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Cryo-EM structures of Escherichia coli Ec86 retron complexes reveal architecture and defence mechanism

Nature Microbiology volume 7, pages 1480–1489 (2022)Cite this article

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Abstract

First discovered in the 1980s, retrons are bacterial genetic elements consisting of a reverse transcriptase and a non-coding RNA (ncRNA). Retrons mediate antiphage defence in bacteria but their structure and defence mechanisms are unknown. Here, we investigate the Escherichia coli Ec86 retron and use cryo-electron microscopy to determine the structures of the Ec86 (3.1 Å) and cognate effector-bound Ec86 (2.5 Å) complexes. The Ec86 reverse transcriptase exhibits a characteristic right-hand-like fold consisting of finger, palm and thumb subdomains. Ec86 reverse transcriptase reverse-transcribes part of the ncRNA into satellite, multicopy single-stranded DNA (msDNA, a DNA-RNA hybrid) that we show wraps around the reverse transcriptase electropositive surface. In msDNA, both inverted repeats are present and the 3′ sides of the DNA/RNA chains are close to the reverse transcriptase active site. The Ec86 effector adopts a two-lobe fold and directly binds reverse transcriptase and msDNA. These findings offer insights into the structure–function relationship of the retron–effector unit and provide a structural basis for the optimization of retron-based genome editing systems.

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Fig. 1: Retron Ec86 genetic system and overall structure of the effector-bound Ec86 complex.
Fig. 2: Structure of Ec86 reverse transcriptase.
Fig. 3: Structure of Ec86 msDNA.
Fig. 4: Intermolecular interactions between retron-Ec86 reverse transcriptase and msDNA.
Fig. 5: Ec86 effector adopts a bilobed fold and directly interacts with msDNA.
Fig. 6: Ec86 effector–reverse transcriptase interactions and a proposed mechanistic model for the Ec86 system.

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Data availability

The atomic coordinates and EM density for the reported structures of the retron Ec86 complex (PDB: 7V9U; Electron Microscopy Data Bank (EMDB): EMD-31827) and the effector-bound Ec86 complex (PDB: 7XJG; EMDB: EMD-33226) have been deposited in the PDB (www.rcsb.org) and the EMDB (www.ebi.ac.uk/pdbe/emdb/). All data needed to evaluate the conclusions in the paper are shown in the article and/or the supplementary materials. Source data are provided with this paper. Additional data related to this paper may be requested from the authors.

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Acknowledgements

We thank the cryo-EM Facility of Hubei University for providing cryo-EM and computational support and the Center for Protein Research and Public Laboratory of Electron Microscopy, Huazhong Agricultural University, for technical support; X. Yan and J. Wang for help with cryo-EM data collection; Y. Wang and E. Sun for technical support with cloning and the phage plaque assays; J. Yan for technical assistance with the mass spectrometry experiments. We thank J. Li of the Mass Spectrometry System at the National Facility for Protein Science in Shanghai, Shanghai Advanced Research Institute, Chinese Academy of Science for data collection and analysis. This work was supported by funds from the National Key R&D Programme of China (nos. 2018YFA0507700, 2020YFA0908400), the National Natural Science Foundation of China (nos. 32070174, 31900930) and the Foundation of Hubei Hongshan Laboratory (no. 2021hszd013). Z.G. acknowledges the support provided by the National Postdoctoral Programme for Innovative Talents (no. BX2021108). Z.G., C.W. and Y.N. thank the BaiChuan fellowship of the College of Life Science and Technology, Huazhong Agricultural University, for funding support.

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  1. These authors contributed equally: Yanjing Wang, Zeyuan Guan, Chen Wang, Yangfan Nie.

