003e, have been shown to provide immunity by DNA-guided DNA interference\u0026nbsp;\u003csup\u003e[4, 12, 13]\u003c/sup\u003e. Moreover, the structures of representative long-A pAgos have been determined\u0026nbsp;\u003csup\u003e[14, 15]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eApproximately 60% of pAgos are classified as highly divergent short pAgos, which lack nuclease activity due to the absence of the DEDX catalytic tetrad in their PIWI domain\u003csup\u003e\u0026nbsp;\u003c/sup\u003e\u003csup\u003e[7, 8]\u003c/sup\u003e. Recent studies have revealed that short pAgos from many bacteria constitute defense systems in association with effector proteins composed of an N-terminal enzymatic domain and a C-terminal APAZ (analog of PAZ) domain\u0026nbsp;\u003csup\u003e[7, 16, 17]\u003c/sup\u003e. The APAZ domain is homologous to the N domain rather than PAZ domain of long pAgos. These characterized N-terminal enzymatic domains include the NADase domains TIR (Toll-interleukin-1 receptor) from SPARTA system and SIR2 (sirtuin 2) from SPARSA system, as well as nuclease domain DREN (DNA and RNA effector nuclease) from SPARDA system\u003csup\u003e\u0026nbsp;\u003c/sup\u003e\u003csup\u003e[17-21]\u003c/sup\u003e. Unlike long-A pAgos, which directly recognize and cleave target nucleic acids, SPARTA and SPARSA systems activate their NADase domains upon target DNA recognition. This leads to the hydrolysis of cellular nicotinamide adenine dinucleotide (NAD⁺) and subsequently triggers cell death, thereby preventing viral infection and plasmids invasion\u0026nbsp;\u003csup\u003e[18, 22, 23]\u003c/sup\u003e. For SPARDA system, target DNA binding activates associated nuclease, leading to degradation of cellular DNA and subsequent cell death or dormancy\u0026nbsp;\u003csup\u003e[24]\u003c/sup\u003e. Structural and biochemical studies have provided a more in-depth understanding of the working mechanism for guide and target recognition and activation of short pAgos-associated systems \u003csup\u003e[21, 24-26]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eHowever, long-B pAgos have been studied much less extensively. Although their structural compositions are similar to that of long-A pAgos, long-B pAgos lack nuclease activity. Recent studies have shown that the gene neighborhood of long-B pAgos encode pAgo-associated proteins, which can be functionally connected with their corresponding long-B pAgos\u0026nbsp;\u003csup\u003e[7, 27]\u003c/sup\u003e. These pAgo-associated proteins include nucleases from BPAN (long-B prokaryotic Argonaute nuclease) systems, SIR2-like proteins from BPAS (long-B prokaryotic Argonaute Sir2) systems and trans-membrane proteins from BPAM (long-B prokaryotic Argonaute trans-membrane) systems\u0026nbsp;\u003csup\u003e[27]\u003c/sup\u003e. The study has shown that these systems can defend against invading plasmids but have not been demonstrated to provide detectable protection against phage infection. Moreover, the mechanisms of action of the long-B pAgos-associated systems remain elusive.\u003c/p\u003e\n\u003cp\u003eIn this study, we found that the \u003cem\u003eEc\u003c/em\u003eBPAN system from \u003cem\u003eEscherichia coli\u003c/em\u003e (\u003cem\u003eE. coli\u003c/em\u003e) provides strong protection against bacteriophage. We further discovered that RNA-guided \u003cem\u003eEc\u003c/em\u003eAgo recognizes target DNA and subsequently recruits \u003cem\u003eEc\u003c/em\u003ebAgaN to form an 8:8 complex with activated nuclease activity, which nonspecifically degrades double-stranded DNA (dsDNA). Our work provides the first mechanistic insights into a long-B pAgo-associated defense system.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eThe anti-phage function and nuclease activation of \u003cem\u003eEc\u003c/em\u003eBPAN system\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eEc\u003c/em\u003eBPAN system consists of two proteins, \u003cem\u003eEc\u003c/em\u003eAgo and the nuclease \u003cem\u003eEc\u003c/em\u003ebAgaN (Fig. 1a). The previous study has demonstrated that the \u003cem\u003eEc\u003c/em\u003eBPAN system can act as a prokaryotic defense system against plasmids via abortive infection\u0026nbsp;\u003csup\u003e[27]\u003c/sup\u003e. However, whether the \u003cem\u003eEc\u003c/em\u003eBPAN system can provide defense against phages remains to be explored. In this study, we tested the resistance of the \u003cem\u003eEc\u003c/em\u003eBPAN system against six \u003cem\u003eE. coli\u0026nbsp;\u003c/em\u003ephages, including T7, T4, T5, \u0026lambda;, lab02 and lab05 (Fig. 1b). We measured the efficiency of plaque formation (EOP) of these phages on \u003cem\u003eE. coli\u003c/em\u003e cells expressing the\u003cem\u003e\u0026nbsp;Ec\u003c/em\u003eBPAN system. The results showed that the \u003cem\u003eEc\u003c/em\u003eBPAN system provided significant protection against phage lab02 (~10 000 fold), and decreased the plaque size formed by phage lab05, while showing no reduction in plaque formation for the other five phages (Supplementary information, Fig. S1). We further examined the anti-phage activity of \u003cem\u003eEc\u003c/em\u003eAgo and \u003cem\u003eEc\u003c/em\u003ebAgaN individually and found that neither protein alone exhibited detectable anti-phage activity, indicating that both components are required for the full defensive function of the \u003cem\u003eEc\u003c/em\u003eBPAN system (Fig. 1c). To further validate the cooperative function of these two proteins, \u003cem\u003eEc\u003c/em\u003eAgo and \u003cem\u003eEc\u003c/em\u003ebAgaN were expressed and purified for \u003cem\u003ein vitro\u003c/em\u003e functional assays. Consistent with the previous findings\u0026nbsp;\u003csup\u003e[27]\u003c/sup\u003e, efficient DNA cleavage was observed only when \u003cem\u003eEc\u003c/em\u003eAgo, \u003cem\u003eEc\u003c/em\u003ebAgaN, guide RNA and target DNA were all present in the reaction mixture (Fig. 1d), indicating that the interaction between \u003cem\u003eEc\u003c/em\u003eAgo and \u003cem\u003eEc\u003c/em\u003ebAgaN upon RNA-guided target recognition.