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Nucleic-acid-triggered NADase activation of a short prokaryotic Argonaute

Nature volume 625, pages 822–831 (2024)Cite this article

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Abstract

Argonaute (Ago) proteins mediate RNA- or DNA-guided inhibition of nucleic acids1,2. Although the mechanisms used by eukaryotic Ago proteins and long prokaryotic Ago proteins (pAgos) are known, that used by short pAgos remains elusive. Here we determined the cryo-electron microscopy structures of a short pAgo and the associated TIR-APAZ proteins (SPARTA) from Crenotalea thermophila (Crt): a free-state Crt-SPARTA; a guide RNA–target DNA-loaded Crt-SPARTA; two Crt-SPARTA dimers with distinct TIR organization; and a Crt-SPARTA tetramer. These structures reveal that Crt-SPARTA is composed of a bilobal-fold Ago lobe that connects with a TIR lobe. Whereas the Crt-Ago contains a MID and a PIWI domain, Crt-TIR-APAZ has a TIR domain, an N-like domain, a linker domain and a trigger domain. The bound RNA–DNA duplex adopts a B-form conformation that is recognized by base-specific contacts. Nucleic acid binding causes conformational changes because the trigger domain acts as a ‘roadblock’ that prevents the guide RNA 5′ ends and the target DNA 3′ ends from reaching their canonical pockets; this disorders the MID domain and promotes Crt-SPARTA dimerization. Two RNA–DNA-loaded Crt-SPARTA dimers form a tetramer through their TIR domains. Four Crt-TIR domains assemble into two parallel head-to-tail-organized TIR dimers, indicating an NADase-active conformation, which is supported by our mutagenesis study. Our results reveal the structural basis of short-pAgo-mediated defence against invading nucleic acids, and provide insights for optimizing the detection of SPARTA-based programmable DNA sequences.

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Fig. 1: Free-state Crt-SPARTA.
Fig. 2: Nucleic-acid-bound Crt-SPARTA.
Fig. 3: Nucleic-acid-induced conformational changes of Crt-SPARTA.
Fig. 4: Higher-order Crt-SPARTA structures.
Fig. 5: Association of Crt-TIR domains.

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Data availability

The atomic models of the Crt-SPARTA binary complex, Crt-SPARTA–gRNA–tDNA quaternary complex, dimer Conf-1 Crt-SPARTA–gRNA–tDNA complexes, dimer Conf-2 and tetrameric Crt-SPARTA–gRNA–tDNA complexes have been deposited into the Protein Data Bank with accession codes 8ISY, 8ISZ, 8IT0, 8K9G and 8IT1, and the corresponding 3D cryo-EM density maps have been deposited into the Electron Microscopy Data Bank under the accession codes EMD-35700, EMD-35701, EMD-35702, EMD-36986 and EMD-35703, respectively. Publicly available protein atomic models with the following PDB codes were used in the study: 4N76, 7UXU, 7NAI, 4N47, 2BGG, 4F3T, 1U04, 4W5O, 5UZB, 7NAK, 7QQK and 7UN8. Source data are provided with this paper.

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Acknowledgements

We thank X. Huang, B. Zhu, X. Li, L. Chen, T. Niu and other staff members at the Center for Biological Imaging, Core Facilities for Protein Science at the Institute of Biophysics, Chinese Academy of Sciences for support with cryo-EM data collection; D. Sun and other staff members from Beijing National Laboratory for Condensed Matter Physics at the Institute of Physics, Chinese Academy of Sciences for cryo-EM data collection and technical assistance; and J. Wan for support with cryo-EM data processing. This work was supported by the Chinese Academy of Medical Sciences Innovation Fund for Medical Sciences (2021-I2M-1-037 to S.C. and X.G.); the National Natural Science Foundation of China (81971985 and 82272308 to X.G.; 81572005 to S.C.); the National Key Research and Development Program of China (2019YFC0840602 to X.G.); and the National Natural Science Foundation of China/RGC Joint Research Scheme (82261160398, N_HKU767/22).

