Structural characterization of the molecular platform for type III secretion system assembly


Type III secretion systems (TTSSs) are multi-protein macromolecular ‘machines’ that have a central function in the virulence of many Gram-negative pathogens by directly mediating the secretion and translocation of bacterial proteins (termed effectors) into the cytoplasm of eukaryotic cells1. Most of the 20 unique structural components constituting this secretion apparatus are highly conserved among animal and plant pathogens and are also evolutionarily related to proteins in the flagellar-specific export system. Recent electron microscopy experiments have revealed the gross ‘needle-shaped’ morphology of the TTSS2,3,4, yet a detailed understanding of the structural characteristics and organization of these protein components within the bacterial membranes is lacking. Here we report the 1.8-Å crystal structure of EscJ from enteropathogenic Escherichia coli (EPEC), a member of the YscJ/PrgK family whose oligomerization represents one of the earliest events in TTSS assembly5. Crystal packing analysis and molecular modelling indicate that EscJ could form a large 24-subunit ‘ring’ superstructure with extensive grooves, ridges and electrostatic features. Electron microscopy, labelling and mass spectrometry studies on the orthologous Salmonella typhimurium PrgK within the context of the assembled TTSS support the stoichiometry, membrane association and surface accessibility of the modelled ring. We propose that the YscJ/PrgK protein family functions as an essential molecular platform for TTSS assembly.

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Figure 1: Stoichiometric analysis of S. typhimurium NC base components.
Figure 2: EscJ structure and intermolecular interactions.
Figure 3: EscJ forms a superhelix in the crystal but is anchored to the inner membrane in vivo.
Figure 4: Modelling and surface electrostatic analysis of the EscJ ring.
Figure 5: Surface mapping of S. typhimurium NC with limited biotinylation and MALDI–TOF MA.


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We thank A. L. Lovering, C. P. C. Chiu and P. I. Lario for discussions; H. Law, K. Hayakawa, Y. Luo and Y. Wu for involvement in the early stages of the project; and the staff at the Advanced Light Source beamline 8.2.1 for data collection time and assistance. C.K.Y. is supported by fellowships from the Natural Sciences and Engineering Research Council of Canada and the Michael Smith Foundation for Health Research. N.C.J.S. and B.B.F. thank the Howard Hughes Medical Institute International Scholar Program, Canadian Institutes of Health Research and the Canadian Bacterial Diseases Network for funding. Funding for this project also came from grants from the NIH to S.I.M.Author Contributions C.K.Y completed the structural determination, analysis and modelling of EscJ, M.V. assisted in purification and crystallization of EscJ, R.A.P. developed the EscJ purification procedure, and E.A.F. did the original cloning of EscJ under the supervision of N.C.J.S. T.G.K. and H.B.F. performed the EM, labelling, and mass spectrometry experiments on Salmonella NCs under the supervision of S.I.M, and N.A.T. performed the EscJ localization and complementation assays under the supervision of B.B.F.

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Correspondence to Natalie C. J. Strynadka.

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Competing interests

Coordinates and observed structure factors have been deposited to the Protein Data Bank under accession code 1YJ7. Reprints and permissions information is available at The authors declare no competing financial interests.

Supplementary information

Supplementary Table S1-S3

Table 1. Data collection and refinement details. Crystallographic data collection, structure determination and refinement details; Table 2. Identification of lysine-biotinylated tryptic peptides of PrgK by MALDI-TOF mass spectrometry. List of all tryptic peptides and modified lysine residues from limited biotinylation-mass spectrometry analysis of PrgK; Table 3. Identification of lysine-biotinylated tryptic peptides of PrgH by MALDI-TOF mass spectrometry. List of all tryptic peptides and modified lysine residues from limited biotinylation-mass spectrometry analysis of PrgH. (DOC 69 kb)

Supplementary Figure S1

Structure-based sequence alignment of EscJ with members of YscJ/PrgK family and flagellar FliF. (PDF 50 kb)

Supplementary Figure S2

PrgK isolated from the needle complex is palmitoylated. Two SDS-PAGE images showing palmitoylation of PrgK. (PDF 304 kb)

Supplementary Figure S3

Effects of triple mutation (E62A/K63A/E64A) on the structure and function of EscJ. A gel from EPEC secretion assay together with two detailed structural figures showing the triple mutation (E62A/K63A/E64A) does not affect function and structure of EscJ. (PDF 2452 kb)

Supplementary Figure Legends (DOC 26 kb)

Supplementary Video S1

EscJ ring model. A movie file illustrating the surface rendered representation of the EscJ ring model. (MP4 2574 kb)

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Yip, C., Kimbrough, T., Felise, H. et al. Structural characterization of the molecular platform for type III secretion system assembly. Nature 435, 702–707 (2005).

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