Abstract

Export of proteins through type III secretion systems is critical for motility and virulence of many major bacterial pathogens. Three putative integral membrane proteins (FliP, FliQ, FliR) are suggested to form the core of an export gate in the inner membrane, but their structure, assembly and location within the final nanomachine remain unclear. Here, we present the cryoelectron microscopy structure of the Salmonella Typhimurium FliP–FliQ–FliR complex at 4.2 Å. None of the subunits adopt canonical integral membrane protein topologies, and common helix-turn-helix structural elements allow them to form a helical assembly with 5:4:1 stoichiometry. Fitting of the structure into reconstructions of intact secretion systems, combined with cross-linking, localize the export gate as a core component of the periplasmic portion of the machinery. This study thereby identifies the export gate as a key element of the secretion channel and implies that it primes the helical architecture of the components assembling downstream.

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Change history

  • 17 July 2018

    In the version of this article initially published, the PDB code associated with the study was given as 6F2E but should have been 6F2D in Table 1 and the data availability statement. The error has been corrected in the HTML and PDF versions of the article.

  • 23 July 2018

    In the version of this paper originally published, the link for Supplementary Video 2 incorrectly led to Supplementary Video 1. This has been corrected in the HTML version of the paper.

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Acknowledgements

We thank E. Johnson and A. Costin of the Central Oxford Structural Microscopy and Imaging Centre and all staff of the ARC Centre of Advanced Imaging, Monash University, for assistance with data collection. We thank H. Elmlund (Monash) for assistance with data collection, processing strategies and access to SIMPLE code ahead of release. For collection of preliminary data, we acknowledge Diamond for access and support of the Cryo-EM facilities at the UK National Electron Bio-Imaging Centre (eBIC), proposal EM15354, funded by the Wellcome Trust, MRC and BBSRC. We thank T. Marlovits, Centre for Structural Systems Biology, Hamburg, for providing PrgK (SctJ) antisera. The Central Oxford Structural Microscopy and Imaging Centre is supported by the Wellcome Trust (201536), The EPA Cephalosporin Trust, the Wolfson Foundation and a Royal Society/Wolfson Foundation Laboratory Refurbishment Grant (WL160052). Work performed in the laboratory of S.M.L. was supported by a Wellcome Trust Investigator Award (100298) and an MRC programme grant (M011984). Work performed in the lab of C.V.R. is funded by a Wellcome Trust Investigator Award (104633), an ERC Advanced Grant ENABLE (641317) and an MRC programme grant (N020413). J.G. is a Junior Research Fellow of The Queen’s College, Oxford. Work performed in the laboratory of S.W. was supported by the Alexander von Humboldt Foundation in the framework of the Sofja Kovalevskaja Award endowed by the Federal Ministry of Education and Research (BMBF) and by the Deutsche Forschungsgemeinschaft (DFG) as part of the Collaborative Research Center (SFB) 766 Bacterial Cell Envelope, project B14.

Author information

Author notes

    • Patrizia Abrusci

    Present address: Structural Genomics Consortium, University of Oxford, Oxford, UK

  1. These authors contributed equally: Lucas Kuhlen, Patrizia Abrusci, Steven Johnson.

Affiliations

  1. Sir William Dunn School of Pathology, University of Oxford, Oxford, UK

    • Lucas Kuhlen
    • , Patrizia Abrusci
    • , Steven Johnson
    • , Justin Deme
    • , Joseph Caesar
    •  & Susan M. Lea
  2. Department of Chemistry, University of Oxford, Oxford, UK

    • Lucas Kuhlen
    • , Joseph Gault
    •  & Carol V. Robinson
  3. Central Oxford Structural Microscopy and Imaging Centre, University of Oxford, Oxford, UK

    • Justin Deme
    • , Joseph Caesar
    •  & Susan M. Lea
  4. Section of Cellular and Molecular Microbiology, Interfaculty Institute of Microbiology and Infection Medicine (IMIT), University of Tübingen, Tübingen, Germany