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Author notes

  1. These authors contributed equally: Xiaopan Gao, Kun Shang, Kaixiang Zhu, Linyue Wang, Zhixia Mu

Authors and Affiliations

  1. NHC Key Laboratory of Systems Biology of Pathogens, Institute of Pathogen Biology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China

    Xiaopan Gao, Kaixiang Zhu, Linyue Wang, Zhixia Mu, Bo Qin & Sheng Cui

  2. Key Laboratory of Pathogen Infection Prevention and Control, Peking Union Medical College, Ministry of Education, Beijing, China

    Xiaopan Gao, Kaixiang Zhu, Linyue Wang, Zhixia Mu, Bo Qin & Sheng Cui

  3. Beijing National Laboratory for Condensed Matter Physics, Institute of Physics, Chinese Academy of Sciences, Beijing, China

    Kun Shang, Xingke Fu, Hongtao Zhu & Wei Ding

  4. Medical School, Yan’an University, Yan’an, China

    Kun Shang

  5. National Clinical Laboratory on Tuberculosis, Beijing Key Laboratory for Drug-resistant Tuberculosis Research, Beijing Chest Hospital, Capital Medical University, Beijing Tuberculosis and Thoracic Tumor Institute, Beijing, China

    Xia Yu

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Contributions

S.C. and X.G. designed the study. X.G., K.S., K.Z., L.W. and X.F. prepared grids. S.C., W.D., H.Z. and X.G. determined the cryo-EM structures and performed the model building. S.C., X.G., H.Z. and W.D. created figures. S.C., X.G., H.Z. and W.D. wrote and revised the paper. X.G., K.S., K.Z., L.W., Z.M., B.Q. and X.Y. performed protein expression and purification, mutagenesis studies and NADase assays. X.G., H.Z., W.D., L.W., K.Z., K.S. and S.C. analysed the data. All authors reviewed the results and approved the final version of the manuscript.

Corresponding authors

Correspondence to Hongtao Zhu, Wei Ding or Sheng Cui.

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Extended data figures and tables

Extended Data Fig. 1 Single-particle cryo-EM analysis of the Crt-SPARTA heterodimer and the Crt-SPARTA–gRNA–tDNA quaternary complex.

a, Workflow of cryo-EM data processing for Crt-SPARTA heterodimer. b, Left, representative cryo-EM micrograph of Crt-SPARTA heterodimer from 7,330 movies was shown; Right, representative 2D averaged classification from the particles used for final reconstruction. c, Gold-standard Fourier shell correlation (FSC) curves of the final Crt-SPARTA heterodimer. The blue line indicates the 0.143 cut-off criterion, indicating a nominal resolution of 3.27 Å. d, Local resolution cryo-EM density map calculated using cryoSPARC, blue to red indicates high to low resolution. e, Single-particle cryo-EM image processing workflow of Crt-SPARTA–gRNA–tDNA quaternary complex. f, Left, representative micrograph of Crt-SPARTA–gRNA–tDNA quaternary complex from 7,080 movies was shown; Right, representative 2D averaged class images from the particles used for final reconstruction. g, Gold-standard Fourier shell correlation (FSC) curves. The map-to-map FSC curve was calculated between the two independently refined half-maps after masking (blue line), and the overall resolution was determined by the gold-standard FSC = 0.143 criterion. h, Local map resolution of the final structure, blue to red indicates high to low resolution.

Extended Data Fig. 2 Structure comparison of short pAgo, long pAgo, eAgo and Crt-SPARTA, and structural alignment of conserved Ago domains of Crt-SPARTA with TtAgo and human Ago2.

a, The domain architectures of short, long pAgos and/or eAgos. b, Structural comparison of Crt-SPARTA and the three representative Ago proteins: short AfAgo (PDB:2BGG), human Ago2(PDB:4F3T) and long PfAgo (PDB:1U04). The different domains are coloured according to the schematic in a. The additional trigger and TIR domain in Crt-SPARTA are coloured in blue and marine, respectively. c–f, Superimpositions of the individual domains of Crt-SPARTA with the TtAgo (yellow, PDB: 4N76) and human Ago2 (light blue, PDB: 4F3T). c, Comparison between N-like domains. d, Comparison between linker domains; the missing PAZ domain in Crt-SPARTA is labelled with a red dotted line circle. e, Comparison between MID domains; the unique insertion in MID domain of Crt-SPARTA is labelled with a red dotted line circle. f, Comparison between PIWI domains.

