Abstract
Escherichia coli Septu system, an anti-phage defense system, comprises two components: PtuA and PtuB. PtuA contains an ATPase domain, while PtuB is predicted to function as a nuclease. Here we show that PtuA and PtuB form a stable complex with a 6:2 stoichiometry. Cryo-electron microscopy structure of PtuAB reveals a distinctive horseshoe-like configuration. PtuA adopts a hexameric arrangement, organized as an asymmetric trimer of dimers, contrasting the ring-like structure by other ATPases. Notably, the three pairs of PtuA dimers assume distinct conformations and fulfill unique roles in recruiting PtuB. Our functional assays have further illuminated the importance of the oligomeric assembly of PtuAB in anti-phage defense. Moreover, we have uncovered that ATP molecules can directly bind to PtuA and inhibit the activities of PtuAB. Together, the assembly and function of the Septu system shed light on understanding other ATPase-containing systems in bacterial immunity.
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Data availability
Accession numbers for PtuA and PtuAB are as follows: (coordinates of atomic models: 8SUX, 8EE4, 8EE7 and 8EEA, deposited to Protein Data Bank), and (density map: EMD-40779, EMD-28045, EMD-28048 and EMD-28049, deposited to Electron Microscopy Data Bank). All data needed to evaluate the conclusions are present in the paper.
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Acknowledgements
Grid screening was performed at OSU CEMAS with the assistance of G. Grandinetti and Y. Narui. Cryo-EM data were collected with the assistance of A. D. Wier, T. J. Edwards, T. Fox and J. Wang at the National Cancer Institute Cryo-Electron Microscopy Center supported by grants from the NIH National Institute of General Medical Sciences (GM103310). A. Rish was supported by an NIH T32 (GM118291-05 and GM144293-01). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. Source data are provided with this paper.
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Q.C., Y.Y. and T.-M.F. conceived the project. Y.L., Z.S., X.-Y.Y., M.Z. and J.X. performed molecular cloning and biochemical purification of the PtuA, PtuB, PtuAB and all the mutants. Y.L. and M.Z. performed the ATPase assay, plasmids nicking assays, bacterial growth assay and phage genomic DNA quantification assay. Z.S. and T.-M.F. prepared grids, determined the cryo-EM structures and built the models. S.P.C., J.G. and M.K. performed the native mass spectrometry analysis and I.A.M. did the mass photometry analysis under the supervision of V.H.W. T.-M.F., Y.Y., Q.C., A.D.R. and C.C. analyzed the data. T.-M.F. wrote the manuscript with inputs from all the authors.
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Nature Structural & Molecular Biology thanks Jun-Jie Liu, Qian Yin and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Sara Osman was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team. Peer reviewer reports are available.
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Extended data
Extended Data Fig. 1 Sequence Alignment of PtuA from Different Species.
Sequence alignment of PtuA from Escherichia coli (Genbank ID AIL15948.1), Proteus mirabilis (Genbank ID AGS60026.1), Pseudomonas veronii (Genbank ID KRP82805.1), and Yersinia enterocolitica (Genbank ID AHM71342.1) with secondary structural elements labeled below. NTD was highlighted in blue, and CTD was highlighted in green. Walker A motif, Walker B motif, and a signature motif critical for coordinating ATP were highlighted in red, yellow, and green, respectively.
Extended Data Fig. 2 Analysis and Purification of PtuA and PtuB.
a, Sequence alignment of PtuB from Escherichia coli (Genbank ID AIL15172.1), Janthinobacterium agaricidamnosum (Genbank ID CDG84671.1), Yersinia enterocolitica (Genbank ID AHM71343.2), and Pseudomonas veronii (Genbank ID KRP82804.1) with secondary structural elements labelled below. Catalytic residues of PtuB were highlighted in yellow. b, Diagram of phylogenetic trees of PtuB from different species. c, SDS-PAGE analysis of PtuA expression and purification via Ni2+ affinity column. The experiment was replicated at least three times. d, SDS-PAGE analysis of PtuB expression and purification via Ni2+ affinity column. Bands of PtuB were highlighted in a red box. The experiment was replicated at least three times.
