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Structural basis for usher activation and intramolecular subunit transfer in P pilus biogenesis in Escherichia coli

Nature Microbiology volume 3, pages 1362–1368 (2018)Cite this article

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Abstract

Chaperone–usher pathway pili are extracellular proteinaceous fibres ubiquitously found on Gram-negative bacteria, and mediate host–pathogen interactions and biofilm formation critical in pathogenesis in numerous human diseases1. During pilus assembly, an outer membrane macromolecular machine called the usher catalyses pilus biogenesis from the individual subunits that are delivered as chaperone–subunit complexes in the periplasm. The usher orchestrates pilus assembly using all five functional domains: a 24-stranded transmembrane β-barrel translocation domain, a β-sandwich plug domain, an amino-terminal periplasmic domain and two carboxy-terminal periplasmic domains (CTD1 and CTD2)2,3,4,5,6. Despite extensive structural and functional characterization, the mechanism by which the usher is activated to initiate pilus biogenesis is unknown. Here, we present the crystal structure of the full-length PapC usher from Escherichia coli in complex with its cognate PapDG chaperone–subunit complex in a pre-activation state, elucidating molecular details of how the usher is specifically engaged by allosteric interactions with its substrate preceding activation and how the usher facilitates the transfer of subunits from the amino-terminal periplasmic domain to the CTDs during pilus assembly. This work elucidates the intricate workings of a molecular machine that catalyses chaperone–usher pathway pilus assembly and opens the door for the development of potent inhibitors to block pilus biogenesis.

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Fig. 1: Structure of the PapC–PapDG ternary complex.
Fig. 2: Comparison of the pre-activation PapC–PapDG and post-activation FimD–FimCH structures.
Fig. 3: Tripartite interface between PapD and the NTD and CTD2 of PapC.
Fig. 4: PapC NTD and CTD2 mutants are defective for usher activation.

Data availability

The data that support the findings of this study are available from the corresponding authors upon request. Atomic coordinates and structure factors for the reported crystal structure have been deposited into the Protein Data Bank under accession code 6CD2.

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Acknowledgements

We thank the staff of beamline 24-ID-E at APS for assistance, especially K. Rajashankar, K. Perry and N. Sukumar. This work used NE-CAT beamlines (GM103403), a Pilatus detector (RR029205) and an Eiger detector (OD021527) at the APS (DE-AC02-06CH11357). This work was supported by grants from the NIH (R01AI029549 and R01AI048689 to S.J.H.) and National Science Foundation (DGE-1745038 to N.S.O.), as well as start-up funds from Washington University School of Medicine (to P.Y.).

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Author notes

  1. These authors contributed equally: Peng Yuan, Scott J. Hultgren.

Authors and Affiliations

  1. Department of Molecular Microbiology, Washington University in St Louis, St Louis, MO, USA

    Natalie S. Omattage, Jerome S. Pinkner, Karen W. Dodson & Scott J. Hultgren

  2. Center for Women’s Infectious Disease Research, Washington University in St Louis, St Louis, MO, USA

    Natalie S. Omattage, Jerome S. Pinkner, Karen W. Dodson & Scott J. Hultgren

  3. Department of Cell Biology and Physiology, Washington University in St Louis, St Louis, MO, USA

    Zengqin Deng & Peng Yuan

  4. Center for the Investigation of Membrane Excitability Diseases, Washington University in St Louis, St Louis, MO, USA

    Zengqin Deng & Peng Yuan

  5. Department of Chemistry, Umeå University, Umeå, Sweden

    Fredrik Almqvist

  6. Umeå Center for Microbial Research, Umeå University, Umeå, Sweden

    Fredrik Almqvist

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  1. Natalie S. Omattage
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Contributions

N.S.O. expressed and purified protein, and produced and crystallized the PapC PapDG complex. N.S.O. and Z.D. collected X-ray data and determined the structure. P.Y. supervised the crystallography work. J.S.P. expressed and purified protein. S.J.H., P.Y., N.S.O. and Z.D. designed the in vitro functional and biochemical assays, and N.S.O. performed them. S.J.H., P.Y., K.W.D., F.A., Z.D. and N.S.O. wrote the manuscript with input from all authors.

Corresponding authors

Correspondence to Peng Yuan or Scott J. Hultgren.

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Omattage, N.S., Deng, Z., Pinkner, J.S. et al. Structural basis for usher activation and intramolecular subunit transfer in P pilus biogenesis in Escherichia coli. Nat Microbiol 3, 1362–1368 (2018). https://doi.org/10.1038/s41564-018-0255-y

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