Abstract
Although eukaryotic and long prokaryotic Argonaute proteins (pAgos) cleave nucleic acids, some short pAgos lack nuclease activity and hydrolyse NAD(P)+ to induce bacterial cell death1. Here we present a hierarchical activation pathway for SPARTA, a short pAgo consisting of an Argonaute (Ago) protein and TIR–APAZ, an associated protein2. SPARTA progresses through distinct oligomeric forms, including a monomeric apo state, a monomeric RNA–DNA-bound state, two dimeric RNA–DNA-bound states and a tetrameric RNA–DNA-bound active state. These snapshots together identify oligomerization as a mechanistic principle of SPARTA activation. The RNA–DNA-binding channel of apo inactive SPARTA is occupied by an auto-inhibitory motif in TIR–APAZ. After the binding of RNA–DNA, SPARTA transitions from a monomer to a symmetric dimer and then an asymmetric dimer, in which two TIR domains interact through charge and shape complementarity. Next, two dimers assemble into a tetramer with a central TIR cluster responsible for hydrolysing NAD(P)+. In addition, we observe unique features of interactions between SPARTA and RNA–DNA, including competition between the DNA 3′ end and the auto-inhibitory motif, interactions between the RNA G2 nucleotide and Ago, and splaying of the RNA–DNA duplex by two loops exclusive to short pAgos. Together, our findings provide a mechanistic basis for the activation of short pAgos, a large section of the Ago superfamily.
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Data availability
Accession numbers for apo monomeric MapSPARTA, RNA–DNA-bound monomeric MapSPARTA, symmetric MapSPARTA dimer, asymmetric MapSPARTA dimer, MapSPARTA tetramer and MapSPARTA tetramer with NAD+ are as follows: coordinates of atomic models: 8FEX, 8SQU, 8SP0, 8SP3, 8FFI and 8SPO, deposited to the Protein Data Bank (PDB); and density maps: EMD-29033, EMD-40679, EMD-40713, EMD-40672, EMD-40673, EMD-29043 and EMD-40680, deposited to the Electron Microscopy Data Bank (EMDB). All data needed to evaluate the conclusions are present in the paper. Source data are provided with this paper.
References
Koopal, B., Mutte, S. K. & Swarts, D. C. A long look at short prokaryotic Argonautes. Trends Cell Biol. 33, 605–618 (2023).
Koopal, B. et al. Short prokaryotic Argonaute systems trigger cell death upon detection of invading DNA. Cell 185, 1471–1486 (2022).
Swarts, D. C. et al. The evolutionary journey of Argonaute proteins. Nat. Struct. Mol. Biol. 21, 743–753 (2014).
Vaucheret, H. Plant ARGONAUTES. Trends Plant Sci. 13, 350–358 (2008).
Nakanishi, K. Anatomy of four human Argonaute proteins. Nucleic Acids Res. 50, 6618–6638 (2022).
Peters, L. & Meister, G. Argonaute proteins: mediators of RNA silencing. Mol. Cell 26, 611–623 (2007).
Lisitskaya, L., Aravin, A. A. & Kulbachinskiy, A. DNA interference and beyond: structure and functions of prokaryotic Argonaute proteins. Nat. Commun. 9, 5165 (2018).
Ryazansky, S., Kulbachinskiy, A. & Aravin, A. A. The expanded universe of prokaryotic Argonaute proteins. mBio 9, e01935-18 (2018).
Kuzmenko, A. et al. DNA targeting and interference by a bacterial Argonaute nuclease. Nature 587, 632–637 (2020).
Swarts, D. C. et al. DNA-guided DNA interference by a prokaryotic Argonaute. Nature 507, 258–261 (2014).
Zander, A. et al. Guide-independent DNA cleavage by archaeal Argonaute from Methanocaldococcus jannaschii. Nat. Microbiol. 2, 17034 (2017).
Li, W. et al. A programmable pAgo nuclease with RNA target preference from the psychrotolerant bacterium Mucilaginibacter paludis. Nucleic Acids Res. 50, 5226–5238 (2022).
Swarts, D. C. et al. Argonaute of the archaeon Pyrococcus furiosus is a DNA-guided nuclease that targets cognate DNA. Nucleic Acids Res. 43, 5120–5129 (2015).
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).
Hegge, J. W. et al. DNA-guided DNA cleavage at moderate temperatures by Clostridium butyricum Argonaute. Nucleic Acids Res. 47, 5809–5821 (2019).
Jolly, S. M. et al. Thermus thermophilus Argonaute functions in the completion of DNA replication. Cell 182, 1545–1559 (2020).
