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Structural insight into the assembly of the type II secretion system pilotin–secretin complex from enterotoxigenic Escherichia coli

Nature Microbiology volume 3, pages 581–587 (2018)Cite this article

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Abstract

Secretin is a large outer-membrane channel found in secretion systems of Gram-negative bacteria, facilitating the last step for transfer of proteins into the extracellular environment. In the type II secretion system, a lipoprotein called pilotin is essential to bind and target its corresponding secretin to the outer membrane. However, there is only limited structural information available about the interaction and assembly of the pilotin–secretin complex. Here we report the first near-atomic-resolution structure of a full-length Vibrio-type pilotin–secretin (AspS–GspD) complex from enterotoxigenic Escherichia coli by cryo-electron microscopy, which reveals the detailed assembly mode of the full-length pilotin–secretin complex. The AspS subunits attach to the secretin channel surface with a 15:15 stoichiometric ratio to GspD subunits, and insert their amino terminus into the outer membrane. The AspS subunits interact with all three secondary structural elements of the S domain of GspD, including strong interaction with the carboxy-terminal α-helix and weak interactions with another two elements, an α-helix and a loop. These structural and biochemical details provide a deeper insight to pilotin–secretin interaction and their assembly mode.

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Fig. 1: Multimeric state of the ETEC AspS–GspD complex.
Fig. 2: Cryo-electron microscopy density maps of the AspS–GspD complex.
Fig. 3: Interactions between AspS and the S domain.
Fig. 4: Influences of GspD C-terminal mutations to the complex state of AspS–GspD.

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Acknowledgements

This work was supported by funds from The National Key Research and Development Program (2016YFA0501102 and 2016YFA0501902 to X.L.), National Natural Science Foundation of China (31570730 to X.L.), Advanced Innovation Center for Structural Biology (to X.L.), Tsinghua-Peking Joint Center for Life Sciences (to X.L.) and One-Thousand Talent Program by the State Council of China (to X.L.). We thank C. Lin for providing assistance with protein preparation. We thank Tsinghua University Branch of the China National Center for Protein Sciences Beijing for providing facility support in protein preparation, cryo-electron microscopy and computation. We are grateful to Tsinghua-Nikon imaging center and Tsinghua Imaging Core Facility for providing technical support and to Yanli Zhang for assistance with confocal microscopy and image processing.

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  1. These authors contributed equally: Meng Yin and Zhaofeng Yan

Authors and Affiliations

  1. Key Laboratory of Protein Sciences (Tsinghua University), Ministry of Education, Beijing, China

    Meng Yin, Zhaofeng Yan & Xueming Li

  2. School of Life Sciences, Tsinghua University, Beijing, China

    Meng Yin, Zhaofeng Yan & Xueming Li

  3. Tsinghua-Peking Joint Center for Life Sciences, Beijing, China

    Meng Yin & Xueming Li

  4. Advanced Innovation Center for Structural Biology, Tsinghua University, Beijing, China

    Meng Yin, Zhaofeng Yan & Xueming Li

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  1. Meng Yin
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  2. Zhaofeng Yan
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Contributions

M.Y., Z.Y. and X.L. designed all experiments. M.Y. and Z.Y. performed all the cryo-electron microscopy and biochemistry experiments. All authors contributed to the data analysis, manuscript preparation and revision.

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Correspondence to Xueming Li.

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Yin, M., Yan, Z. & Li, X. Structural insight into the assembly of the type II secretion system pilotin–secretin complex from enterotoxigenic Escherichia coli. Nat Microbiol 3, 581–587 (2018). https://doi.org/10.1038/s41564-018-0148-0

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