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Target ssDNA activates the NADase activity of prokaryotic SPARTA immune system

Nature Chemical Biology volume 20, pages 503–511 (2024)Cite this article

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Abstract

Argonaute proteins (Agos), which use small RNAs or DNAs as guides to recognize complementary nucleic acid targets, mediate RNA silencing in eukaryotes. In prokaryotes, Agos are involved in immunity: the short prokaryotic Ago/TIR–APAZ (SPARTA) immune system triggers cell death by degrading NAD+ in response to invading plasmids, but its molecular mechanisms remain unknown. Here we used cryo-electron microscopy to determine the structures of inactive monomeric and active tetrameric Crenotalea thermophila SPARTA complexes, revealing mechanisms underlying SPARTA assembly, RNA-guided recognition of target single-stranded DNA (ssDNA) and subsequent SPARTA tetramerization, as well as tetramerization-dependent NADase activation. The small RNA guides Ago to recognize its ssDNA target, inducing SPARTA tetramerization via both Ago- and TIR-mediated interactions and resulting in a two-stranded, parallel, head-to-tail TIR rearrangement primed for NAD+ hydrolysis. Our findings thus identify the molecular basis for target ssDNA-mediated SPARTA activation, which will facilitate the development of SPARTA-based biotechnological tools.

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Fig. 1: Overall architecture of monomeric C. thermophila SPARTAgRNA–target ssDNA complex.
Fig. 2: Recognition of the gRNA–ssDNA target duplex by the SPARTA complex.
Fig. 3: Overall architecture of the tetrameric SPARTAgRNA–ssDNA complex.
Fig. 4: Target ssDNA binding induces conformational changes in TIR domains.
Fig. 5: Formation of a SPARTA tetramer is mediated by pAgo and TIR domain interactions.
Fig. 6: Proposed mechanistic model of prokaryotic SPARTA complex activation in response to invading plasmid DNA.

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Data availability

The cryo-EM density maps have been deposited in the Electron Microscopy Data Bank (EMDB) under accession number EMD-35419 (monomeric SPARTAgRNA–ssDNA target complex); EMD-35420 (tetrameric SPARTAgRNA–ssDNA target complex in state 1); EMD-35421 (tetrameric SPARTAgRNA–ssDNA target complex in state 2) and EMD-36843 (SPARTAgRNA binary complex). The atomic coordinates have been deposited in the PDB with accession number PDB-8IFK (monomeric SPARTAgRNA–ssDNA target complex); PDB-8IFL (tetrameric SPARTAgRNA–ssDNA target complex in state 1); PDB-8IFM (tetrameric SPARTAgRNA–ssDNA target complex in state 2) and PDB-8K34 (SPARTAgRNA binary complex). Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request. Source data are provided with this paper.

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Acknowledgements

We thank the staff at Southern University of Science and Technology (SUSTech) Cryo-EM Center for assistance in data collection on the SUSTech Titan KRIOS cryo-electron microscope. This work was supported by the National Natural Science Foundation of China (grant no. 32270050 to N.J.), Guangdong and Shenzhen Natural Science Foundation (grant no. 2023A1515012420 and JCYJ20220530114409022 to N.J.), Key Project of Shenzhen Science and Technology Innovation Commission (grant no. JCYJ20220818100616034) and the Guangdong Provincial Science and Technology Innovation Council Grant (2017B030301018).

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Author notes

  1. These authors contributed equally: Jun-Tao Zhang, Xin-Yang Wei.

Authors and Affiliations

  1. Department of Biochemistry, School of Medicine, Southern University of Science and Technology, Shenzhen, China

    Jun-Tao Zhang, Xin-Yang Wei, Ning Cui & Ning Jia

  2. Department of Medical Neuroscience, School of Medicine, Southern University of Science and Technology, Shenzhen, China

    Ruilin Tian

  3. Key University Laboratory of Metabolism and Health of Guangdong, Southern University of Science and Technology, Shenzhen, China

    Ruilin Tian & Ning Jia

  4. Shenzhen Key Laboratory of Cell Microenvironment, Guangdong Provincial Key Laboratory of Cell Microenvironment and Disease Research, Southern University of Science and Technology, Shenzhen, China

    Ning Jia

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Contributions

J.-T.Z. and X.-Y.W. undertook biochemical studies, from sample preparation and purification, and biochemical assays. J.-T.Z also performed cryo-EM data collection data processing and structure refinement. N.C. contributed to the cryo-EM sample preparation, and R.T. participated in the helpful discussions regarding this project. N.J. directed the research. N.J. and J.-T.Z. wrote the manuscript with input from other authors.