Extended Data Fig. 3 Ribbon models of Crt-TIR, Crt-pPAZ and Crt-Ago and secondary-structure diagrams.

a, Top, cryo-EM density map of Crt-SAPARTA heterodimer (white) with the TIR domain highlighted in light blue. Bottom, ribbon model of Crt-TIR using the same colour code; secondary structures are labelled. b, Secondary-structure diagram of Crt-TIR; TIR core domain is highlighted with a dashed box. c, Top, cryo-EM density map of Crt-SAPARTA heterodimer (white) with the N-like domain highlighted in cyan, linker domain highlighted in grey and trigger domain highlighted in dark blue. Bottom, ribbon model of Crt-pPAZ with the secondary structures labelled. d, Secondary-structure diagram of Crt-pPAZ; linker domain is composed of L0, L1 and L2, which are indicated by dashed regions. e, Top, cryo-EM density map of Crenotalea thermophila (Crt) SAPARTA heterodimer; Crt-Ago is highlighted by pink (MID) and magenta (PIWI), whereas the rest of the complex is coloured white. Bottom, ribbon model of Crt-Ago with the secondary structures labelled. f, Secondary-structure diagram of Crt-Ago.

Extended Data Fig. 4 Superimposition of Crt-SPARTA with TtAgo and human Ago2.

a, The structure of the Crt-SPARTA heterodimer is superimposed with the structure of TtAgo complexed with gDNA and tDNA (PDB: 4N47) b, The structure of the Crt-SPARTA heterodimer is superimposed with the structure of human Argonaute 2 bound to a gRNA and tRNA (human Ago2, PDB: 4W5O). Crt-SPARTA is coloured by chain. Crt-TIR-pPAZ is coloured light blue, Crt-Ago is coloured magenta; individual domains of Crt-SPARTA are labelled. TtAgo and human Ago2 are coloured grey and the guide DNA or RNA and target DNA or RNA are coloured orange and red, respectively. Crt-pPAZ lacks the PAZ domain but retains an N-like and a linker domain, topologically equivalent to the N and L1–L2 domains of TtAgo and human Ago2. Crt-pPAZ contains a unique C-terminal trigger domain unseen in previous Ago structures. The trigger domain seals the exit of the target DNA–RNA-binding tunnel.

Extended Data Fig. 5 Preparation of the Crt-SPARTA heterodimer and the quaternary protein–nucleic acid complex containing the target DNA and the guide RNA.

a, Schematic of Crt-SPARTA system protein domains, and the guide RNA and target DNA used in sample preparation. b, Purification of the Crt-SPARTA–gRNA–tDNA system. When Crt-SPARTA was incubated with guide RNA and target ssDNA in a 1:1.2:1.2 molar ratio and subjected to size-exclusion chromatography, Crt-SPARTA forms Crt-SPARTA–gRNA–tDNA monomer and Crt-SPARTA–gRNA–tDNA tetramer. The absorbance at A280 (protein) and absorbance at A260 (nucleic acid) are indicated by the blue line and the red line, respectively. c, SDS–PAGE analysis of the elution fractions corresponding to the gel filtration chromatography shown in b. The peak fractions containing the target complex were used for cryo-EM analysis. Data are representative of three independent experiments. d, Elution peaks of the co-eluted nucleic acids shown in b are detected by 15% urea–PAGE. Data are representative of three independent experiments.

Extended Data Fig. 6 Structural details of the interactions between Crt-SPARTA and nucleic acids.

a, The nucleic acid substrates of Crt-SPARTA form an unusual B-form duplex. The structure of Crt-SPATA–RNA–DNA hybrid duplex (coloured green and yellow) is superimposed to an ideal B-form duplex (coloured grey, left) and an ideal A-form duplex (coloured grey, right) using the software Matchmaker. b, Magnified view of the nucleic-acid-binding pocket of Crt-SPARTA–RNA–DNA complex with superimposed TtAgo–DNA–DNA complex (PDB: 4N47). Guide RNA (green) of Crt-SPARTA superimposed with the guide DNA of TtAgo (black) are shown with ribbon models. The trigger domain (blue) is close to the guide RNA but there are no direct contacts between them. The superimposed guide DNA of TtAgo extends near the 5’-phosphate-binding pocket in Crt-PIWI, whereas the 5′ terminus of Crt-guide RNA is around five base steps behind. c, Target DNA (yellow) of Crt-SPARTA with superimposed template DNA of TtAgo (black); TtAgo template DNA extends around five base steps ahead of Crt-SPARTA template DNA. The trigger domain recognizes the 3′ terminus of Crt-SPARTA template DNA, whereas the 3′ terminus of the superimposed TtAgo DNA template strand is around five base steps ahead. d–g, Magnified views of the Crt-SPARTA–nucleic acid interactions. Residues and nucleotides participating in the interaction are shown as stick models and labelled. d, Nucleic acid recognition by the lasso unit. e, Nucleic acid recognition by the Crt-TIR-pPAZ domain. f, Nucleic acid recognition by the Crt-PIWI RNase H domain. g, Nucleic acid recognition by the Crt-PIWI-Box domain.