Authors and Affiliations

  1. State Key Laboratory of Agricultural Microbiology, Hubei Hongshan Laboratory, Huazhong Agricultural University, Wuhan, China

    Yanjing Wang, Yangfan Nie, Yibei Chen, Zhaoyang Qian, Yongqing Cui, Han Xu, Pan Tao, Ming Sun & Tingting Zou

  2. National Key Laboratory of Crop Genetic Improvement, Hubei Hongshan Laboratory, Huazhong Agricultural University, Wuhan, China

    Zeyuan Guan, Chen Wang, Qiang Wang, Fen Zhao, Delin Zhang, Ping Yin & Shuangxia Jin

  3. State Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei Collaborative Innovation Center for Green Transformation of Bio-Resources, Hubei Key Laboratory of Industrial Biotechnology, School of Life Sciences, Hubei University, Wuhan, China

    Shan Wu

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Contributions

T.Z. conceived the project. Y.W., Z.G., C.W., Y.N. and T.Z. designed the experiments. Y.W., Y.N., Y.Chen., Z.Q., Y.Cui., H.X., Q.W. and F.Z. performed the experiments. Q.W. and S.W. prepared the cryo-EM samples and collected the cryo-EM data. Z.G. determined the structures. All authors analysed the data and contributed to manuscript preparation. D.Z., P.T., M.S., P.Y. and S.J. contributed to the data analysis and discussion. Y.W., C.W. and T.Z. wrote the manuscript.

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Correspondence to Shan Wu or Tingting Zou.

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Extended data

Extended Data Fig. 1 Purification of the Ec86 and effector-bound Ec86 complexes.

a, Purification of the Ec86 complex is indicated by a representative gel filtration chromatography, SDS-PAGE, and 12 % urea-PAGE. The peaks containing the target complex are illustrated by a black arrow and grey shadow. Fractions in lane 5 was used for cryo-EM sample preparation. b, Purification of the effector-bound Ec86 complex similar to the processes described in (a). Fractions in lane 6 was used for cryo-EM sample preparation. Lane 1, homemade DNA ladders. The msDNA (DNA-RNA hybrid) migrates slower than DNA. Images in (a) and (b) are representatives of three independent experiments.

Source data

Extended Data Fig. 2 Cryo-EM single particle analysis of the retron-Ec86 binary and retronEc86-NDT ternary complex.

a, Top panel, representative cryo-EM micrograph images of the Ec86 and effector-bound Ec86 complexes. Typical particles of the complex are marked by white circles. Bottom panel, flowchart for the cryo-EM data processing. See Methods for detailed information. b, Left panel, FSC curves between the cryo-EM density maps and the atomic models of the retron-Ec86 complex. Middle panel, angular distribution for the final reconstruction. Right panel, local map resolutions of the final structures. c, Left, FSC curves between the cryo-EM density maps and the atomic models of the retron-Ec86-NDT complex. Middle panel, angular distribution for the final reconstruction. Right panel, local map resolutions of the final structures.

Extended Data Fig. 3 Overall structure of the Ec86 complex and comparison with the effector-bound Ec86 complex.

a, Cryo-EM map and atomic model of the Ec86 complex in cartoon. msrRNA, burly wood; msdDNA, light green; RT, slate blue; IRs, gold. b, Superposition of effector-bound Ec86 with Ec86. Right panel, the close-up view of the conformational change of the DSL region.

Extended Data Fig. 4 In vitro and in vivo biochemical assays for the Ec86 and Ec86 defence unit.

a, Interaction interface between the two RT-msDNA protomers in the Ec86 complex. Top panel, atomic model of the Ec86 complex in cartoon. Bottom panel, close-up views of the interface illustrated by the box. b, The sequence alignment of Ec86 RT α1 region with the corresponding regions in Eco9 and Sen2 RTs. The blue asterisk illustrates the Ec86 RT R13 residue. c, Purification of the Ec86 msDNA-RTWT, Ec86 msDNA-RTR13A, Eco9-msDNA-RTWT and Sen2-msDNA-RTWT indicated by representative gel filtration chromatography. The absorbance at A280 (protein) and absorbance at A260 (nucleic acid) are indicated by solid line and dotted line, respectively. The shift of the elution peaks containing the target msDNA-RT complex is illustrated by gray shadows. The elution fractions corresponding to the gel filtration chromatography are subjected to SDS-PAGE and Coomassie brilliant blue staining. d, Elution peaks of the co-eluted nucleic acids are detected by 12 % urea-PAGE. e, The in vitro pull down of effector with wildtype or mutant Ec86 RTs. Images in (c-e) are representatives of three independent experiments. f, Serial dilution plaque assays shown for T5 phage on E. coli MG1655 strain transformed with plasmids encoding wild-type or mutated Ec86 systems. Images are representative of two replicates.