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Correspondence to Ning Jia.

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Nature Chemical Biology thanks Dinshaw Patel and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 In vitro assembly of the SPARTAgRNA-ssDNA target complex.

(a) Size exclusion chromatography and SDS-PAGE profiles of the purified SPARTA complex. Data are representative of three independent experiments. (b) Size exclusion chromatography profile of the SPARTAgRNA and SPARTAgRNA-ssDNA complexes.

Source data

Extended Data Fig. 2 CryoEM reconstruction of monomeric and tetrameric SPARTA complexes in two different assembly states.

(a, d and g) Fourier Shell Correlation curve of the monomeric SPARTA complex (a) and the tetrameric SPARTA complex in assembly state 1(d) and state 2 (g). (b, e and h) Direction distribution plot of the monomeric SPARTA complex (b) and the tetrameric SPARTA complex in assembly state 1 (e) and state 2 (h). (c, f and i) Final three-dimensional reconstructed map of the monomeric SPARTA complex (c) and the tetrameric SPARTA complex in assembly state 1 (f) and state 2 (i), colored according to local resolution.

Extended Data Fig. 3 CryoEM densities of each domain and the gRNA- ssDNA target duplex in the monomeric SPARTAgRNA-target ssDNA complex structure.

Densities for indicated regions are shown in the context of the atomic model.

Extended Data Fig. 4 Structure and sequence comparisons of the SPARTA complex with related proteins.

(a) Structural comparison of the prokaryotic R. sphaeroides RsAgo complex (PDB 5AWH, in grey) and monomeric SPARTAgRNA-ssDNA complex (in color). (b) Multiple sequence alignment of the conserved catalytic tetrad motif in the PIWI domain of C. thermophila SPARTA, NP_036286.2 (Homo sapiens AGO2), WP_011011654.1 (Pyrococcus furiosus Argonaute) and WP_011174533.1 (Thermus thermophilus Argonaute). The catalytic motif DEDX is indicated with red font. (c) Structural comparison of the catalytic tetrad between T. thermophilus TtAgo (grey, PDB 3F73) and C. thermophila SPARTA (dark green). (d) Structural alignment of the R. sphaeroides RsAgo N domain, L1, L2 (PDB 5AWH) and C. thermophila SPARTA APAZ domain. (e) The C. thermophila SPARTA TIR domain (magenta) structurally resembles the H. sapiens MyD88 TIR domain (grey, PDB 7BEQ). (f) Ribbon representation of the SPARTA TIR domain. The conserved structural elements (αA–αE, βA–βE) and the BB loop critical for NADase activity are labeled.

Extended Data Fig. 5 TIR domain assembly patterns.

(a) Ribbon (left) and surface (right) comparision of tetrameric SPARTA complex in two different states. Tetrameric SPARTA in the second state can transfer to the first state via 180° rotation horizontally. (b) Ribbon and schematic representations of TIR domain assembly patterns in H. sapiens MyD88 (PDB 7BEQ), H. sapiens SARM1 (PDB 6O0Q) and AbTIR (PDB 7UXU).

Extended Data Fig. 6 Overall architecture of the SPARTAgRNA binary complex.

(a and b) Direction distribution plot (a) and Fourier Shell Correlation curve (b) of the SPARTAgRNA complex. (c and d) Surface (c) and ribbon (d) representations of the SPARTAgRNA complex. (e) CryoEM densities of the guide RNA in the SPARTAgRNA complex. (f) CryoEM densities of the sensor loop and sensor helix in the SPARTAgRNA complex. (g) Structural comparison of the SPARTAgRNA complex with the protomer2 of tetrameric SPARTA complex. Vector length correlates with the domain movement scale. (h) Structural comparison between the SPARTAgRNA binary and the protomer 2 of the tetrameric SPARTAgRNA-ssDNA complex. The C-tail region of SPARTAgRNA binary is indicated.

