Structural insight into the assembly and working mechanism of helicase-primase D5 from Mpox virus | Nature Structural & Molecular Biology

Oct 14, 2024

Nature Structural & Molecular Biology volume 31, pages 68–81 (2024)Cite this article

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The Mpox pandemic, caused by the Mpox virus (or monkeypox virus, MPXV), has gained global attention. The D5 protein, a putative helicase-primase found in MPXV, plays a vital role in viral replication and genome uncoating. Here we determined multiple cryo-EM structures of full-length hexameric D5 in diverse states. These states were captured during ATP hydrolysis while moving along the single-stranded DNA (ssDNA) track. Through comprehensive structural analysis combined with the helicase activity system, we revealed that when the primase domain is truncated or the interaction between the primase and helicase domains is disrupted, the double-stranded DNA (dsDNA) unwinds into ssDNA, suggesting a critical regulatory role of the primase domain. Two transition states bound with ssDNA substrate during unwinding reveals that two ATP molecules were consumed to drive DNA moving forward two nucleotides. Collectively, our findings shed light on the molecular mechanism that links ATP hydrolysis to the DNA unwinding in poxviruses.

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Cryo-EM maps and molecular models have been deposited in the EM Data Bank and PDB, respectively. Accession codes are listed here and in Table 1. Atomic coordinates and cryo-EM density maps of D5 protein in ATP-ADP-apo-ssDNA form IS1 (PDB 8HWA whole map EMD-35051), ATP-ADP-apo-ssDNA form IS2 (PDB 8HWB whole map EMD-35052), apo form (PDB 8HWC whole map EMD-35053), ADP form (PDB 8HWD whole map: EMD-35054), ATP-ADP form (PDB 8HWE whole map EMD-35055), ADP-ssDNA form (PDB 8HWF whole map EMD-35056), ATP-γS-ADP-ssDNA form (PDB 8HWG whole map EMD-350567) and apo-ssDNA form (PDB 8HWH whole map EMD-350568) have been deposited to the PDB (http://www.rcsb.org) and the Electron Microscopy Data Bank (https://www.ebi.ac.uk/pdbe/emdb/), respectively. All other data will be made available upon request. Source data are provided with this paper.

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We thank the Cryo-EM Facility of Southern University of Science and Technology (SUSTech) for providing the facility support. We thank X. Ma, L. Zhang and P. Li at the Cryo-EM Center of SUSTech for technical support in EM data acquisition. We thank Z. Liu for technical support on computing environment. This work was funded by the Science, Technology and Innovation Commission of Shenzhen Municipality (grant no. JSGG20220226085550001 to R.Y.) and the National Natural Science Foundation of China (grant no. 82202517 to R.Y.).

These authors contributed equally: Yaning Li, Jing Zhu.

Department of Biochemistry, School of Medicine, Key University Laboratory of Metabolism and Health of Guangdong, Institute for Biological Electron Microscopy, Southern University of Science and Technology, Shenzhen, China

Yaning Li, Jing Zhu, Yingying Guo & Renhong Yan

Center for Infectious Disease Research, Westlake Laboratory of Life Sciences and Biomedicine, Key Laboratory of Structural Biology of Zhejiang Province, School of Life Sciences, Westlake University, Hangzhou, China

Yaning Li

Beijing Advanced Innovation Center for Structural Biology, Tsinghua-Peking Joint Center for Life Sciences, School of Life Sciences, Tsinghua University, Beijing, China

Yaning Li

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R.Y. conceived the project. R.Y., Y.G., Y.L. and J.Z. designed the experiments. Y.L. did the molecular cloning, protein purification, cryo-EM data collection and processing, and model building. Y.G. and J.Z. did the protein purification, cryo-EM data collection and ATPase assay, DNA-binding assay and the helicase assay. All authors contributed to data analysis. R.Y. and Y.G. wrote the paper.

Correspondence to Yingying Guo or Renhong Yan.

The authors declare no competing interests.