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOverall structure of the \u003cem\u003eEc\u003c/em\u003eBPAN complex bound to guide RNA and target DNA\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo further validate the interaction between\u0026nbsp;\u003cem\u003eEc\u003c/em\u003eAgo and\u003cem\u003e\u0026nbsp;Ec\u003c/em\u003ebAgaN, we incubated \u003cem\u003eEc\u003c/em\u003ebAgaN with \u003cem\u003eEc\u003c/em\u003eAgo alone, \u003cem\u003eEc\u003c/em\u003eAgo loaded with guide RNA, \u003cem\u003eEc\u003c/em\u003eAgo bound to target DNA, or \u003cem\u003eEc\u003c/em\u003eAgo preloaded with both guide RNA and target DNA, followed by size-exclusion chromatography (SEC) analysis. The results showed that only \u003cem\u003eEc\u003c/em\u003eAgo preloaded with both guide RNA and target DNA was able to interact with \u003cem\u003eEc\u003c/em\u003ebAgaN to form a larger complex (Fig. 2a and Supplementary information, Fig. S2). The complex fraction was collected, and its structure was resolved by cryo-electron microscopy (cryo-EM) at\u0026nbsp;a resolution of\u0026nbsp;3.27\u0026nbsp;\u0026Aring; (Fig. 2b, c and Supplementary information, Fig. S3 and Table S1). The well-defined cryo-EM density allowed us to build an atomic model with high confidence.\u0026nbsp;The overall structure of\u0026nbsp;\u003cem\u003eEc\u003c/em\u003eBPAN\u0026nbsp;complex\u0026nbsp;shows\u0026nbsp;a distinct \u0026ldquo;bowl-shaped\u0026rsquo;\u0026rsquo;\u0026nbsp;architecture, composed of the \u003cem\u003eEc\u003c/em\u003eAgo-guide RNA-target DNA complex and \u003cem\u003eEc\u003c/em\u003ebAgaN in an 8:8 stoichiometric ratio. Within this arrangement, eight \u003cem\u003eEc\u003c/em\u003eAgo ternary complex molecules are arranged in a circular pattern to form the body of the bowl, while eight \u003cem\u003eEc\u003c/em\u003ebAgaN molecules are interconnected to constitute the bottom of the bowl.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInteraction between \u003cem\u003eEc\u003c/em\u003eAgo and guide RNA-target DNA duplex\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe overall structure of\u0026nbsp;\u003cem\u003eEc\u003c/em\u003eAgo\u0026nbsp;is homologous to that of\u0026nbsp;\u003cem\u003eRs\u003c/em\u003eAgo (C\u0026alpha; RMSD=2.173 \u0026Aring;) (Supplementary information, Fig. S4a). Based on the structure assembly, \u003cem\u003eEc\u003c/em\u003eAgo is divided into N domain (1-92), linker 1 (92-155), PAZ domain (155-218), linker 2 (218-308), MID domain (308-484) and PIWI domain (484-731) (Fig. 3a). \u003cem\u003eEc\u003c/em\u003eAgo adopts conserved domain architecture found in other Ago proteins\u0026nbsp;\u003csup\u003e[14]\u003c/sup\u003e, however, its PAZ domain is significantly smaller than the canonical PAZ domain and comprises approximately 60 residues that adopts a hairpin-like structure (Supplementary information, Fig. S4b).\u0026nbsp;The guide RNA-target DNA duplex is stably positioned between the N-PAZ and MID-PIWI lobes (Fig. 3b). Notably, the 5\u0026rsquo;-end of guide RNA remains unpaired with 3\u0026rsquo;-end of target DNA. The nucleotides at positions 20-21 of guide RNA strand, as well as the terminal nucleotide C21\u0026rsquo; of target DNA are missing. Meanwhile, positions 2-19 of both strands engage in canonical Watson-Crick base pairing (Fig. 3c).\u003c/p\u003e\n\u003cp\u003eThe structure of \u003cem\u003eEc\u003c/em\u003eAgo bound guide RNA-target DNA complex reveals a network of intermolecular hydrogen-bond interactions between \u003cem\u003eEc\u003c/em\u003eAgo and guide RNA-target DNA duplex, particularly around 5\u0026rsquo;\u0026nbsp;end of the guide RNA and nucleotides at positions 2-13 of guide RNA-target DNA duplex (Fig. 3c). In contrast, there is almost no intermolecular interaction between nucleotides at positions 14-20 of guide RNA-target DNA duplex and \u003cem\u003eEc\u003c/em\u003eAgo. The 5\u0026rsquo;-phosphate group of guide strand is stably anchored within MID pocket and forms direct hydrogen bonds with conserved four-amino acids motif X-K-Q-K (Y426, K430, Q441, K469) and D442 from MID domain (Fig. 3d). Furthermore, the 5\u0026rsquo;-phosphate group of guide RNA coordinates to a Mg\u003csup\u003e2+\u003c/sup\u003e ion, which is simultaneously coordinated by the carboxylate group of C-terminal residue L731 of \u003cem\u003eEc\u003c/em\u003eAgo (Fig. 3d). To examine the role of the 5\u0026rsquo;-phosphate group of guide RNA in mediating interactions with MID domain, we performed an \u003cem\u003ein vitro\u003c/em\u003e gnomic DNA degradation assay. Our results showed that the removal of the 5\u0026rsquo;-phosphate group from guide RNA significantly reduced DNA cleavage activity (Fig. 3e), suggesting that \u003cem\u003eEc\u003c/em\u003eAgo preferentially loads 5\u0026rsquo;- phosphorylated guide RNA for target DNA recognition.