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. 2024 Feb 28;52(4):2012-2029.
doi: 10.1093/nar/gkad1248.

Target DNA-dependent activation mechanism of the prokaryotic immune system SPARTA

Giada Finocchio  1 , Balwina Koopal  2 , Ana Potocnik  2 , Clint Heijstek  2 , Adrie H Westphal  2 , Martin Jinek  1 , Daan C Swarts  2
Affiliations

Target DNA-dependent activation mechanism of the prokaryotic immune system SPARTA

Giada Finocchio et al. Nucleic Acids Res. .

Abstract

In both prokaryotic and eukaryotic innate immune systems, TIR domains function as NADases that degrade the key metabolite NAD+ or generate signaling molecules. Catalytic activation of TIR domains requires oligomerization, but how this is achieved varies in distinct immune systems. In the Short prokaryotic Argonaute (pAgo)/TIR-APAZ (SPARTA) immune system, TIR NADase activity is triggered upon guide RNA-mediated recognition of invading DNA by an unknown mechanism. Here, we describe cryo-EM structures of SPARTA in the inactive monomeric and target DNA-activated tetrameric states. The monomeric SPARTA structure reveals that in the absence of target DNA, a C-terminal tail of TIR-APAZ occupies the nucleic acid binding cleft formed by the pAgo and TIR-APAZ subunits, inhibiting SPARTA activation. In the active tetrameric SPARTA complex, guide RNA-mediated target DNA binding displaces the C-terminal tail and induces conformational changes in pAgo that facilitate SPARTA-SPARTA dimerization. Concurrent release and rotation of one TIR domain allow it to form a composite NADase catalytic site with the other TIR domain within the dimer, and generate a self-complementary interface that mediates cooperative tetramerization. Combined, this study provides critical insights into the structural architecture of SPARTA and the molecular mechanism underlying target DNA-dependent oligomerization and catalytic activation.