Extended Data Fig. 7 Conformational changes of the SPARTAgRNA complex upon target ssDNA binding.

(a) Structural comparison between the SPARTAgRNA binary and the protomer 2 of the tetrameric SPARTAgRNA-ssDNA complex. The sensor loop and sensor helix regions are indicated. (b and d) Effect of sensor loop and sensor helix mutants together with the C-tail deletion mutant on NADase activity. Data are representative of three independent experiments. Data are presented as mean values ± s.d. (c) SDS-PAGE profiles of SPARTA mutants. ΔC-tail indicates deletion of the C-tail in the APAZ domain. KHR and KE mutants indicate mutation of residues K354, H358, R361 in the APAZ domain and K325, E327 in the PIWI domain to alanines, respectively. Loop GS linker indicates the replacement of Y321PIWI to Y328PIWI with a GS linker. A MBP tag is fused to the N terminal of TIR-APAZ subunit in all SPARTA mutants. Data are representative of three independent experiments.

Source data

Extended Data Fig. 8 Structural comparison of the MID and TIR domains of SPARTA and other proteins.

(a) Structural comparison of the MID domains of the monomeric SPARTA complex (grey) and the tetrameric SPARTA complex (yellow). (b) Multiple sequence alignment of the loop in the SPARTA MID domain and other Agos. The aligned sequences are from WP_109649955.1 (Maribacter polysiphoniae Argonaute, MapAgo), ABP72561.1 (Rhodobacter sphaeroides Argonaute, RsAgo), WP_011011654.1 (Pyrococcus furiosus Argonaute, PfAgo), NP_036286.2 (human AGO2), WP_010880937.1 (Aquifex aeolicus Argonaute, AaAgo), WP_011174533.1 (Thermus thermophilus Argonaute, TtAgo), WP_014295921.1 (Marinitoga piezophila Argonaute, MpAgo). (c) Structural alignment of the loop in the SPARTA MID domain with T. thermophilus TtAgo (grey, PDB 3HVR). (d) Docking of NAD+ into the TIR domains of tetrameric SPARTA, and superimposition of SPARTA TIRs with AbTIR (PDB 7UXU). (e) The putative NAD+ binding pocket in the SPARTA TIR domains superimposed with AbTIR.

Extended Data Fig. 9 Size exclusion chromatography analysis of SPARTA mutants.

(a) Size exclusion chromatography (left) and SDS-PAGE profiles (right) of SPARTA mutants. E133-D137 (GSGSG) indicates the replacement of loop (133-EERVD-137) with a GSGSG linker in the MID domain. G42P and E50A indicate substitution of residues G42 and E50 in the TIR domain into proline and alanine, respectively. A MBP tag is fused to the N terminal of TIR-APAZ subunit in all SPARTA mutants. Red asterisk indicates the individual MBP tag. Data are representative of three independent experiments. (b) The left panel indicates size exclusion chromatography analysis of SPARTA E77A mutant. Then the tetrameric SPARTAE77A-gRNA-ssDNA complex was reloaded for a second round of size exclusion chromatography analysis (right panel).

Source data

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Supplementary Fig. 1 and Tables 1 and 2.

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Source Data Figs. 2c, 3a and 5c,e and Extended Data Fig. 7b,d

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Source Data Extended Data Fig. 1

Unprocessed gels of Extended Data Fig. 1a.

Source Data Extended Data Fig. 7

Unprocessed gels of Extended Data Fig. 7c.

Source Data Extended Data Fig. 9

Unprocessed gels of Extended Data Fig. 9a.

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Zhang, JT., Wei, XY., Cui, N. et al. Target ssDNA activates the NADase activity of prokaryotic SPARTA immune system. Nat Chem Biol 20, 503–511 (2024). https://doi.org/10.1038/s41589-023-01479-z

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