\u003c/p\u003e\n\u003cp\u003eThe nucleotides A1 of guide RNA and T1\u0026rsquo; of target DNA are each inserted into their respective binding pockets (Fig. 3d, f). The nucleotide A1 of guide RNA strand is stabilized by stacking against Y426 in the MID domain, a conserved interaction observed among Argonaute proteins\u0026nbsp;\u003csup\u003e[28]\u003c/sup\u003e. Alanine substitution of Y426 impaired the cleavage activity (Fig. 3g). Furthermore, the nucleotide A1 forms a hydrogen bond with side chain of residue N424 situated on the \u0026lsquo;\u0026lsquo;specificity loops\u0026rsquo;\u0026rsquo;, a region known to discriminate different terminal nucleotides\u0026nbsp;\u003csup\u003e[9, 29]\u003c/sup\u003e. To further probe the specificity of first RNA-DNA base pair recognized by \u003cem\u003eEc\u003c/em\u003eAgo, we assessed the cleavage activity using different RNA-DNA base pair combinations at this position. The results showed that substituting A\u0026middot;T with U\u0026middot;A reduced cleavage activity by approximately 30%, while replacing it with G\u0026middot;C or C\u0026middot;G led to a more pronounced reduction of about 60% in genomic DNA cleavage (Fig. 3e), suggesting that \u003cem\u003eEc\u003c/em\u003eAgo exhibits a bias toward A at the position 1 of guide RNA.\u003c/p\u003e\n\u003cp\u003eThe\u0026nbsp;terminal nucleotide T1\u0026rsquo; of the target DNA inserts into a hydrophilic pocket formed by E309, Y358, Y457, N460 and Y687, and forms polar contact with the side chain of N460 from the MID domain. Alanine substitution of N460 impaired the cleavage activity (Fig. 3g). To determine whether \u003cem\u003eEc\u003c/em\u003eAgo has a preference for thymine at the position 1\u0026rsquo; of target DNA, we measured the genomic DNA cleavage activity after substituting T1\u0026rsquo; with A\u0026rsquo;, or G\u0026rsquo;, or C\u0026rsquo; in the target DNA. The results showed that the replacement of T1\u0026rsquo; with any other nucleotides had no significant effect on cleavage activity (Fig. 3e). Moreover, similar interaction patterns involving N460 were observed when the 3\u0026rsquo;-terminal nucleotide was A\u0026rsquo;, or G\u0026rsquo;, or C\u0026rsquo; (Supplementary information, Fig. S5a), further supporting that \u003cem\u003eEc\u003c/em\u003eAgo lacks a strong base preference at the position 1\u0026rsquo; of target DNA.\u003c/p\u003e\n\u003cp\u003eMismatches between guide and target strands have been demonstrated to impair the cleavage efficiency in several pAgos\u0026nbsp;\u003csup\u003e[20, 30, 31]\u003c/sup\u003e. In this study, to evaluate the effect of mismatches on\u0026nbsp;cleavage activity\u0026nbsp;of the \u003cem\u003eEc\u003c/em\u003eBPAN system, we designed a series of target DNA, each containing a single mismatched nucleotide at position 2-21 relative to guide RNA (Supplementary information Table S2) and tested their cleavage reaction by \u003cem\u003eEc\u003c/em\u003eBPAN system. As shown in Supplementary information, Fig. S5b, single mismatches at positions 2-10, 12, and the 3\u0026rsquo;-end of the guide-target duplex exhibited minimal impact on cleavage efficiency. In contrast, single mismatches at positions 11, or 13-16 impaired the cleavage activity. Notably, the cleavage activity of \u003cem\u003eEc\u003c/em\u003eBPAN\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003esystem is not affected by mismatches in the 5\u0026rsquo;-seed region (position 2-8) but is impaired by mismatches in the 3\u0026rsquo; supplementary guide region (position 13-16).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePositioning of putative catalytic pocket residues in \u003cem\u003eEc\u003c/em\u003eAgo\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe cleavage-competent prokaryotic Agos contain a catalytic pocket with a conserved DEDD tetrad in the PIWI domain. \u003cem\u003eEc\u003c/em\u003eAgo exhibits two substitutions in the DEDD tetrad (G492, E532, Q568, D700 as shown in Supplementary information, Fig. S6a) and Q568 forms a hydrogen bond with phosphate group of A10\u0026rsquo; of target DNA (Fig. 3c). Ago-mediated cleavage requires insertion of the conserved \u0026ldquo;glutamate finger\u0026rdquo; into the catalytic pocket to form the functional DEDX tetrad\u0026nbsp;\u003csup\u003e[32]\u003c/sup\u003e. In \u003cem\u003eTt\u003c/em\u003eAgo (\u003cem\u003eThermus thermophilus\u003c/em\u003e), a glutamate residue (E512) undergoes conformational transition from unplugged state in complex with guide strand to plugged-in state in complex with guide and target strands\u0026nbsp;(Supplementary information, Fig. S6b, c), resulting in the cleavage of target\u0026nbsp;\u003csup\u003e[11]\u003c/sup\u003e. The structure of guide RNA-target DNA-bound \u003cem\u003eEc\u003c/em\u003eAgo in\u0026nbsp;the\u0026nbsp;quaternary complex\u0026nbsp;shows that E532, the glutamate residue corresponding to E512 (\u003cem\u003eTt\u003c/em\u003eAgo) and E569 (\u003cem\u003eRs\u003c/em\u003eAgo) (Supplementary information, Fig. S6), is located away from the catalytic pocket and adopts an unplugged conformation. This structural feature probably explains that \u003cem\u003eEc\u003c/em\u003eAgo complex with guide RNA and target DNA lacks of DNA cleavage activity (Fig. 2d).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStructural basis of \u003cem\u003eEc\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003ebAgaN\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;octamer\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eEc\u003c/em\u003ebAgaN is annotated as a member of the RecB-like protein family. Structural analysis reveals that \u003cem\u003eEc\u003c/em\u003ebAgaN comprises two distinct domains connected by a flexible, unstructured linker (Fig. 4a). Although \u003cem\u003eEc\u003c/em\u003ebAgaN\u0026nbsp;shares low sequence identity with known structures, its C-terminal domain is highly identical to structurally characterized PD-(D/E)XK nucleases. The C-terminal domain exhibits a conserved \u0026alpha;-\u0026beta;-\u0026alpha; fold architecture, featuring a four-stranded mixed \u0026beta;-sheet sandwiched by two \u0026alpha;-helices on both sides. The nuclease domain harbors\u0026nbsp;a conserved D-EVK motif (D298, E309, V310 and K311) (Supplementary information, Fig. S7a), which is essential for the nuclease