    • Tobias Dietsche
    • , Mehari Tesfazgi Mebrhatu
    •  & Samuel Wagner
  5. Proteome Center Tübingen, University of Tübingen, Tübingen, Germany

    • Tariq Ganief
    •  & Boris Macek
  6. German Center for Infection Research, Partner-site Tübingen, Tübingen, Germany

    • Samuel Wagner

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Contributions

All authors contributed to editing of manuscript and figures. L.K. performed experiments: strain and plasmid construction, complex purification, nMS, cryo-EM grid optimization, cryo-EM data analysis and model building and analysis. P.A. performed experiments: strain and plasmid construction, complex purification, initial cryo-EM grid optimization and cryo-EM data analysis. S.J. performed experiments and designed and supervised experiments for characterization of protein complexes, cryo-EM data analysis, structure determination and analysis, and wrote the first draft of manuscript and figures with S.M.L. J.G. performed and supervised nMS experiments. J.D. performed experiments: cryo-EM grid optimization and data collection. J.C. performed experiments: optimized computational analysis of cryo-EM data. T.D. performed experiments: strain and plasmid construction, in vivo photo-cross-linking. M.T.M. performed experiments: NC purification, chemical cross-linking. T.G. performed experiments for nMS of DSS cross-linked samples and analyzed data. B.M. analyzed DSS nMS data and wrote the DSS nMS methods section. S.W. analyzed data for the in vivo photo-cross-linking, chemical cross-linking and DSS nMS, and wrote the first draft of photo- and chemical-cross-linking sections. C.V.R. supervised design and implementation of nMS experiments. S.M.L. performed and supervised experiments for cryo-EM grid optimization and data collection, data and structure analysis, and wrote the first draft of paper with S.J.

Competing interests

The authors declare no competing interests.

Corresponding author

Correspondence to Susan M. Lea.

Integrated supplementary information

  1. Supplementary Figure 1 Characterization of the S. Typhimurium flagellar PQR.

    (a) High energy dissociation spectrum of the FliPQR complex from S. Typhimurium extracted, purified and sprayed in DDM detergent. nMS followed by high energy collisional activation (200 V HCD, 150 V in source dissociation) causes dissociation of the PQR assembly and formation of many different subcomplexes containing combinations of P and R subunits. The extracted subunit spectra are displayed in color below the raw data after processing in Unidec. No subcomplexes containing Q could be observed implying it is less tightly associated. This suggests that R is integrated into the PR core complex and Q is more peripherally bound. (b) SEC-MALS analysis on a Superdex200 column of FliPQR purified in LMNG co-expressed with FlhB and FlhA. Molecular weight of the protein component across the peak is represented as a red line and the portion of the peak used for cryo-EM is highlighted in purple. The inset shows SDS-PAGE of the sample confirming the presence of all three components plus FlhB (levels of which vary across the peak) which is seen in two fragments in the gel annotated FlhBN and FlhBC for N and C terminal fragments respectively.

  2. Supplementary Figure 2 Single-particle cryo-EM of FliPQR in LMNG.

    (a) Representative micrograph of the FliPQR complex. (b) Selected reference-free 2D class averages. (c) Gold-standard FSC curve of the final map calculated using a soft-edged mask. (d) Local resolution estimates of the final map calculated using ResMap (Swint-Kruse & Brown, 2005). On the left is the overall map with representative slices through the map shown on the right. (e) Examples of good (left), average (middle) and bad (right) density.

  3. Supplementary Figure 3

    Flow chart summarizing the processing of the Titan Krios dataset.

