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.
Subscribe to Journal
Get full journal access for 1 year
only $3.90 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Ghosh, P. Process of protein transport by the type III secretion system. Microbiol. Mol. Biol. Rev. 68, 771–795 (2004)
Marlovits, T. C. et al. Structural insights into the assembly of the type III secretion needle complex. Science 306, 1040–1042 (2004)
Kubori, T. et al. Supramolecular structure of the Salmonella typhimurium type III protein secretion system. Science 280, 602–605 (1998)
Blocker, A. et al. Structure and composition of the Shigella flexneri ‘needle complex’, a part of its type III secreton. Mol. Microbiol. 39, 652–663 (2001)
Kimbrough, T. G. & Miller, S. I. Assembly of the type III secretion needle complex of Salmonella typhimurium . Microbes Infect. 4, 75–82 (2002)
Kimbrough, T. G. & Miller, S. I. Contribution of Salmonella typhimurium type III secretion components to needle complex formation. Proc. Natl Acad. Sci. USA 97, 11008–11013 (2000)
Galan, J. E. & Collmer, A. Type III secretion machines: bacterial devices for protein delivery into host cells. Science 284, 1322–1328 (1999)
Sukhan, A., Kubori, T., Wilson, J. & Galan, J. E. Genetic analysis of assembly of the Salmonella enterica serovar Typhimurium type III secretion-associated needle complex. J. Bacteriol. 183, 1159–1167 (2001)
Crago, A. M. & Koronakis, V. Salmonella InvG forms a ring-like multimer that requires the InvH lipoprotein for outer membrane localization. Mol. Microbiol. 30, 47–56 (1998)
Aizawa, S. I. Flagellar assembly in Salmonella typhimurium . Mol. Microbiol. 19, 1–5 (1996)
Burghout, P. et al. Structure and electrophysiological properties of the YscC secretin from the type III secretion system of Yersinia enterocolitica . J. Bacteriol. 186, 4645–4654 (2004)
Linderoth, N. A., Simon, M. N. & Russel, M. The filamentous phage pIV multimer visualized by scanning transmission electron microscopy. Science 278, 1635–1638 (1997)
Nouwen, N. et al. Secretin PulD: association with pilot PulS, structure, and ion-conducting channel formation. Proc. Natl Acad. Sci. USA 96, 8173–8177 (1999)
Jones, C. J., Macnab, R. M., Okino, H. & Aizawa, S. Stoichiometric analysis of the flagellar hook–(basal-body) complex of Salmonella typhimurium . J. Mol. Biol. 212, 377–387 (1990)
Sekiya, K. et al. Supermolecular structure of the enteropathogenic Escherichia coli type III secretion system and its direct interaction with the EspA-sheath-like structure. Proc. Natl Acad. Sci. USA 98, 11638–11643 (2001)
Suzuki, H., Yonekura, K. & Namba, K. Structure of the rotor of the bacterial flagellar motor revealed by electron cryomicroscopy and single-particle image analysis. J. Mol. Biol. 337, 105–113 (2004)
Thomas, J., Stafford, G. P. & Hughes, C. Docking of cytosolic chaperone-substrate complexes at the membrane ATPase during flagellar type III protein export. Proc. Natl Acad. Sci. USA 101, 3945–3950 (2004)
Leslie, A.G.W. Recent changes to the MOSFLM package for processing film and image plate data. Joint CCP4 + ESF-EAMCB Newsl. Protein Crystallogr. no. 26 (1992).
Evans, P. R. Data reduction. Proc. CCP4 Study Weekend on Data Collection and Processing 114–122, (1993)
Terwilliger, T. C. & Berendzen, J. Automated MAD and MIR structure solution. Acta Crystallogr. D 55, 849–861 (1999)
Terwilliger, T. C. Maximum-likelihood density modification. Acta Crystallogr. D 56, 965–972 (2000)
McRee, D. E. XtalView/Xfit—A versatile program for manipulating atomic coordinates and electron density. J. Struct. Biol. 125, 156–165 (1999)
Brunger, A. T. et al. Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr. D 54, 905–921 (1998)
Murshudov, G. N. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D 53, 240–255 (1997)
Kabsch, W. A solution for the best rotation to relate two sets of vectors. Acta Crystallogr. A 32, 922–923 (1976)
Nicholls, A., Sharp, K. A. & Honig, B. Protein folding and association: insights from the interfacial and thermodynamic properties of hydrocarbons. Proteins 11, 281–296 (1991)
Kraulis, P. J. MOLSCRIPT: A program to produce both detailed and schematic plots of protein structures. J. Appl. Crystallogr. 24, 946–950 (1991)
Merritt, E. A. B. & Bacon, D. J. Raster3D: photorealistic Molecular Graphics. Methods Enzymol. 277, 505–524 (1997)
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.
Coordinates and observed structure factors have been deposited to the Protein Data Bank under accession code 1YJ7. Reprints and permissions information is available at npg.nature.com/reprintsandpermissions. The authors declare no competing financial interests.
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)
Structure-based sequence alignment of EscJ with members of YscJ/PrgK family and flagellar FliF. (PDF 50 kb)
PrgK isolated from the needle complex is palmitoylated. Two SDS-PAGE images showing palmitoylation of PrgK. (PDF 304 kb)
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)
EscJ ring model. A movie file illustrating the surface rendered representation of the EscJ ring model. (MP4 2574 kb)
About this article
Cite this article
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). https://doi.org/10.1038/nature03554
PLOS Pathogens (2020)
HilD, HilC, and RtsA Form Homodimers and Heterodimers To Regulate Expression of the Salmonella Pathogenicity Island I Type III Secretion System
Journal of Bacteriology (2020)
Journal of Chemical Information and Modeling (2020)
Current Opinion in Structural Biology (2020)
An account ofin silicoidentification tools of secreted effector proteins in bacteria and future challenges
Briefings in Bioinformatics (2019)