activity\u0026nbsp;\u003csup\u003e[27]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThe previous study \u003csup\u003e[27]\u003c/sup\u003e and our SEC analysis both showed that \u003cem\u003eEc\u003c/em\u003ebAgaN forms a dimer in solution (Supplementary information, Fig. S2). Consistently, the \u003cem\u003eEc\u003c/em\u003ebAgaN dimeric state was observed in our structure. Within \u003cem\u003eEc\u003c/em\u003ebAgaN dimer, two copies stacked against each other by two interfaces: Interface I and Interface II, accompanied by cross-linking between the linker regions (Fig. 4a). At the Interface I, hydrogen bonds are formed between residues D14 and Y15\u0026rsquo;, as well as between N212 and Q189\u0026rsquo; (residues marked with a prime [\u0026rsquo;] denote those from the opposing protomer). At the Interface II, a more extensive hydrogen-bonding network is observed. Specifically, Q259, H265, N268, D283 and Y271 form a hydrogen-bond interactions with W258\u0026rsquo;, E245\u0026rsquo;, T243\u0026rsquo; R241\u0026rsquo;, and K240\u0026rsquo;. Alanine substitution of D14/Y15/Q189 impaired the cleavage activity. A multi-site alanine substitution mutant (designated M1: K240A, R241A, T243A, E245A, W258A, Q259A, H265A, N268A, Y271A and D283A) almost completely abolished the cleavage activity (Fig. 4b), highlighting the critical role of these interfaces in the \u003cem\u003eEc\u003c/em\u003ebAgaN dimer for the activation of \u003cem\u003eEc\u003c/em\u003eBPAN system.\u003c/p\u003e\n\u003cp\u003eFurthermore, four \u003cem\u003eEc\u003c/em\u003ebAgaN dimer units further assemble into an octamer (Fig. 4c). Hydrogen-bond interactions are observed between the C-terminal regions of these dimers, involving the following residue pairs: I281 and K361, Q288 and Y340, Q292 and E358, Q357 and S382, N286 pair and S332 pair. Alanine substitution of S332/Y340, N286/Q288/Q292 and Q357/E358/K361 reduced the cleavage activity (Fig. 4b). Notably, the D14A/Y15A/Q189A and M1 mutations from the \u003cem\u003eEc\u003c/em\u003ebAgaN dimer interface and N286A/Q288A/Q292A mutation from the \u003cem\u003eEc\u003c/em\u003ebAgaN octamer interface disrupted the formation of the \u003cem\u003eEc\u003c/em\u003eBPAN 8:8 complex, resulting in smaller complexes compared to the wild-type \u003cem\u003eEc\u003c/em\u003eBPAN system (Fig. 4d). Taken together, these findings highlight the important role of \u003cem\u003eEc\u003c/em\u003ebAgaN-\u003cem\u003eEc\u003c/em\u003ebAgaN interactions in \u003cem\u003eEc\u003c/em\u003eBPAN 8:8 complex and demonstrate that the assembly of \u003cem\u003eEc\u003c/em\u003eBPAN 8:8 complex is indispensable for the activation of its nuclease activity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInteraction between \u003cem\u003eEc\u003c/em\u003eAgo and\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eEc\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003ebAgaN is essential for the\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eformation of the\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eEc\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003eBPAN 8:8 complex\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStructural analysis uncovers that guide-directed target recognition by \u003cem\u003eEc\u003c/em\u003eAgo\u0026nbsp;recruits\u0026nbsp;the nuclease\u003cem\u003e\u0026nbsp;Ec\u003c/em\u003ebAgaN\u0026nbsp;via\u0026nbsp;a protruding loop (segments 81-90) from N-terminal domain of\u0026nbsp;\u003cem\u003eEc\u003c/em\u003ebAgaN. This loop inserts into the interface between MID and PIWI domains of \u003cem\u003eEc\u003c/em\u003eAgo, forming an extensive hydrogen-bonding network (Fig. 5a). Specifically, residues A82, D86 and P89 of \u003cem\u003eEc\u003c/em\u003ebAgaN form hydrogen bonds with R552, S524 and Y318 of \u003cem\u003eEc\u003c/em\u003eAgo, respectively. In addition, hydrogen bonds also are observed between R77, S94, H134 of \u003cem\u003eEc\u003c/em\u003ebAgaN and N554, D559, R321 and F323 of \u003cem\u003eEc\u003c/em\u003eAgo. To validate the functional significance of this interface, we introduced alanine substitutions at these critical residues. These mutations lead to strong reduction in defense against invading DNA as assessed by bacteriophage infection assay (Fig. 5b). Consistently, the same mutations also reduced the genomic DNA cleavage activity \u003cem\u003ein vitro\u003c/em\u003e (Fig. 5c).\u003c/p\u003e\n\u003cp\u003eTo investigate the role of interaction between \u003cem\u003eEc\u003c/em\u003eAgo and\u0026nbsp;\u003cem\u003eEc\u003c/em\u003ebAgaN\u0026nbsp;in the\u0026nbsp;formation of the\u0026nbsp;\u003cem\u003eEc\u003c/em\u003eBPAN 8:8 complex, we replaced this protruding loop of\u0026nbsp;\u003cem\u003eEc\u003c/em\u003ebAgaN with a (GGGGS)₂ linker and subsequently performed \u003cem\u003ein vitro\u003c/em\u003e genomic DNA cleavage activity assay and SEC analysis. The results confirmed that the substitution of this loop with (GGGGS)₂ linker impaired the cleavage activity and prevented the assembly of the \u003cem\u003eEc\u003c/em\u003eBPAN complex (Fig. 5c, d). Thus, our structural and biochemical data demonstrate that the specific \u003cem\u003eEc\u003c/em\u003eAgo-\u003cem\u003eEc\u003c/em\u003ebAgaN interaction, mediated by the \u003cem\u003eEc\u003c/em\u003ebAgaN loop, is a critical prerequisite for the formation of\u0026nbsp;stable \u003cem\u003eEc\u003c/em\u003eBPAN 8:8 complex\u0026nbsp;and the consequent activation of its nuclease function.