Extended Data Fig. 3 Structure Determination of PtuA.
a, Workflow of PtuA 3D reconstruction using CryoSPARC. b, Local resolutions of the reconstructions of PtuA. Resolutions are color-coded by scale bars. c, Fourier shell correlation (FSC) curve of 3D reconstructed PtuA.
Extended Data Fig. 4 Cryo-EM Density of PtuA and Structure comparison.
a, Representative segments of PtuA cryo-EM density map with the final atomic model (2.0 σ). b, Overlaid structures of PtuA (green and cyan) and Rad50 dimers (Grey). c, Residues on the dimeric interface of PtuA were highlighted in sticks. d,Gel filtration profiles of PtuA wild type (blue) and L81R mutant (gold), showing that L81R disrupted the PtuA hexamer.
Extended Data Fig. 5 Comparison of ATP Binding Sites.
a, Overlaid structures of the central dimer (cyan and green) and the side dimer (yellow and magenta), revealing a larger cleft at the ATP binding site of the side dimer. ATP is highlighted as sticks. b, Key residues for ATP coordination are highlighted as sticks. Key residues are far away from the potential ATP binding site in the side dimer.
Extended Data Fig. 6 ATP Catalysis.
a, ATP catalytic site of Rad50, highlighting an extensive hydrogen bond network formed by key residues and magnesium (sphere). b, ATP catalytic site of PtuA. Key residues are not well aligned to form hydrogen bonds with ATP. c, Comparison of ATP molecules in PtuA (cyan) and Rad50 (yellow), revealing the unique configuration of ATP in PtuA. d, Overlaid structures of ATP catalytic sites in PtuA (cyan) and Rad50 (yellow).
Extended Data Fig. 7 Structural Reconstruction of PtuA from the PtuAB Dataset.
a, A representative cryo-EM image of the PtuA and PtuB complex. Thousands of images were collected. b, Workflow of PtuA 3D reconstruction using CryoSPARC. c, Fourier shell correlation (FSC) curve of 3D reconstructed PtuA. d, Local resolutions of the reconstructions of PtuA. Resolutions are color-coded by scale bars.
Extended Data Fig. 8 Cryo-EM Structure Reconstruction of the PtuA and PtuB complex.
a, Workflow of the PtuAB complex 3D structure reconstruction using CryoSPARC. b, Initial cryo-EM map of the PtuAB complex at a resolution of 5 Å. c, FSC curve of the focused refined cryo-EM map of the PtuAB complex. d, Ribbon diagram of the full-length PtuA, revealing that PtuA CTD is composed of three α-helices.
Extended Data Fig. 9 Structural Comparison of PtuB and Cas9.
a, Overlaid structures of PtuB (pink) and the HNH domain of Cas9 (green) revealing their similarity. b, Overlaid catalytic cores of PtuB (pink) and the NHN domain of Cas9 (green) with catalytic residues highlighted in sticks.
Extended Data Fig. 10 Anti-phage Immune Defense by the PtuA and PtuB Complex.
a, Bacteria growth curves with T2 phage infection at different MOI. b, Bacteria growth curves with T5 phage infection at different MOI. c, PtuA/PtuB_H72A failed to confer anti-phage protection as evaluated by bacterial growth assay. The experiment was replicated more than three times. d, PtuA L81R/PtuB failed to confer anti-phage protection as evaluated by bacterial colony-forming assay. The experiment was replicated more than three times.
Supplementary information
Supplementary Information
The supplementary file contains all the primers, information of kits, and gene ID information.
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Source Data Figs. 1, 3, 6 and 7, and Extended Data Figs. 2 and 10
Unprocessed gels.
Source Data Fig. 4
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Source Data Fig. 7
Statistical source data.
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Li, Y., Shen, Z., Zhang, M. et al. PtuA and PtuB assemble into an inflammasome-like oligomer for anti-phage defense. Nat Struct Mol Biol 31, 413–423 (2024). https://doi.org/10.1038/s41594-023-01172-8
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DOI: https://doi.org/10.1038/s41594-023-01172-8
