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Auto-inhibition and activation of a short Argonaute-associated TIR-APAZ defense system

Nature Chemical Biology volume 20, pages 512–520 (2024)Cite this article

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Abstract

Short prokaryotic Ago accounts for most prokaryotic Argonaute proteins (pAgos) and is involved in defending bacteria against invading nucleic acids. Short pAgo associated with TIR-APAZ (SPARTA) has been shown to oligomerize and deplete NAD+ upon guide-mediated target DNA recognition. However, the molecular basis of SPARTA inhibition and activation remains unknown. In this study, we determined the cryogenic electron microscopy structures of Crenotalea thermophila SPARTA in its inhibited, transient and activated states. The SPARTA monomer is auto-inhibited by its acidic tail, which occupies the guide-target binding channel. Guide-mediated target binding expels this acidic tail and triggers substantial conformational changes to expose the Ago–Ago dimerization interface. As a result, SPARTA assembles into an active tetramer, where the four TIR domains are rearranged and packed to form NADase active sites. Together with biochemical evidence, our results provide a panoramic vision explaining SPARTA auto-inhibition and activation and expand understanding of pAgo-mediated bacterial defense systems.

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Fig. 1: Auto-inhibition of SPARTA by an acidic C-terminal tail.
Fig. 2: Guide-target duplex binding activates SPARTA.
Fig. 3: Ago–Ago interaction induced by guide-target binding is the prerequisite for SPARTA activation.
Fig. 4: TIR rearrangement and packing shapes NAD+ active sites in SPARTA tetramer.
Fig. 5: Proposal for the mechanism of SPARTA auto-inhibition and activation.

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Data availability

Atomic coordinates, maps and structure factors of the reported cryo-EM structures have been deposited in the PDB under accession codes 8J84 (SPARTA-apo), 8J9G (monomeric SPARTA bound with guide-target, state 1: Monomer-GT1), 8J8H (monomeric SPARTA bound with guide-target, state 2: Monomer-GT2), 8J9P (SPARTA dimer bound with guide-target) and 8JAY (SPARTA tetramer bound with guide-target) and in the Electron Microscopy Data Bank under accession codes EMD-36059 (SPARTA-apo), 36095 (monomeric SPARTA bound with guide-target, state 1: Monomer-GT1), 36070 (monomeric SPARTA bound with guide-target, state 2: Monomer-GT2), 36114 (SPARTA dimer bound with guide-target) and 36138 (SPARTA tetramer bound with guide-target). The structures used for comparison in this study are available under PDB accession codes 5GQ9, 5UZB and 6O0Q. Source data are provided with this paper.

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Acknowledgements

We would like to thank the Instrument Analysis Center at Shanghai Jiao Tong University for cryo-EM data collection. This work is supported by the National Key Research and Development Program of China (2018YFA0902000, Y.X.), STI2030-Major Projects (2021ZD0203400, Y.X.), the National Natural Science Foundation of China (32271330, M.C.; 31970547, Y.X.; 32000889, M.C.; 82321005, Y.X.), the Natural Science Foundation of Jiangsu Province (BK20190552, Y.X.), the Project Program of the State Key Laboratory of Natural Medicines, China Pharmaceutical University (SKLNMZZ202014, Y.X.) and the Fundamental Research Funds for the Central Universities (2632023GR17, Z.L.).

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Author notes

  1. These authors contributed equally: Lijie Guo, Pingping Huang, Zhaoxing Li.

Authors and Affiliations

  1. State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing, China

    Lijie Guo, Pingping Huang, Zhaoxing Li, Purui Yan, Meirong Chen & Yibei Xiao

  2. Department of Pharmacology, School of Pharmacy, China Pharmaceutical University, Nanjing, China

    Lijie Guo, Pingping Huang, Zhaoxing Li, Purui Yan, Meirong Chen & Yibei Xiao

  3. Department of Chemical Biology, School of Life Sciences, Southern University of Science and Technology, Shenzhen, China

    Young-Cheul Shin

  4. Department of Biochemistry, School of Life Science and Technology, China Pharmaceutical University, Nanjing, China

    Meiling Lu

  5. Chongqing Innovation Institute of China Pharmaceutical University, Chongqing, China

    Yibei Xiao

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Contributions

L.G. and P.H. purified proteins and performed in vitro reconstitution of SPARTA complexes. Z.L. and M.C. determined cryo-EM structures. L.G. performed NAD+ hydrolysis assay, gel filtration assay and EMSA assay to biochemically characterize SPARTA. P.Y. prepared the SPARTA mutants. M.C., Y.X., L.G., P.H., Z.L., Y.C.S. and M.L. analyzed the data. P.H. prepared the figures. M.C. and Y.X. conceived experiments, supervised the work and wrote the manuscript. All authors discussed the results and contributed to the final manuscript.