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDimerization of the \u003cem\u003eEc\u003c/em\u003eAgo ternary complex upon \u003cem\u003eEc\u003c/em\u003ebAgaN recruitment is\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;essential for\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eEc\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003eBPAN\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eactivation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn the \u003cem\u003eEc\u003c/em\u003eBPAN 8:8 complex, eight \u003cem\u003eEc\u003c/em\u003ebAgaN molecules assemble into an octamer by forming four \u003cem\u003eEc\u003c/em\u003ebAgaN dimers, whereas the eight \u003cem\u003eEc\u003c/em\u003eAgo ternary complexes bound with\u0026nbsp;guide RNA-target DNA duplex\u0026nbsp;organize into four independent \u003cem\u003eEc\u003c/em\u003eAgo ternary dimers (Fig. 5e). Each \u003cem\u003eEc\u003c/em\u003eAgo molecule interacts specifically with one \u003cem\u003eEc\u003c/em\u003ebAgaN molecule. Interestingly, within each \u003cem\u003eEc\u003c/em\u003eAgo ternary dimer, one \u003cem\u003eEc\u003c/em\u003eAgo protomer binds to one protomer of an \u003cem\u003eEc\u003c/em\u003ebAgaN dimer, while the other \u003cem\u003eEc\u003c/em\u003eAgo protomer interacts with an adjacent \u003cem\u003eEc\u003c/em\u003ebAgaN dimer (Supplementary information, Fig. S7b). This cross-linking pattern connects the four \u003cem\u003eEc\u003c/em\u003eAgo dimers through the \u003cem\u003eEc\u003c/em\u003ebAgaN octamer, ultimately resulting in a globally stable \u003cem\u003eEc\u003c/em\u003eBPAN 8:8 complex.\u003c/p\u003e\n\u003cp\u003eAt the\u0026nbsp;intermolecular interface of \u003cem\u003eEc\u003c/em\u003eAgo ternary dimer, the carboxylate group of D400 from the MID domain respectively forms\u0026nbsp;hydrogen bonds with the side chains of R552 and imidazole ring of H726 from the PIWI domain of another\u0026nbsp;\u003cem\u003eEc\u003c/em\u003eAgo molecule\u0026nbsp;(Fig. 5f).\u0026nbsp;The same interaction is also observed in the another MID-PIWI interface in the \u003cem\u003eEc\u003c/em\u003eAgo ternary dimer due to symmetry.\u0026nbsp;Mutations of the interface residues impaired DNA cleavage activity (Fig. 5c), suggesting the dimerization of \u003cem\u003eEc\u003c/em\u003eAgo\u0026nbsp;ternary complex\u0026nbsp;is\u0026nbsp;indispensable\u0026nbsp;for activating the DNase activity of \u003cem\u003eEc\u003c/em\u003eBPAN system.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWorking model of the EcBPAN system\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBased on our findings and the previous study\u0026nbsp;\u003csup\u003e[27]\u003c/sup\u003e, we propose a working model for \u003cem\u003eEc\u003c/em\u003eBPAN system (Fig. 6). Upon phage or plasmid invasion, \u003cem\u003eEc\u003c/em\u003eAgo binds to the transcripts derived from the invader and subsequently recognizes the complementary target DNA. This recognition induces a conformational change in \u003cem\u003eEc\u003c/em\u003eAgo, enabling the recruitment of an \u003cem\u003eEc\u003c/em\u003ebAgaN dimer and further assembly of an active \u003cem\u003eEc\u003c/em\u003eBPAN 8:8 complex with nuclease activity. The process involves target DNA recognition, \u003cem\u003eEc\u003c/em\u003eAgo dimerization, and\u003cem\u003e\u0026nbsp;Ec\u003c/em\u003ebAgaN octamerization. The activated\u0026nbsp;\u003cem\u003eEc\u003c/em\u003eBPAN complex degrades the genome DNA, leading to host cell death and thereby conferring population-level immunity against the invading elements.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eLong-B pAgos are often associated with different types of effector proteins, including nuclease, NADase and transmembrane protein, and have been shown to confer bacterial immunity against invading plasmids\u0026nbsp;\u003csup\u003e[27]\u003c/sup\u003e. Here, we characterized the anti-phages activity of bacterial long-B pAgo-\u003cem\u003eEc\u003c/em\u003ebAgaN\u0026nbsp;nuclease (\u003cem\u003eEc\u003c/em\u003eBPAN) system and determined its structure in complex with guide and target nucleic acids, thereby revealing\u0026nbsp;the molecular mechanism underlying \u003cem\u003eEc\u003c/em\u003eBPAN system activation.\u003c/p\u003e\n\u003cp\u003ePrevious studies have primarily focused on elucidating the mechanisms of long-A and short pAgos. Long-A pAgos possess intrinsic nuclease activity and primarily function as single proteins against invading DNA. Structural studies have shown that most long-A pAgos act as monomers upon target recognition, while a few dimerize for activation\u003csup\u003e\u0026nbsp;\u003c/sup\u003e\u003csup\u003e[28]\u003c/sup\u003e. In contrast, short pAgos lack catalytic activity and usually associate with APAZ-containing effector proteins to form heterodimers. Upon recognition of target nucleic acids, these heterodimers further oligomerize or assemble into filamentous structures to achieve activation\u0026nbsp;\u003csup\u003e[19, 21, 26, 33, 34]\u003c/sup\u003e. Long-B pAgos share a similar overall domain architecture with long-A pAgos but are catalytically inactive. However, unlike the short pAgo systems, the associated effector proteins in long-B pAgo systems lack an APAZ domain and cannot directly form stable heterodimers with the pAgos. In this study, we uncovered the molecular mechanism of a long-B pAgo defense system for the first time. We found that upon target DNA recognition, long-B pAgo recruits its cognate nuclease dimer to assemble into a bowl-shaped 8:8 supramolecular complex, leading to activation of its nuclease activity. The action of the mechanism of this system is distinct from the mechanisms of both long-A and short pAgo systems and expands our understanding of the functional diversity of pAgo-mediated defense systems.