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Figures

Graphical Abstract
Graphical Abstract
Figure 1.
Figure 1.
Molecular architecture of the monomeric apo-SPARTA complex. (A) Schematic diagram of the domain organization of the BabTIR-APAZ and BabAgo proteins. TIR, Toll-interleukin-1 receptor-like; APAZ, analog of PIWI-AGO-ZWILLE domain; Ct, C-terminal tail; MID, Middle; PIWI, P-element induced (PIWI, P-element induced wimpy testis domain.). (B) Cryo-electron microscopic (cryo-EM) densities of the BabSPARTA complex. Domains are colored according to the scheme in (A). (C) Cartoon representation of the overall structure of the Apo-BabSPARTA complex. Domains are colored according to the scheme in (A). (D) Close-up view of MID-TIR interactions. (E) Close-up view of PIWI-APAZ interactions. (F and G) Multiple residues at the PIWI-APAZ interface contribute to heterodimerization of TIR-APAZ and pAgo. 6xHis-MBP-BabTIR-APAZ or mutants thereof (F) were co-expressed in E. coli with BabAgo or mutants thereof (G) and proteins were purified using amylose affinity chromatography. Graphs show the percentage of the BabAgo/BabTIR-APAZ ratio normalized against the BabAgo/BabTIR-APAZ ratio of the WT proteins. Expression and consecutive pull-down experiments were performed in triplicates and data points reflect individual replicates. See also Supplementary Figure S3.
Figure 2.
Figure 2.
The C-terminal tail of TIR-APAZ blocks the nucleic acid binding cleft of the monomeric SPARTA complex. (A) Close-up view of the interactions of the C-terminal tail with the MID and APAZ domains. (B) Total NAD (NAD+ + NADH) level in E. coli cultures expressing MapSPARTA, catalytic mutant E77ATIR-APAZ, or C-terminal tail truncation mutant Δ417–450TIR-APAZ in the absence or presence of a highly transcribed high copy number plasmid (pUC-mRFPΔRBS), determined 4 h after induction of MapSPARTA expression. The averages of three biological replicates are shown, error bars indicate standard deviations. ***P < 0.001. (C) Growth curves of E. coli cultures expressing MapSPARTA, catalytic mutant E77ATIR-APAZ, or C-terminal tail truncation mutant Δ417–450TIR-APAZ in the absence or presence of pUC-mRFPΔRBS. Cultures were started with pre-cultures in which MapSPARTA expression was induced 4 h earlier. The averages of three biological replicates are shown, shaded areas indicate standard deviations. (D) In vitro NADase activity of WT and C-terminally truncated BabSPARTA in the presence of varying concentrations of target ssDNA. Total fluorescence was measured over time after the addition of ϵ-NAD+. The averages of three technical replicates are shown, shaded areas indicate standard deviations. (E) Affinity of WT and C-terminally truncated SPARTA for guide RNA. Equilibrium dissociation constants (Kd) were determined by measuring fluorescence polarization of a 3′-ATTO532-labelled guide RNA in the presence of varying concentrations of SPARTA or SPARTA mutant Δ417–450TIR-APAZ. Relative Fluorescence Polarization (Supplementary Figure S7F) was used to calculate the Kd. The data points in the graph reflect averages of three technical replicates and error bars indicate standard deviations.
Figure 3.
Figure 3.
Molecular architecture of the activated tetrameric SPARTA complex. (A) Cryo-electron microscopy (cryo-EM) densities of the tetramer of guide RNA/target DNA duplex-bound BabSPARTA complexes. SPARTA protein domains are colored according to the scheme Figure 1A, the guide/target duplex is colored according to the scheme in panel (A). (B) Cartoon representation of the overall structure of the tetrameric BabSPARTA complex. (C) Schematic representation of the interactions of SPARTA with the guide RNA-target DNA duplex. Transparent and dotted rectangles indicate structurally disordered nucleotides. Hydrogen-bonding and electrostatic interactions are indicated with dotted lines. (D) Vector map displaying observed domain movement between the apo-BabSPARTA complex and each of the BabSPARTA protomers in the activated BabSPARTA complex. Vectors are colored corresponding to the domain coloring used in other panels.
Figure 4.
Figure 4.
RNA-guided target DNA binding induces pAgo-pAgo interactions. (A) Guide RNA/target DNA duplex binding is associated with conformational changes of the sensor loop and other segments within the PIWI domain. (B–E) Structural rearrangements facilitate the formation of polar interactions and salt bridges between MID and PIWI domain residues. See also Supplementary Figure S10. (F) Mutational analyses reveal that pAgo-pAgo interaction residues are crucial for catalytic activation of SPARTA. The total NAD (NAD+ + NADH) level was determined in E. coli cultures expressing MapSPARTA, catalytic mutant E77ATIR-APAZ, or MapSPARTA with mutations at the pAgo-pAgo interface, in presence of a highly transcribed high copy number plasmid (pUC-mRFPΔRBS). The averages of three biological replicates are shown, error bars indicate standard deviations. *p < 0.05, **p < 0.01; ***p < 0.001. See also Supplementary Figure S11. (G–I) SPARTA activation is increased by target sites co-localized in cis. BabSPARTA was mixed with guide RNA and ssDNA targets that contain a single target site (split target) or two target sites (chained target) complementary to the guide RNA, with equal abundance of binding sites in every condition (G). After addition of ϵ-NAD+ fluorescence was measured over time. SPARTA shows increased NADase activity with chained targets compared to split targets and chained targets in which one of the target sites is scrambled (H). SPARTA activation by chained targets is influenced by the linker length that connect the two target sites (I). In panel H and I, measurements are corrected for a control without ssDNA target, and normalized to the minimum and maximum of each individual reaction. The averages of three technical replicates are shown, shadings indicate standard deviations.
Figure 5.
Figure 5.
TIR domain repositioning facilitates tetramerization and catalytic activation. (A) Comparison of the TIR domain orientation between two SPARTA protomers interacting after tetramerization. The TIR domain in protomer A1 (in pink) is rotated 177° with respect to the TIR domain in protomer B1 (teal), with helix αF (bright orange) functioning as a hinge. Alignment performed with respect to the pAgo-APAZ moieties (grey). (B) Alignment of the TIR domains of the inactive apo monomeric SPARTA complex structure (green) with the TIR domains of two asymmetric SPARTA protomers from the activated tetrameric SPARTA structure (orange and teal). The overall TIR domain structure is maintained, except for the rotation of the αF helix (bright orange) in protomer A1 (orange) of the activated tetrameric SPARTA structure. (C) Overview of the assembly of the four TIR domains (TIR_A1/B1/A2/B2) in the activated tetrameric SPARTA structure. (D) TIR-TIR interfaces and key residues involved in TIR-TIR dimerization, TIR tetramerization, and TIR catalytic activity. (E, F) TIR-TIR interface residues are crucial for catalytic activation of SPARTA. The total NAD (NAD+ + NADH) level (E) or OD600 nm (F) was determined in E. coli cultures expressing MapSPARTA, catalytic mutant E77ATIR-APAZ, or MapSPARTA with mutations at the TIR-TIR interfaces, in presence of a highly transcribed high copy number plasmid (pUC-mRFPΔRBS). The MapSPARTA and the E77ATIR-APAZ mutant controls in the right panel are identical to those in Figure 1 (same experiment). The averages of three biological replicates are shown, error bars indicate standard deviations. *P < 0.05, **P < 0.01; ***P < 0.001. See also Supplementary Figure S13.
Figure 6.
Figure 6.
Schematic model of guide RNA-mediated target binding and catalytic activation of SPARTA.
Figure 7.
Figure 7.
Comparison of oligomerization modes of enzymatic and scaffolding TIR domains found in bacteria, animals, and plants. Representative structures of TIR containing proteins with different origin, assembly geometry and function. Schematics of the main interaction interfaces are represented using triangles of different colors for different TIR-helices positions. (AbTir TIR, PDB: 7UXU; SfTIR-STING, PDB: 7UN8; MkTIR-SAVED, PDB: 7QQK; hSARM1 TIR, PDB: 7NAK; hMAL TIR, PDB: 5UZB; hMyD88 TIR, PDB: 6I3N; NbTIR-dsDNA, PDB: 7 × 5K; AtNLR-RPP1, PDB: 7CRC; NbRoq1-XopQ, PDB: 7JLV).
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References

    1. Bobadilla Ugarte P., Barendse P., Swarts D.C. Argonaute proteins confer immunity in all domains of life. Curr. Opin. Microbiol. 2023; 74:102313. - PubMed
    1. Swarts D.C., Makarova K., Wang Y., Nakanishi K., Ketting R.F., Koonin E.V., Patel D.J., Van Der Oost J. The evolutionary journey of argonaute proteins. Nat. Struct. Mol. Biol. 2014; 21:743–753. - PMC - PubMed
    1. Burroughs A.M., Ando Y., Aravind L. New perspectives on the diversification of the RNA interference system: insights from comparative genomics and small RNA sequencing. Wiley Interdiscip. Rev. RNA. 2014; 5:141–181. - PMC - PubMed
    1. Meister G. Argonaute proteins: functional insights and emerging roles. Nature Publishing Group. 2013; 14:447. - PubMed
    1. Jolly S.M., Gainetdinov I., Jouravleva K., Zhang H., Strittmatter L., Bailey S.M., Hendricks G.M., Dhabaria A., Ueberheide B., Zamore P.D. Thermus thermophilus argonaute functions in the completion of DNA replication. Cell. 2020; 182:1545–1559. - PMC - PubMed

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