Nature Structural & Molecular Biology thanks Hauke Hillen and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available. Primary Handling Editor: Katarzyna Ciazynska, in collaboration with the Nature Structural & Molecular Biology team. Peer reviewer reports are available.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

The hallmark motifs (Walker a and b) and the Arginine finger residues are shown by yellow, green and black dashed box, respectively. Various colors are used to distinguish the primase domain (blue color), Zn-binding motif (orange color), collar-domain (yellow color), AAA+ helicase domain (green color) and C-terminal domain (purple color). Gene and protein ID are as follows: MPXV 2022 West African strain (Genebank: ON563414.3), MPXV_Zaire_96_I_16 strain (Genebank: NP_536529.1), MPXV_UK_P3 strain (Genebank: YP_010377099.1), VACV (Genebank: AGB75830.1), VARV (Genebank: ABG44074.1). Sequences were aligned by MultAlin and ESPript 3.0.

a, Represent micrographs and 2D class averages of D5. Scale bar of 2D, 10 nm. A total of 2237 micrographs were collected. b and c, Flowchart for cryo-EM data processing. Please refer to the ‘Data Processing’ in Methods section for details. (c) Local resolution map for the 3D reconstruction of the overall structure. d, FSC curve. The resolution was estimated with the gold-standard Fourier shell correlation 0.143 criterion with high-resolution noise substitution. e-h, Euler angle distribution in the final 3D reconstruction of class 1–4. i and j, FSC curve of the refined model versus the overall structure of class 1/2 that it is refined against (black); of the model refined against the first half map versus the same map (red); and of the model refined against the first half map versus the second half map (green). The small difference between the red and green curves indicates that the refinement of the atomic coordinates did not suffer from overfitting.

a-c, Represent micrographs and 2D class averages of D5 Apo, ADP, and ATP-ADP form. Scale bar of 2D, 10 nm. A total of 3504, 1514 and 1004 micrographs were collected, respectively. d and e, Flowchart for cryo-EM data processing. Please refer to the ‘Data Processing’ in Methods section for details. e, Local resolution map for the 3D reconstruction of the overall structure. f, FSC curve. The resolution was estimated with the gold-standard Fourier shell correlation 0.143 criterion with high-resolution noise substitution. g, Euler angle distribution in the final 3D reconstruction. h, FSC curve of the refined model versus the overall structure that it is refined against (black); of the model refined against the first half map versus the same map (red); and of the model refined against the first half map versus the second half map (green). The small difference between the red and green curves indicates that the refinement of the atomic coordinates did not suffer from overfitting.

a-c, Represent micrographs and 2D class averages of D5-ADP-ssDNA, ATP-γS -ADP-ssDNA, and Apo-ssDNA form. Scale bar of 2D, 10 nm. A total of 4594, 1431 and 201 micrographs were collected, respectively. d and e, Flowchart for cryo-EM data processing. Please refer to the ‘Data Processing’ in Methods section for details. e, Local resolution map for the 3D reconstruction of the overall structure. f, FSC curve. The resolution was estimated with the gold-standard Fourier shell correlation 0.143 criterion with high-resolution noise substitution. g, Euler angle distribution in the final 3D reconstruction. h, FSC curve of the refined model versus the overall structure that it is refined against (black); of the model refined against the first half map versus the same map (red); and of the model refined against the first half map versus the second half map (green). The small difference between the red and green curves indicates that the refinement of the atomic coordinates did not suffer from overfitting.

a, Cryo-EM density maps of protein are shown at threshold of 7 σ. b, Cryo-EM density map of ssDNA is shown at threshold of 5 σ. c-e, Cryo-EM density maps of ligand and the binding pocket are shown at threshold of 7 σ. f, Density maps show the C-terminal motif could interact with the helicase domain in neighboring protomer.

a, The organization of four layers of D5. Left: Structure of one protomer of full-length D5 is showed by cartoon. The color strategy is consistent with Fig.1a. Right: Overall structures of D5 is showed by surface and the color strategy is consistent with Fig.1d. b, Structural analysis of N-terminal primase domain of D5. Shown here is cartoon presentation of domain-colored cryo-EM structures of N-terminal primase domain of D5 and structural alignment between RRM in protomer A and human PrimPol (PDB ID: 5L2X). RMSD between 44 pruned atom pairs is 1.210 Å and across all 207 pairs is 9.768 Å. Insets: In top panel, structural similarity between RRM in protomer A (red) and human PrimPol (cyan) indicates the similar functions and potential ATP binding pocket in N-terminal primase domain of MPVX. Bottom panel shows the density at potential ATP binding pocket allow us to build an ATP molecular. Cryo-EM density map is shown at threshold of 7 σ.