\u003c/p\u003e\n\u003cp\u003eRecently, a small subset of long-A pAgo systems has been found to contain an associated Cas4 family nuclease, which cooperates with the pAgo protein to defend against invading DNA\u0026nbsp;\u003csup\u003e[35]\u003c/sup\u003e. In addition, the newly reported DdmDE defense system features a catalytically inactive pAgo-like protein, DdmE, that recognizes target DNA and recruits a helicase-nuclease, DdmD, to eliminate invading plasmids\u003csup\u003e\u0026nbsp;\u003c/sup\u003e\u003csup\u003e[36-38]\u003c/sup\u003e. Although these recently discovered pAgo-based defense mechanisms differ from the long-B pAgo system characterized in this study, they collectively highlight the mechanistic diversity of pAgo-mediated strategies against foreign DNA elements. In our study, the long-B pAgo interacts with the N-terminal domain of its associated nuclease, a domain that is not found in other effector proteins of long-B pAgo systems, such as NADase and transmembrane protein. This is distinct from the APAZ domain found in diverse effector partners of short pAgo systems, suggesting that the activation and regulation of other long-B pAgo systems may differ from the one in this study. Future investigations into these long-B pAgo systems may bring new insights into pAgo-based defense mechanism.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eEc\u003c/em\u003ebAgaN contains an N-terminal \u0026alpha;-helical domain and a C-terminal\u0026nbsp;PD-(D/E)XK nuclease domain. Under normal physiological conditions, \u003cem\u003eEc\u003c/em\u003ebAgaN exists as an autoinhibited dimer. Upon target DNA recognition by\u0026nbsp;\u003cem\u003eEc\u003c/em\u003eAgo, the N-terminal domain of \u003cem\u003eEc\u003c/em\u003ebAgaN interacts with\u0026nbsp;\u003cem\u003eEc\u003c/em\u003eAgo, which in turn promotes octamerization of its C-terminal nuclease domains and subsequent activation. Notably, no direct interaction is observed between the N-terminal and C-terminal domains in the active state. The N-terminal domain, which has no homologs in any structurally characterized proteins, appears to have specifically evolved within the long-B pAgo systems as a sophisticated molecular \u0026ldquo;switch\u0026rdquo; to regulate the activity of the C-terminal nuclease domain. Under normal physiological conditions, this N-terminal domain likely maintains the nuclease in an inactive conformation by inhibiting octamer formation. The interaction between the N-terminal domain and \u003cem\u003eEc\u003c/em\u003eAgo upon target recognition releases this autoinhibition, allowing the C-terminal nuclease domain to assemble into an active octamer. Attempts to predict the dimeric structure of \u003cem\u003eEc\u003c/em\u003ebAgaN using AlphaFold3 were unsuccessful. Determination of the\u0026nbsp;\u003cem\u003eEc\u003c/em\u003ebAgaN dimer structure in the future will be critical for elucidating how the N-terminal domain regulates the activation of the C-terminal nuclease domain.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003eBacterial strains and phages\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eE. coli\u003c/em\u003e strains DH5\u0026alpha; and BL21 (DE3) were used for plasmid construction and protein expression,\u0026nbsp;respectively. \u003cem\u003eE. coli\u003c/em\u003e C600, which kindly provided by Professor Ruichao Li\u0026nbsp;\u003csup\u003e[39]\u003c/sup\u003e, and\u003cem\u003e\u0026nbsp;E. coli\u003c/em\u003e MG1655 were used to assess the anti-phage activity of the \u003cem\u003eEc\u003c/em\u003eBPAN system. All bacterial strains were cultured in LB medium. Phages T4, T5, and T7 were kindly provided by Professor Bin Zhu\u0026nbsp;\u003csup\u003e[40]\u003c/sup\u003e. Phage \u0026lambda; was kindly provided by Professor Lianrong Wang\u0026nbsp;\u003csup\u003e[41]\u003c/sup\u003e. Two additional phages, lab02 and lab05, were isolated from a laboratory sink. Lab02 shares 98.9% nucleotide identity with \u003cem\u003eE. coli\u003c/em\u003e phage TR2 (GenBank accession: OP251154), and lab05 shares 99.9% identity with \u003cem\u003eE. coli\u003c/em\u003e phage phiD2-2 (GenBank accession: OR861627).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePlasmid construction and mutagenesis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe full-length \u003cem\u003eEc\u003c/em\u003eAgo and \u003cem\u003eEc\u003c/em\u003ebAgaN were respectively synthesized by GeneGreat Biological Engineering Co. Ltd. (Wuhan, China) and inserted into pET28a vector for protein expression. For site-directed mutagenesis, the plasmid fragments (Supplementary information, Table S3) were constructed by inverse polymerase chain reaction using the primers with mutant sites.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eAll constructs were verified by DNA sequencing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhage plaque assays\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe \u003cem\u003eEc\u003c/em\u003eBPAN operon was cloned into the pQE80L vector under the control of T5 promoter. For assays examining \u003cem\u003eEc\u003c/em\u003eBPAN activity against different phages, \u003cem\u003eE. coli\u003c/em\u003e MG1655 strains carrying plasmids expressing \u003cem\u003eEc\u003c/em\u003eBPAN systems, or the empty vector control were grown overnight at 37℃ in LB medium supplemented with 100 \u0026mu;g/mL ampicillin. Phage resistance was assessed using a double-layer agar method, with 1.5% LB agar as the bottom layer and 0.5% LB agar containing bacterial cells as the top layer. \u003cem\u003eEc\u003c/em\u003eBPAN expression was induced with 0.01 mM IPTG. Serial 10-fold dilutions of phages were spotted onto the plates and incubated at 37℃ for the indicated times. For assays evaluating the anti-phage activity of \u003cem\u003eEc\u003c/em\u003eBPAN mutants against phage lab02, \u003cem\u003eE. coli\u003c/em\u003e C600 strains were used to express \u003cem\u003eEc\u003c/em\u003eBPAN and its mutants upon induction with 0.01 mM IPTG. Serially diluted lab02 phage samples were spotted onto the plates and incubated at 37℃ for ~7 h.