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Cryo-EM structure of the ssDNA-activated SPARTA complex

Cell Research volume 33, pages 731–734 (2023)Cite this article

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The well-resolved electron density enabled us to build a 5′-phosphorylated 19-nt gRNA and a 24-nt target ssDNA, located at the central basic channel formed by Ago and the APAZ domain of TIR-APAZ. The remaining 2 nt at the C-terminus of the gRNA and 7 nt at the N-terminus of the target ssDNA are disordered (Supplementary information, Fig. S3a). The gRNA is fully complementary to the target ssDNA spanning positions 3–19 to form a heteroduplex (A(3):dT(–3)–A(19):dT(–19)), leaving the 5′-end U(1) of gRNA inserted into the MID domain and the 3′-end dC(–2)–dA(5) of target ssDNA extending towards the solvent (Supplementary information, Fig. S5). The 5′-phosphorylated U(1) makes extensive hydrogen bonds with residues Arg152, His207, Lys211, Gln222, Lys263 and the C-terminal carboxyl group of Ago (Fig. 1d). These results are consistent with the observation that the SPARTA complex is preferentially activated by the binding of 5′-phosphorylated RNA-guided target ssDNA, and that gRNA 3′-end binding is not necessary for SPARTA activation.4 The MID (residues Arg72, Arg225, Thr228, Arg243, Lys247), PIWI (residues Lys286, Asn325, Gln327, Lys362, Arg364, Lys390, Lys395, Asn439, Asn466, Asn468, Arg481, Lys485) and APAZ (residues Arg201, Arg209, Lys211, Arg257, Asn270, Ser288, Lys313, Lys315, Lys328, His340, Ser359, Arg361, Lys366) domains make extensive hydrogen bonds with the backbone phosphates of the gRNA–ssDNA heteroduplex (Supplementary information, Fig. S5). An important discovery is that two pairs of spatially neighboring residues (His358:Arg362 and Glu260:Arg263) on the APAZ domain anchor into the minor groove of the gRNA–ssDNA heteroduplex at each end, and form hydrogen bonding interactions with the ribose of G(6)–C(7), and A(16) of gRNA, respectively (Fig. 1e, f). Furthermore, these residues contribute hydrogen bonds with the nucleobases of G(6):dC(–6), C(7):dG(–7), U(15):dA(–15) and A(16):dT(–16) base pairs (Fig. 1e, f). Our result supports the observation that mismatches between the gRNA and target ssDNA at guide positions 5–10 and 14–15 attenuated the activity of SPARTA complex.4 We speculated that the unique structural feature of APAZ domain could facilitate the RNA-guided base pairing with the target ssDNA and monitor possible mismatches, compensating for the loss of PAZ domain in the SPARTA complex. As the SPARTA complex could be activated upon guide-mediated target binding, we investigated whether the residues involved in the recognition of the gRNA–ssDNA heteroduplex play a critical role in the activation of SPARTA complex. We constructed eleven mutants, including AgoK211A, AgoQ222A, AgoK286A, AgoN325A, AgoK395A, AgoN439A, AgoN466A, AgoR481A, TIR-APAZR201A, TIR-APAZR263A, and TIR-APAZH358A. Consistent with the structural observation, these mutations resulted in modest or severe reductions in the NADase activity of SPARTA complex (Fig. 1g), suggesting that guide-mediated target binding may be the trigger for the activation of SPARTA complex.

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Fig. 1: Cryo-EM structure of the SPARTA–gRNA–ssDNA complex.

Data availability

The atomic coordinate of the SPARTA–gRNA–ssDNA complex has been deposited to the Protein Data Bank under the accession number 8J7S. The corresponding map has been deposited in the Electron Microscopy Data Bank under the accession number EMD-36047.

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Acknowledgements

We thank the EM and NMR platform at School of Life Science and Technology of Harbin Institute of Technology and the cryo-EM facility, the high-performance computing center core facility of Westlake University for providing supports. This research was funded by the National Natural Science Foundation of China (31825008 and U21A20276), the Tencent Foundation through the XPLORER PRIZE, the New Cornerstone Science Foundation, and Heilongjiang Touyan Team (HITTY-20190034) to Z.H., Postdoctoral Scientific Research Fund of Heilongjiang Province (AUGA4120002723) to Y.Z., and the Fellowship of China National Postdoctoral Program for Innovative Talents (AUGA4150000720) to M.G.

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  1. These authors contributed equally: Minghui Guo, Yuwei Zhu, Zhiying Lin.

Authors and Affiliations

  1. HIT Center for Life Sciences, School of Life Science and Technology, Harbin Institute of Technology, Harbin, Heilongjiang, China

    Minghui Guo, Yuwei Zhu, Zhiying Lin, Dehui Yang, Anqi Zhang, Changyou Guo & Zhiwei Huang

  2. Westlake Center for Genome Editing, Westlake Laboratory of Life Sciences and Biomedicine, School of Life Sciences, Westlake University, Hangzhou, Zhejiang, China

    Zhiwei Huang

  3. New Cornerstone Science Laboratory, Shenzhen, Guangdong, China

    Zhiwei Huang

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  1. Minghui Guo
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Contributions

Z.H. conceived and supervised the whole project. M.G. and Z.L. prepared the protein samples for negative-staining and cryo-EM analysis. M.G., Z.L., A.Z. and C.G. did negative-staining, prepared the cryo-EM grids and collected the cryo-EM data. M.G., Z.L. and D.Y. did functional experiments. Y.Z. and Z.H. performed cryo-EM data processing and model building. Y.Z. wrote the manuscript with contributions from all authors.

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Correspondence to Zhiwei Huang.

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Guo, M., Zhu, Y., Lin, Z. et al. Cryo-EM structure of the ssDNA-activated SPARTA complex. Cell Res 33, 731–734 (2023). https://doi.org/10.1038/s41422-023-00850-y

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