a-d, Helicase activity of D5 (Apyrase) and truncation D5238-785 (Apyrase). When ATP acts as an energy substrate, D5full length (Apyrase) has weak helicase activity, while truncation D5238-785 (Apyrase) shows apparent increasing in helicase activity. Whereas, ADP can’t catalyze the reaction. When apyrase treated proteins were pre-incubated with ADP, the helicase activity was reduced. However, for D5full length (Apyrase), pre-incubation with Fork DNA before ADP restored its activity to ATP only group, which indicates that ADP may have a regulatory effect on helicase activity of full length D5. e, Free DNA releasing by proteins was delivered quantitative analysis with ImageJ 2.9.0 to evaluate the helicase ability of various proteins. Three replications were taken independently for data analysis and the data has been presented as mean ± standard deviation. The samples derive from the same experiment and that gels were processed in parallel.

Source data

a, ATP hydrolysis activity of D5 and the mutants. WT D5 shows obvious ATPase activity. Comparing with WT D5, R619/620A mutant and M5t (K509A/ T511A/ R514A/ F630A/ R656A) mutant show apparent decreasing in ATPase activity. Also, ATP-γS has a great impact on the ATPase activity of the proteins. b, DNA binding ability of D5 and the mutants. WT D5 has DNA binding activity, and R585A, F588A and R585A/F588A mutants could impair the DNA binding ability of D5. All experiments were performed in triplicates and the free DNA was delivered quantitative analysis to evaluate the DNA binding ability of various proteins. In (a) and (b), **** indicates extremely significant difference (p < 0.0001), ** indicates significant difference (p < 0.01), ns indicates no significance. Three replications were taken independently for data analysis and the data has been presented as mean ± standard deviation. For multiple comparisons, P values were derived from ordinary one-way ANOVA with Šídák′s multiple comparisons test. The default parament settings were applied to multiple comparisons. P values< 0.05 (two side) were considered significant. Graphs were prepared in Graphpad prism (Version 9.0).

Source data

a, Here shows the cryo-EM map and model of D5 ATP-γS-ADP-ssDNA form. Density of D5 is colored light blue and cartoon presentation of D5 and DNA are colored gray and black, respectively. Alignments among N-terminal primase domain of D5 ATP-γS-ADP ssDNA form and D5 ATP-ADP-Apo ssDNA form IS1/2 shows that DNA in ATP-γS-ADP ssDNA form is consistent with it in IS1 (b) but not IS2 (c). RMSD between 3139 Cα pairs of D5 IS1 and D5 ATP-γS-ADP-ssDNA form, D5 IS2 and D5 ATP-γS-ADP-ssDNA form are 1.118 Å and 4.840 Å, respectively.

a, The structural comparison of D5 bound to different ligands shows there is no shift between collar domains and dramatic shifts in AAA+ helicase domain. Right panel is the diagram to show the shifts angles in AAA+ helicase domain. AAA+ helicase domain of D5-ATP form (yellow), D5-ADP tight form (green), D5-Apo bound DNA form (purple), D5-ADP loose form (blue), and Apo (magenta) are looser in turn. b, The interaction network of ADP in D5-ADP loose form. To make the shift more clearly, superpositions of ATP form and ADP tight form, ADP tight form and Apo bound DNA form, Apo bound DNA form and ADP loose form, and ADP loose form and Apo are showed from left to right. RMSD between 378 cα pairs (323 to 700 amino acid) of the chain in ATP form and ADP tight form, ADP tight form and Apo bound DNA form, Apo bound DNA form and ADP loose form, and ADP loose form and Apo are 1.791, 2.931, 4.220, and 1.990. c, The interaction network of ADP in D5-ADP loose form. d, Structural comparison of D5 of MPXV and E1 of papillomavirus. Middle: Structural alignment of D5 and E1. D5 are colored blue and E1 are colored green. D5 and E1 are showed at left and right panel. Collar domain are colored light blue (D5) and light green (E1), and AAA+ helicase domains are colored dark blue (D5) and dark green (E1).

Supplementary Table 1.

Unprocessed gels.

Statistical source data.

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Li, Y., Zhu, J., Guo, Y. et al. Structural insight into the assembly and working mechanism of helicase-primase D5 from Mpox virus. Nat Struct Mol Biol 31, 68–81 (2024). https://doi.org/10.1038/s41594-023-01142-0

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Received: 04 March 2023

Accepted: 27 September 2023

Published: 04 January 2024

Issue Date: January 2024

DOI: https://doi.org/10.1038/s41594-023-01142-0

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