Olovnikov, I., Chan, K., Sachidanandam, R., Newman, D. K. & Aravin, A. A. Bacterial argonaute samples the transcriptome to identify foreign DNA. Mol. Cell 51, 594–605 (2013).
Makarova, K. S., Wolf, Y. I. & Koonin, E. V. Comprehensive comparative-genomic analysis of type 2 toxin–antitoxin systems and related mobile stress response systems in prokaryotes. Biol. Direct 4, 19 (2009).
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 5, 141–181 (2014).
Zaremba, M. et al. Short prokaryotic Argonautes provide defence against incoming mobile genetic elements through NAD+ depletion. Nat. Microbiol. 7, 1857–1869 (2022).
Zeng, Z. et al. A short prokaryotic Argonaute activates membrane effector to confer antiviral defense. Cell Host Microbe 30, 930–943 (2022).
Song, J. J., Smith, S. K., Hannon, G. J. & Joshua-Tor, L. Crystal structure of Argonaute and its implications for RISC slicer activity. Science 305, 1434–1437 (2004).
Nakanishi, K., Weinberg, D. E., Bartel, D. P. & Patel, D. J. Structure of yeast Argonaute with guide RNA. Nature 486, 368–374 (2012).
Elkayam, E. et al. The structure of human argonaute-2 in complex with miR-20a. Cell 150, 100–110 (2012).
Schirle, N. T. & MacRae, I. J. The crystal structure of human Argonaute2. Science 336, 1037–1040 (2012).
Shi, Y. et al. Structural basis of SARM1 activation, substrate recognition, and inhibition by small molecules. Mol. Cell 82, 1643–1659 (2022).
Hogrel, G. et al. Cyclic nucleotide-induced helical structure activates a TIR immune effector. Nature 608, 808–812 (2022).
Morehouse, B. R. et al. Cryo-EM structure of an active bacterial TIR-STING filament complex. Nature 608, 803–807 (2022).
Wang, Y. et al. Nucleation, propagation and cleavage of target RNAs in Ago silencing complexes. Nature 461, 754–761 (2009).
Miyoshi, T., Ito, K., Murakami, R. & Uchiumi, T. Structural basis for the recognition of guide RNA and target DNA heteroduplex by Argonaute. Nat. Commun. 7, 11846 (2016).
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).
Potocnik, A. & Swarts, D. C. Short prokaryotic Argonaute system repurposed as a nucleic acid detection tool. Clin. Transl. Med. 12, e1059 (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 Chimera—a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).
Rosenthal, P. B. & Henderson, R. Optimal determination of particle orientation, absolute hand, and contrast loss in single-particle electron cryomicroscopy. J. Mol. Biol. 333, 721–745 (2003).
Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004).
Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D 66, 213–221 (2010).
Williams, C. J. et al. MolProbity: more and better reference data for improved all-atom structure validation. Protein Sci. 27, 293–315 (2018).
The PyMOL Molecular Graphics System v.2.5 (Schrödinger, 2022).
Pettersen, E. F. et al. UCSF ChimeraX: structure visualization for researchers, educators, and developers. Protein Sci. 30, 70–82 (2021).
Acknowledgements
We thank W. Tang and M. Elowitz for discussions; Q. Lin for help with the collection and processing of structural data; M. Kearse for technical assistance with the NADase assay; and A. Day for technical assistance with binding assays. Grid screening was performed at OSU CEMAS with the assistance of G. Grandinetti and Y. Narui. Cryo-EM data were collected with the assistance of C. Zhang and P. Mitchell at the Stanford-SLAC Cryo-Electron Microscopy Center, supported by grants from the NIH National Institute of Health Common Fund Transformative High Resolution Cryo-Electron Microscopy program, O. Davulcu at Pacific Northwest Center for Cryo-EM and G. Grandinetti and Y. Narui at OSU CEMAS. K.N. is supported by NIH grants R01GM124320 and R01GM138997. S.X. is supported by a postdoctoral fellowship from the Jane Coffin Childs Fund.
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Contributions
T.-M.F. conceived the project. Z.S. and X.-Y.Y. performed mutagenesis, biochemical purification, oligomerization assays and NADase assays. Z.S., X.-Y.Y. and T.-M.F. prepared grids, determined the cryo-EM structures and built the models. Z.S. and W.H. performed the binding assay and analysed the data. D.J.T. supervised the binding assay. T.-M.F., Z.S., X.-Y.Y., S.X. and K.N. analysed the data. T.-M.F. and S.X. wrote the manuscript with input from all authors.