  4. Supplementary Figure 4 Purification and crosslinking of the S. Typhimurium T3SS-1 needle complex base.

    (a) Negative stain electron micrograph of purified bases of an S. Typhimurium strain with an enzymatically inactive InvC (SctN) ATPase. (b) Coomassie-stained SDS PAGE gel showing the protein content of purified bases with and without crosslinking by DSS. (c) Coomassie-stained SDS PAGE gel showing the briefly run sample (same as in (b)) that was used for in-gel digestion and mass spectrometry. (d) Secretion assay showing functionality of PrgK (SctJ)-pBpa mutants used for in vivo photocrosslinking. Type III-dependent secretion into the culture supernatant of the wild type and indicated pBpa mutants of PrgK (SctJ) was assayed by SDS PAGE and immunodetection of the early substrate InvJ and the intermediate substrate SipB. (e) Uncropped originals of Western blots shown in Fig. 4b. In vivo photo-crosslinking studies reveal cross-links between P5Q4R1 and the inner membrane ring component SctJ in the S. Typhimurium injectisome. SctJ tends to crosslink pBpa-indendent to some extent. * indicates these pBpa-independent crosslinks. SctJE138X crosslinks readily to neighbouring SctJE138X. This results in a crosslink-ladder, of which the homodimeric SctJ-SctJ crosslink is indicated by **. Consequently, also SctJ-SctJ-RFLAG crosslinks are readily detected. Due to the less focussed running behavior of SctJ-PFLAG crosslinks on the SDS-gel, SctJ-SctJ-PFLAG crosslinks are less clearly apparent. *** indicates a band likely resulting from a SctJ-Q crosslink. Q is 9 kDa. Since there is no reliable antibody against Q available and since tagging of Q results in loss of function of the protein, a reciprocal validation of this crosslink is not possible at this point. The images result from a dual-color detection using the LiCor Odyssey system. Therefore, the all blue molecular mass marker is only visible in the 700 nm emission channel that was used for detecting the polyclonal rabbit anti-SctJ (PrgK) antibody.

  5. Supplementary Figure 5 Annotated MS/MS spectra for the QYGEETETVKR (SpaP)–VAKDDSLR (InvG) cross-linked peptide.

    Annotated MS/MS spectra for both heavy (a) and light (b) crosslinks. Light (595.8058 m/z) and heavy (598.8228 m/z) DSS crosslinked peptides appear as a quadruply charged doublet with the expected 12.075 Da mass shift (c).

  6. Supplementary Figure 6 Annotated MS/MS spectra for the DKDEIEKPSIF (SpaP)–VAKDDSLR (InvG) cross-linked peptide.

    Annotated MS/MS spectra for both heavy (a) and light (b) crosslinks. Light (591.0604 m/z) and heavy (594.0784 m/z) DSS crosslinked peptides appear as a quadruply charged doublet with the expected 12.075 Da mass shift (c).

  7. Supplementary Figure 7 Alignment of typical flagellar and non-flagellar sequences of P, Q and R annotated with secondary structure as seen in our 3D model.

    FliPQR and SctRST sequences are those of S. Typhimurium.

  8. Supplementary Figure 8 Reinterpretation of earlier tomograms in light of the structure of PQR. Figure taken from Hu et al, 2017.

    (a) Tomogram of wild type S. Typhimurium T3SS. PQR complex is positioned within the image and yellow highlights probable course of inner membrane whilst green denotes density likely contributed by cytoplasmic and membrane domains of A. (b) Tomogram of S. Typhimurium T3SS from a strain lacking expression of A. The PQR complex is shown and yellow indicates the inner membrane which seems distorted to contact the base of the PQR complex in the absence of A.

Supplementary Information

  1. Supplementary Figures

    Supplementary Figures 1–8 and Supplementary Tables 1–5

  2. Reporting Summary

  3. Supplementary Data Set 1

    Native mass spectroscopy files used to generate Fig. 1

  4. Supplementary Video 1

    Animation of fitting of Gremlin models into the experimental cryo-EM map. The movie starts by showing the fitting of the FliP(1) in the unsharpened experimental map. After morphing to the final protein model, the map is replaced by the final sharpened map (see Methods) and side chains are shown. The zoom is then reset and the fitting of the other chains shown in the unsharpened map

  5. Supplementary Video 2

    Morph between closed and open states of the PQR complex. The PQR complex is shown in the context of the coordinates for the rest of the S. Typhimurium injectisome basal body (PDB 5TCR). The closed state is the structure seen by cryo-EM; the open state is modeled on the basis of the initial Gremlin models as described in the Methods

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https://doi.org/10.1038/s41594-018-0086-9

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