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProtein expression and purification\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe recombinant plasmids used for the purification of \u003cem\u003eEc\u003c/em\u003eAgo and \u003cem\u003eEc\u003c/em\u003ebAgaN and their variants were transformed into \u003cem\u003eE. coli\u003c/em\u003e BL21 (DE3) cells, plated onto agar plate with 50 \u0026mu;g/mL kanamycin. Cells were added into LB medium and grown to an optical density reached 0.6 at 600 nm, and then induced by adding 0.3 mM isopropyl-\u003cem\u003e\u0026beta;\u003c/em\u003e-D-thiogalactopyranoside at 16 \u0026deg;C for 18 h. Cells were harvested by centrifugation (5000 rpm for 8 min at room temperature) and then resuspended in buffer A (25 mM Tris, 300 mM NaCl, pH 7.5, 20 mM imidazole). The lysate was clarified by centrifugation (12,000 rpm\u003cem\u003e\u0026nbsp;\u003c/em\u003efor 1 h at 4 \u0026deg;C) after disruption by a French press with 1000 bar (JuNeng Nano \u0026amp; Bio Technology Co. Ltd., Guangzhou, China). The His-tagged protein was loaded onto a Ni-NTA column pre-equilibrated in buffer A and eluted in buffer B (25 mM Tris, 300 mM NaCl, pH 7.5, 200 mM imidazole). Eluted protein was collected and concentrated in buffer C (25 mM Tris-HCl, 300 mM NaCl, pH 7.5). For \u003cem\u003eEc\u003c/em\u003eAgo, concentrated protein was loaded onto\u0026nbsp;5-mL HiTrap Heparin HP column (Cytiva) pre-equilibrated in buffer D (25\u0026nbsp;mM Tris-HCl, pH 7.5) and eluted with a linear gradient of buffer E (25 mM Tris-HCl, 1 M NaCl, pH 7.5).\u0026nbsp;Eluted \u003cem\u003eEc\u003c/em\u003eAgo protein was pooled and concentrated in buffer C. The purity of \u003cem\u003eEc\u003c/em\u003eAgo and \u003cem\u003eEc\u003c/em\u003ebAgaN were confirmed by SDS-PAGE.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eIn vitro\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;activity assays\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe oligonucleotides sequences are listed in Supplementary information, Table S2. The genomic DNA\u0026nbsp;were extracted from\u003cem\u003e\u0026nbsp;E. coli\u0026nbsp;\u003c/em\u003eDH5a by Bacteria Genomic DNA Kit (Tsingke, Wuhan). Unless otherwise specified, 500 nM \u003cem\u003eEc\u003c/em\u003eAgo or its mutant was first incubated with 500 nM guide RNA at 37℃ for 15 min in buffer C with\u0026nbsp;5 mM MgCl\u003csub\u003e2\u003c/sub\u003e and\u0026nbsp;5 mM MnCl\u003csub\u003e2\u003c/sub\u003e. Then 500 nM target DNA\u0026nbsp;was added and incubated\u0026nbsp;at 37℃\u0026nbsp;for\u0026nbsp;additional\u0026nbsp;15 min. Finally, ~300 ng\u0026nbsp;genomic DNA and 50\u0026nbsp;nM\u0026nbsp;\u003cem\u003eEc\u003c/em\u003ebAgaN or its mutant were added into the reaction mixtures, followed by\u0026nbsp;incubation\u0026nbsp;at 37℃\u0026nbsp;for\u0026nbsp;1 h. The\u0026nbsp;samples were treated with 2 mg/mL proteinase K and stopped by mixing loading dye and analyzed by agarose gel electrophoresis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eEc\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003eBPAN complex assembly\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGuide and target nucleic acids were dissolved with\u0026nbsp;buffer C. \u003cem\u003eEc\u003c/em\u003eAgo was first incubated with 21 nt\u0026nbsp;long 5\u0026rsquo;-phosphorylated guide RNA in buffer C containing 5 mM MgCl\u003csub\u003e2\u003c/sub\u003e for 15 min,\u0026nbsp;then 21 nt long target DNA was added and incubated for 15 min. Finally, the\u0026nbsp;\u003cem\u003eEc\u003c/em\u003ebAgaN was supplemented into the reaction mixture and then incubated for\u0026nbsp;15 min. The molar ratio of \u003cem\u003eEc\u003c/em\u003eAgo, guide, target and\u0026nbsp;\u003cem\u003eEc\u003c/em\u003ebAgaN was 1:1:1:1. All incubations were performed at room temperature. The acquired complex was applied to a size exclusion column (Superose 6 increase 10/300 GL, Cytiva) pre-equilibrated with\u0026nbsp;25 mM Tris-HCl, 150 mM NaCl, pH 7.5,\u0026nbsp;5 mM MgCl\u003csub\u003e2\u0026nbsp;\u003c/sub\u003eand 1 mM DTT. The protein fractions at different elution time were characterized by SDS-PAGE and collected for subsequent structural analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCryo-EM data acquisition and\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eprocessing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo prepare cryo-EM samples, 4 \u0026micro;L sample of \u003cem\u003eEc\u003c/em\u003eBPAN\u0026nbsp;complex\u0026nbsp;was applied onto glow-discharged holey carbon grids (Quantifoil Cu R1.2/1.3; 300 mesh). The grids were blotted for 5 seconds with force 2 in 100% humidity at 4 ℃, and then plunged into liquid ethane using a Vitrobot. Cryo-EM data collection was operated at 300 kV Titan Krios electron microscope equipped with a K3 summit electron direct detector. Micrographs were recorded in super-resolution mode at 105 000\u0026times; magnification (0.425 \u0026Aring;/pixel).\u0026nbsp;Each stack of 40 frames was exposed for 2.5 s with\u0026nbsp;a defocus range from\u0026nbsp;-1.0 \u0026micro;m to -2.2\u0026thinsp;\u0026mu;m, and the total dose of about\u0026nbsp;30 e\u003csup\u003e\u0026nbsp;\u0026minus;\u003c/sup\u003e/ \u0026Aring;\u003csup\u003e2\u003c/sup\u003e. 21 538 movies were collected in total in three individual times.