Source data

Extended Data Fig. 5 Superposition of Ec86 RT with other RTs.

a-d, Superposition of Ec86 RT with (a) LtrA of group IIA intron (PDB:5HHJ; RMSD = 5.4 Å), (b) RT of group IIC intron (GsI-IIC RT, PDB:6AR3; RMSD = 4.8 Å), (c) HIV-1 RT (PDB: 4PQU; RMSD = 5.3 Å) and (d) telomerase RT (TERT, PDB: 3DU5; RMSD = 4.3 Å). Palm domain, slate blue; fingers domain, cyan; thumb domain, violet; N-terminal extension (NTE), red; the insertion in fingers domain (IFD), light blue; DNA endonuclease (EN) domain, lime green; DNA binding (D) domain, pale yellow; connection, light pink; RNaseH, deep salmon; telomerase RNA-binding domain (TRBD), orange.

Extended Data Fig. 6 Sequence alignment of Ec86-RT with other retron RTs.

Seven universal reverse transcriptase regions (RT1-7) are indicated by black boxes. The secondary structural elements are labelled above the sequences. The general catalytic sites were illustrated by red solid squares. The conserved residues involving in RT-msDNA interaction were illustrated by orange stars.

Extended Data Fig. 7 RT-msDNA interactions in Ec86 complex.

Middle panel, overall structure of one Ec86 protomer. a, The X and Y regions in Ec86 RT. The X region (dodger blue) contains a long loop (between α4 and α5) and α5 and interacts with the RNA segment of DNA-RNA duplex and a short loop (between α5 and β3). The Y region (light sky blue) contains a long loop (between β6–α11) and α11. The conserved residues N105, A106, and H109 in X region and VTG (243-245) in Y region are close to the catalytic core YADD. b, Interaction between ssRb and the palm-finger domain. c, The interactions between ssDa region and palm domain. d, The interactions of ssDb with palm domain.

Extended Data Fig. 8 The IRs of msDNA.

a, Cryo-EM density map (grey mesh) of the IRa1-IRa2 dsRNA. b, The IR can be detected on the gel when the Ec86 complex was subjected to 12 % urea-PAGE. Lanes 1-6, RNA marker. The last three lanes (8-10) were merged as one lane. Image is a representative of two independent experiments.

Source data

Extended Data Fig. 9 Effector in the effector-bound Ec86 complex.

a, Schematic diagram of 6EVS-NDT, Ec86-effector and HxlR. The NDT domain and wHTH domain of Ec86-effector are shaded in yellow and pink, respectively. The wHTH domain of Ec86-effector are shaded in pink as the same as the wHTH domain of Ec86-effector. b, Alignment of the NDT domain from the Ec86-effector and BpNDT (PDB: 6EVS, RMSD of 4.984 over 120 Cα atoms). c, Alignment of the wHTH domain from the Ec86-effector and BsHxlR (PDB: 7BZG, RMSD of 5.427 over 80 Cα atoms).

Extended Data Fig. 10 Sequence alignment of the Ec86 effector NDT domain with other NDT domains.

The secondary structural elements are labelled above the sequences. The general catalytic sites were illustrated by red solid squares. Ec86, Ec73, Eco9, and Sen2 indicate the effector proteins from these retron systems. 6EVS and 1F8X indicate the PDB accession code of known NDT proteins.

Supplementary information

Supplementary Information

Cryo-EM data collection and refinement statistics.

Reporting Summary

Source data

Source Data Fig. 5

Unprocessed plate images.

Source Data Fig. 6

Unprocessed plate images.

Source Data Extended Data Fig. 1

Unprocessed SDS–PAGE and urea–PAGE gels.

Source Data Extended Data Fig. 4

Unprocessed SDS–PAGE and urea–PAGE gels and unprocessed plate images.

Source Data Extended Data Fig. 8

Unprocessed urea–PAGE gels.

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Wang, Y., Guan, Z., Wang, C. et al. Cryo-EM structures of Escherichia coli Ec86 retron complexes reveal architecture and defence mechanism. Nat Microbiol 7, 1480–1489 (2022). https://doi.org/10.1038/s41564-022-01197-7

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