Extended Data Fig. 7 Working models.

a, A proposed working model for the guide RNA–target DNA-induced conformational changes of Crt-SPARTA. The process is divided into four steps; steps 1 and 4 are supported by the cryo-EM structures determined in this study, and steps 2 and 3 (encircled by a dashed box) are postulated. b, A working model of the Crt-SPARTA system. The working model describes the process of Crt-SPARTA activation through guide RNA-mediated DNA target binding. Following phage infection or foreign DNA transformation, the autoinhibitory Crt-SPARTA loads guide RNA to recognize a segment in the invading DNAs. Guide RNA-mediated target DNA binding induces conformational changes in the Crt-SPARTA that facilitate dimerization Crt-SPARTA–RNA–DNA complexes. Next, two dimeric Crt-SPARTA–RNA–DNA complexes form tetrameric Crt-SPARTA–RNA–DNA complexes, in which the NAD(P)ase activity of the TIR assemblies is activated, resulting in NAD(P)+ depletion and cell death.

Extended Data Fig. 8 Cryo-EM single-particle analysis of dimeric and tetrameric Crt-SPARTA–RNA–DNA complexes.

a, Flow chart of single-particle cryo-EM image processing for two dimeric and tetrameric Crt-SPARTA–RNA–DNA complexes b, Representative cryo-EM micrographs of two dimeric and tetrameric Crt-SPARTA–gRNA–tDNA complexes from 20,299 movies was shown are shown at 290,000× magnifications; Right, representative reference-free 2D-class averages of two dimeric and tetrameric Crt-SPARTA–gRNA–tDNA complexes. c, The gold-standard Fourier shell correlation (FSC) curves of two dimeric and tetrameric Crt-SPARTA–gRNA–tDNA complexes for the reconstruction are indicated with resolutions at FSC = 0.143 criterion. d, Local map resolution of the two dimeric and tetrameric Crt-SPARTA–gRNA–tDNA complexes, blue to red indicates high to low resolution.

Extended Data Fig. 9 Comparison of the TIR domain observed in MAL-TIR and TIR-SAVED.

a, Structural superimposition of the Crt-TIR (magenta) with MAL-TIR (PDB:5UZB) (yellow), the BCD interface consisting of αB, αC and αD is labelled. b, The TIR domain tetramers observed in SARM1 (PDB: 7NAK), the BE interface and AE interface are labelled. c, The BE interface of a TIR-SAVED dimer composed of the BB loop and DE loop is labelled (PDB:7QQK). d, Structural comparison of the Crt-TIR assembly with bacterial STING-TIR assembly. A head-to-tail-organized Crt-TIR dimer (blue) is superimposed with SfSTING-TIR filaments (coloured by monomer, PDB: 7UN8). The BB loop of TIR domains, the NADase activate site and the DD loop extending from the opposite TIR filament are labelled. The direction of SfSTING-TIR filaments is indicated by arrows.

Extended Data Table 1 Cryo-EM data collection, refinement and validation statistics
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Supplementary information

Supplementary Information

This file contains Supplementary Figures 1–5, Supplementary Tables 1-4 and legend for Supplementary Video 1

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Supplementary Video 1

Binding of guide RNA–target DNA hybrid duplex induce conformational changes in Crt-SPARTA

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Gao, X., Shang, K., Zhu, K. et al. Nucleic-acid-triggered NADase activation of a short prokaryotic Argonaute. Nature 625, 822–831 (2024). https://doi.org/10.1038/s41586-023-06665-6

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