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Nature thanks Andrey Kulbachinskiy and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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Extended data figures and tables
Extended Data Fig. 1 Purification and structural reconstruction of MapSPARTA.
a, Domain arrangement of Homo sapiens Ago2 (an eAgo) and Pyrococcus furiosus Ago (a long pAgo). b, Diagram of the construct for MapSPARTA expression. TIR-APAZ and short pAgo are cloned into a poly-cistron for expression. c, Gel filtration profile of MapSPARTA in apo state purification. d, SDS–PAGE of samples from gel filtration in c, showing the purity of MapSPARTA. e, Gel filtration profile of MapSPARTA in complex with DNA–RNA duplex, revealing a monomeric peak and a tetrameric peak. f, Workflow for the 3D reconstruction of MapSPARTA in apo state using cryoSPARC. g, FSC curve of 3D reconstruction of MapSPARTA in apo state. h, Local resolutions of the reconstructions correlating with the final map in g. Resolutions are colour-coded by scale bars.
Extended Data Fig. 2 Structural comparison.
a, Ribbon diagram of TIR-APAZ with secondary structures labelled. b, Ribbon diagram of pAgo, in which PIWI (yellow) and MID (magenta) form a cleft in the middle. c, Overlaid structures of TIR domains from TIR-APAZ (green) and MyD88 (wheat, PDB ID 7BEQ). d–f, Superimposed structures of MapSPARTA (green) with P. furiosus Ago (PfAgo, magenta, PDB ID 1U04;d), yeast Ago (magenta, PDB ID 4F1N; e), and human Ago2 (magenta, PDB ID 4EI1; f), respectively. The unique auto-inhibitory CTM of MapSPARTA is highlighted in surface representation (green). g, Overlaid structures of the APAZ domain (green) of MapSPARTA and the N domain (magenta), PAZ domain (pink), L1 (magenta) and L2 (orange) domain of PfAgo. h, Overlaid structures of PIWI domains from MapAgo (yellow) and PfAgo (magenta) with catalytic residues in sticks.
Extended Data Fig. 3 Interfaces between TIR-APAZ and pAgo.
a, Interfaces between TIR-APAZ and pAgo with pAgo showing in electrostatic surface and TIR-APAZ in ribbon diagram. b, Surface areas of different interfaces shown in a. c, Interfaces between the APAZ domain and the PIWI domain. Key residues on the interfaces are highlighted in sticks. d, Interfaces between the APAZ domain and the MID domain. Key residues on the interfaces are highlighted in sticks. e, Detailed interactions between the TIR domain and the MID domain. Key residues on the interfaces are highlighted in sticks. f, Representative kinetics data of NAD+ hydrolysis by wild-type and CTM-truncated MapSPARTA. g, Quantification of the catalytic activities of wild-type and CTM-truncated MapSPARTA. Data are mean ± s.d. from three or more replicates as indicated (WT, n = 6; CTM-truncated SPARTA, n = 3).
Extended Data Fig. 4 Reconstruction of the MapSPARTA tetramer, dimer and monomer.
Workflow for the 3D reconstruction of MapSPARTA tetramer, dimer and monomer using cryoSPARC.
Extended Data Fig. 5 Resolutions of the MapSPARTA tetramer.
a–f, Local resolutions and FSC curves of reconstructed MapSPARTA tetramer (a), each protomer of the tetramer (b–e), and TIR domains (f).
Extended Data Fig. 6 Mechanism of TIR tetramerization.
a, Detailed interactions between TIRIA and TIRIIA with interfacial residues in sticks. b, Interface between TIR and TIR with key residues highlighted in sticks. c, TIRIIB engages with TIRIA and TIRIIA through tetramerization interfaces. d, Compared to wild type, V113R eluted as dimers and monomers in the presence of RNA–DNA. e, Compared to wild type, V113R/D106R/D111R eluted as dimers and monomers in the presence of RNA–DNA. f, Representative kinetic data of NAD+ hydrolysis by wild-type and mutant MapSPARTA. g, Gel filtration profile of TIR domain alone, showing that TIR domain eluted as a monomer. h, Compared to wild type, G42R/D44R eluted as monomers in the presence of RNA–DNA. i,j, BB loop in TIR in inactive state (i) and active state (j) fitted into cryo-EM densities at 2.0 σ. k, Overlaid structures of TIR in inactive state (pink) and in active state (blue), revealing conformational changes of the BB loop. l, BB loop conformational changes are crucial for the formation of the asymmetric dimer. Inactive TIR modelled into the asymmetric dimer revealed that the BB loop in the inactive state could clash with the other protomer.