\u003c/p\u003e\n\u003cp\u003eAll the cryo-EM movies were processed using CryoSPARC v4.2.1\u0026nbsp;\u003csup\u003e[42]\u003c/sup\u003e. The basic workflow was described in\u0026nbsp;Supplementary information, Fig. S3. In brief, the collected movies were motion-corrected with Patch Motion Correction and then subjected to contrast transfer function (CTF) estimation with Patch CTF. Micrograph curation was applied to exclude frames with CTF fit \u0026gt;4 \u0026Aring; or ice contamination. Subsequent Particle picking was performed with Blob/Template-based particle peaking and followed by several rounds of Iterative 2D classification, ab\u0026nbsp;initio reconstruction, and heterogeneous refinement. The final step was non-uniform refinement with imposed C2 symmetry.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eModel building and refinement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInitial models generated by AlphaFold2 were rigid-body docked into cryo-EM maps using ChimeraX v1.6.1\u0026nbsp;\u003csup\u003e[43]\u003c/sup\u003e. Manual rebuilding in Coot v0.9.8\u0026nbsp;\u003csup\u003e[44]\u003c/sup\u003e resolved poorly fitted regions, followed by iterative real-space refinement in Phenix v1.20.1\u0026nbsp;\u003csup\u003e[45]\u003c/sup\u003e (global minimization, rotamer optimization). Validation included: MolProbity (Ramachandran outliers \u0026lt;0.5%; clash score \u0026lt;5); EMRinger (rotamer Z-score \u0026gt;3); Map-model FSC (0.143 cutoff). Final structure was visualized in PyMOL v2.5.2 and ChimeraX. Refinement statistics are detailed in\u0026nbsp;Supplementary information, Table S1\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by Project of Technological Innovation Plan in Hubei Province (2024BCA001); Natural Science Foundation of Wuhan City (2024040701010046).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eM.L. and L.M. supervised the study. M.L. and Q.Y. prepared the samples and performed the activity assays. X.Z. and F.H. performed the phage plaque assays.\u0026nbsp;M.L., Q.X. and D.S. collected and processed the cryo-EM data and built the atomic model. M.L. wrote the manuscript. L.M., F.H. and Q.X. reviewed and edited the manuscript. All authors analyzed the data.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC\u003c/strong\u003e\u003cstrong\u003eompeting interest\u003c/strong\u003e\u003cstrong\u003es\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors claim no conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSwarts, D.C., et al. The evolutionary journey of Argonaute proteins. \u003cem\u003eNat. Struct. Mol. Biol\u003c/em\u003e \u003cstrong\u003e21\u003c/strong\u003e, 743-753 (2014).\u003c/li\u003e\n\u003cli\u003eOzata, D.M., Gainetdinov, I., Zoch, A., O\u0026apos;Carroll, D. \u0026amp; Zamore, P.D. PIWI-interacting RNAs: small RNAs with big functions. \u003cem\u003eNat. Rev. Genet.\u003c/em\u003e \u003cstrong\u003e20\u003c/strong\u003e, 89-108 (2019).\u003c/li\u003e\n\u003cli\u003eDing, S.W. RNA-based antiviral immunity. \u003cem\u003eNat. Rev. 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Real-space refinement in PHENIX for cryo-EM and crystallography. \u003cem\u003eActa Crystallogr D Biol Crystallogr\u003c/em\u003e \u003cstrong\u003e74\u003c/strong\u003e, 531-544 (2018).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"State Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei Key Laboratory of Industrial Biotechnology, School of Life Sciences, Hubei University","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"long-B pAgo, anti-phage activity, EcBPAN system, defense mechanism","lastPublishedDoi":"10.21203/rs.3.rs-8163682/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8163682/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eProkaryotic Argonautes (pAgos) are widely distributed and provide immunity against invading DNA. Based on their domain architecture, pAgos are classified into three major groups: long-A, long-B, and short pAgos. Among them, long-B pAgos remain the least understood subgroup. Here, we show that a long-B pAgo-nuclease system (\u003cem\u003eEc\u003c/em\u003eBPAN) from \u003cem\u003eEscherichia coli \u003c/em\u003eprovides the first clear evidence of anti-phage activity in long-B pAgo systems. By combining structural determination, biochemical analyses, and \u003cem\u003ein vivo\u003c/em\u003e phage-resistance assays, we elucidated the activation mechanism of \u003cem\u003eEc\u003c/em\u003eBPAN. We found that RNA-guided \u003cem\u003eEc\u003c/em\u003eAgo recognizes target DNA and subsequently recruits the autoinhibited dimer of its associated nuclease (\u003cem\u003eEc\u003c/em\u003ebAgaN) to form an unprecedented 8:8 pAgo-nuclease complex with robust nonspecific DNase activity. The cryo-EM structure of the activated complex revealed a distinctive bowl-shaped architecture in which the C-terminal nuclease domains of \u003cem\u003eEc\u003c/em\u003ebAgaN form an active octamer, while the N-terminal domains engage four \u003cem\u003eEc\u003c/em\u003eAgo-guide RNA-target DNA ternary dimers in an interleaved manner, thereby relieving autoinhibition. Together, these findings provide the first mechanistic insights into a long-B pAgo defense system and reveal a mode of immune activation fundamentally distinct from those of long-A and short pAgo systems.\u003c/p\u003e","manuscriptTitle":"Distinct mechanism of the long-B prokaryotic Argonaute-mediated nuclease activation in bacterial immunity","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-21 05:03:42","doi":"10.21203/rs.3.rs-8163682/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"851fab98-e439-4bc0-8860-c5cb839e051b","owner":[],"postedDate":"November 21st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-12-18T17:31:02+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-21 05:03:42","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8163682","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8163682","identity":"rs-8163682","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}