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Correspondence to Meirong Chen or Yibei Xiao.

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Nature Chemical Biology thanks Andrey Kulbachinskiy, 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 The reconstitution of SPARTA complexes.

a. NADase activity at 37 °C and 50 °C. The experiments were repeated in triplicates, and the data represents mean values ± s.d. b. The size exclusion chromatography of reconstituted SPARTA complexes.

Source data

Extended Data Fig. 2 Single-particle cryo-EM data processing.

Data processing scheme of SPARTA-apo (a), monomeric SPARTA bound with guide-target (Monomer-GT) (b) and SPARTA tetramer sample (c).

Extended Data Fig. 3 Cryo-EM density maps of the SPARTA complexes.

Maps of SPARTA-apo (a), monomeric SPARTA bound with guide-target (Monomer-GT1) (b), monomeric SPARTA bound with guide-target (Monomer-GT2) (c), SPARTA dimer (d) and SPARTA tetramer (e) are colored by local resolution, and gold-standard FSC of 0.143 resolution graphs is indicated for each complex.

Extended Data Fig. 4 The structure of SPARTA apo form.

a. CrtTIR-APAZ and CrtAgo are shown side-by-side with each domain highlighted. b. The surface representation of APAZ and N domain connected by L1 linker from TtAgo (PDB:5GQ9). c. The overall structure comparison of CrtSPARTA with TtAgo (PDB:5GQ9).

Extended Data Fig. 5 The affinity of SPARTA to RNA guide.

a. The size exclusion chromatography of wild-type CrtSPARTA and C-tail truncated SPARTA (SPARTA_ΔCtail). The absorption at 260 nm and 280 nm is monitored and shown in red and blue, respectively. b. The binding affinity quantified by Electrophoretic Mobility Shift Assay (EMSA). 1 μM guide RNA was incubated with increasing amount of protein. The complex and 21nt free guide were resolved in Native-PAGE. The samples derived from the same experiment and gels were processed in parallel. The intensity of the free guide bands was measured, and the binding affinity was then analyzed using GraphPad Prism 9.3.0. The intensity of target band was quantified from single gel for Kd calculation.

Extended Data Fig. 6 Conformational changes induced by guide-target binding and propagation.

a. The cartoon representation of helixentry withdrawal upon guide-target binding. b. D-loop flipping upon the propagation of guide-target. c. Structure superimposition of SPARTA-apo, Monomer-GT1, Monomer-GT2 and dimer shows consecutive and unidirectional movement of the D-loop. d. The conformational changes likely coupling C-tail/TIR release with guide-target propagation. The structure of Monomer-GT2 (light blue) is superimposed onto Monomer-GT1 (grey), and the regions of substantial conformational changes in Monomer-GT2 are colored in yellow, orange, blue, and magenta.

Extended Data Fig. 7 The size exclusion chromatography of SPARTA mutants.

The mutant of D-loop truncation (a), and the Ago-Ago interface mutant SPARTA_ERDAAA (b) and SPARTA_YKAA (c), incubated with guide RNA and target DNA.

Extended Data Fig. 8 The Cryo-EM maps showing the flexibility of the TIR domain.

Comparison of TIR density in Monomer-GT1 (a), Monomer-GT2 (b), SPARTA dimer (c), and SPARTA tetramer (d).

Extended Data Fig. 9 The typical assemblies of TIR domains.

a. The structure of TIR domain in SPARTA. The secondary structures are indicated. Eukaryotic TIR domains are primarily arranged either as ‘scaffold assembly’ (b) or ‘enzymatic assembly’ (c), corresponding to parallel or anti-parallel two-stranded assembly via distinct interfaces, respectively. The involved interfaces are indicated by the frames.

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Guo, L., Huang, P., Li, Z. et al. Auto-inhibition and activation of a short Argonaute-associated TIR-APAZ defense system. Nat Chem Biol 20, 512–520 (2024). https://doi.org/10.1038/s41589-023-01478-0

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