Extended Data Fig. 7 MapSPARTA with NAD+ and catalytic mechanism of TIR.
a, Workflow for the 3D reconstruction of MapSPARTA tetramer with NAD+. b, Local resolutions of reconstructed MapSPARTA tetramer with NAD+. c, FSC curves of reconstructed MapSPARTA tetramer. d, TIR tetramer in complex with NAD+, revealing two NAD+-binding sites in the tetramer. e, NAD+ fitted into cryo-EM density at 2.0 σ. f, Overlaid structures of TIR domains from MapSPARTA and human SARM1 with a root-mean-square deviation (RMSD) of 1.0 Å. g, Overlaid structures of TIR domains from MapSPARTA and MkTIR-SAVED with an RMSD of 3.5 Å. h, Overlaid structures of TIR domains from MapSPARTA and SfTIR-STING with an RMSD of 5.8 Å. i, Representative kinetic data of NAD+ hydrolysis by MapSPARTA wild type and NAD+-coordinating mutants.
Extended Data Fig. 8 Interfaces between MapSPARTA and the RNA–DNA duplex.
a, Schematic depiction of the detailed interactions between MapSPARTA and RNA–DNA duplex. Residues from the PIWI domain, MID domain and APAZ domain are coloured in yellow, pink and blue, respectively. b, Detailed interactions between the PIWI domain and the RNA–DNA duplex. Residues involved in coordinating the RNA–DNA duplex are highlighted in sticks. DNA and RNA bases are labelled in green and red, respectively. c, Enlarged view of the interface between APAZ and RNA–DNA duplex. Residues involved in interacting with the RNA–DNA duplex are highlighted in sticks. DNA and RNA bases are labelled in green and red, respectively. d, Overlaid structures of apo-MapSPARTA and MapSPARTA with RNA–DNA, revealing the tilting of the negatively charged motif and positively charged pocket crucial for MID–MID interactions mediated dimerization.
Extended Data Fig. 9 Recognition of RNA by MapSPARTA.
a, A magnesium ion fitted into cryo-EM density at 2.5 σ. b–d, Expanded views of the 5′ nucleotide of guide RNA coordinated by residues in pockets of MID domains from MapAgo (b), C. sphaeroides long Ago (PDB ID 5AWH; c), and human Ago2 (PDB ID 4W5T; d). Magnesium ions in spheres are responsible in coordinating guide RNA by interacting with phosphate groups. Residues involved in coordinating guide RNA are highlighted in sticks. e–h, AA (e), UU (f), CC (g) and GG (h) as the first and second RNA nucleotides were modelled into the binding pocket, revealing a similar mechanism of being recognized by MapSPARTA. i,j, The sensorgrams of wild-type SPARTA binding to the chip-immobilized RNAs, UG-RNA (i) and AA-RNA (j), are expressed in shift versus time. The protein concentrations were 50, 25, 12.5, 6.25 and 3.12 nM (from top to bottom). k, Representative kinetic data of NAD+ hydrolysis by MapSPARTA in the presence of UG-RNA or AA-RNA. l, Plotted graphs of MapSPARTA catalysis stimulated by UG-RNA or AA-RNA. Data are mean ± s.d. from more than three replicates as indicated (UG-RNA, n = 6; AA-RNA, n = 4).
Extended Data Fig. 10 Reconstruction of the MapSPARTA monomer in complex with RNA–DNA.
a, Workflow for the 3D reconstruction of MapSPARTA monomer with RNA–DNA. b, Local resolutions and FSC curves of reconstructed MapSPARTA monomer with RNA–DNA from monomeric peak on gel filtration. c, Local resolutions and FSC curves of reconstructed MapSPARTA monomer with RNA–DNA from the tetramer dataset.
Extended Data Fig. 11 RNA–DNA-bound MapSPARTA dimers.
a, Local resolutions of MapSPARTA dimer in mixed states. b, FSC curves of reconstructed MapSPARTA dimer in mixed states. c, Local resolutions of MapSPARTA asymmetric dimer. d, FSC curves of reconstructed MapSPARTA asymmetric dimer. e, Overlaid structures of asymmetric dimer (green) and that (grey) in the tetramer, revealing their similarity. f, Symmetric dimer fitted into cryo-EM map. g, E134R, disrupting MID–MID interactions, eluted as monomers in the presence of RNA–DNA. h, MapSPARTA eluted as monomers in the presence of RNA.
Supplementary information
Supplementary Figures
This file contains 3 figures. Supplementary Figure 1: Uncropped SDS–PAGE from Extended Data Fig. 1d. Supplementary Figure 2: Sequence alignment of short pAgos from different species. Supplementary Figure 3: Sequence alignment of TIR domains.
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Shen, Z., Yang, XY., Xia, S. et al. Oligomerization-mediated activation of a short prokaryotic Argonaute. Nature 621, 154–161 (2023). https://doi.org/10.1038/s41586-023-06456-z
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DOI: https://doi.org/10.1038/s41586-023-06456-z
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