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Structure of the GspK–GspI–GspJ complex from the enterotoxigenic Escherichia coli type 2 secretion system

Nature Structural & Molecular Biology volume 15, pages 462–468 (2008)Cite this article

Abstract

Gram-negative bacteria translocate various proteins including virulence factors across their outer membrane via type 2 secretion systems (T2SSs). T2SSs are thought to contain a pseudopilus, a subcomplex formed by one major and several minor pseudopilins. We report the crystal structure of the complex formed by three minor pseudopilins from enterotoxigenic Escherichia coli. The GspK–GspI–GspJ complex has quasihelical characteristics and an architecture consistent with a localization at the pseudopilus tip. The α-domain of GspK has a previously unobserved fold with an unexpected dinuclear metal binding site. The area surrounding its disulfide bridge is conserved and might interact with other T2SS components or with secreted proteins.

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Figure 1: The ETEC GspK–GspI–GspJ complex.
Figure 2: Calcium binding site and fold of GspK.
Figure 3: The interfaces of ETEC GspK–GspI–GspJ ternary complex.
Figure 4: The helical properties of the ETEC GspK–GspI–GspJ complex.

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References

  1. Sandkvist, M. Type II secretion and pathogenesis. Infect. Immun. 69, 3523–3535 (2001).

    Article  CAS  Google Scholar 

  2. Tauschek, M., Gorrell, R.J., Strugnell, R.A. & Robins-Browne, R.M. Identification of a protein secretory pathway for the secretion of heat-labile enterotoxin by an enterotoxigenic strain of Escherichia coli. Proc. Natl. Acad. Sci. USA 99, 7066–7071 (2002).

    Article  CAS  Google Scholar 

  3. Turner, S.M., Scott-Tucker, A., Cooper, L.M. & Henderson, I.R. Weapons of mass destruction: virulence factors of the global killer enterotoxigenic Escherichia coli. FEMS Microbiol. Lett. 263, 10–20 (2006).

    Article  CAS  Google Scholar 

  4. Sandkvist, M. et al. General secretion pathway (eps) genes required for toxin secretion and outer membrane biogenesis in Vibrio cholerae. J. Bacteriol. 179, 6994–7003 (1997).

    Article  CAS  Google Scholar 

  5. Filloux, A. The underlying mechanisms of type II protein secretion. Biochim. Biophys. Acta 1694, 163–179 (2004).

    Article  CAS  Google Scholar 

  6. Johnson, T.L., Abendroth, J., Hol, W.G. & Sandkvist, M. Type II secretion: from structure to function. FEMS Microbiol. Lett. 255, 175–186 (2006).

    Article  CAS  Google Scholar 

  7. Hobbs, M. & Mattick, J.S. Common components in the assembly of type 4 fimbriae, DNA transfer systems, filamentous phage and protein-secretion apparatus: a general system for the formation of surface-associated protein complexes. Mol. Microbiol. 10, 233–243 (1993).

    Article  CAS  Google Scholar 

  8. Peabody, C.R. et al. Type II protein secretion and its relationship to bacterial type IV pili and archaeal flagella. Microbiology 149, 3051–3072 (2003).

    Article  CAS  Google Scholar 

  9. Mattick, J.S. & Alm, R.A. Response from Mattick and Alm: common architecture of type 4 fimbriae and complexes involved in macromolecular traffic. Trends Microbiol. 3, 411–413 (1995).

    Article  Google Scholar 

  10. Filloux, A., Michel, G. & Bally, M. GSP-dependent protein secretion in Gram-negative bacteria: the Xcp system of Pseudomonas aeruginosa. FEMS Microbiol. Rev. 22, 177–198 (1998).

    Article  CAS  Google Scholar 

  11. Nunn, D.N. & Lory, S. Product of the Pseudomonas aeruginosa gene pilD is a prepilin leader peptidase. Proc. Natl. Acad. Sci. USA 88, 3281–3285 (1991).

    Article  CAS  Google Scholar 

  12. Nunn, D.N. & Lory, S. Cleavage, methylation, and localization of the Pseudomonas aeruginosa export proteins XcpT, -U, -V, and -W. J. Bacteriol. 175, 4375–4382 (1993).

    Article  CAS  Google Scholar 

  13. Parge, H.E. et al. Structure of the fibre-forming protein pilin at 2.6 Å resolution. Nature 378, 32–38 (1995).

    Article  CAS  Google Scholar 

  14. Sauvonnet, N., Vignon, G., Pugsley, A.P. & Gounon, P. Pilus formation and protein secretion by the same machinery in Escherichia coli. EMBO J. 19, 2221–2228 (2000).

    Article  CAS  Google Scholar 

  15. Bleves, S. et al. The secretion apparatus of Pseudomonas aeruginosa: identification of a fifth pseudopilin, XcpX (GspK family). Mol. Microbiol. 27, 31–40 (1998).

    Article  CAS  Google Scholar 

  16. Wolfgang, M., van Putten, J.P., Hayes, S.F. & Koomey, M. The comP locus of Neisseria gonorrhoeae encodes a type IV prepilin that is dispensable for pilus biogenesis but essential for natural transformation. Mol. Microbiol. 31, 1345–1357 (1999).

    Article  CAS  Google Scholar 

  17. Toma, C., Kuroki, H., Nakasone, N., Ehara, M. & Iwanaga, M. Minor pilin subunits are conserved in Vibrio cholerae type IV pili. FEMS Immunol. Med. Microbiol. 33, 35–40 (2002).

    Article  CAS  Google Scholar 

  18. Winther-Larsen, H.C. et al. A conserved set of pilin-like molecules controls type IV pilus dynamics and organelle-associated functions in Neisseria gonorrhoeae. Mol. Microbiol. 56, 903–917 (2005).

    Article  CAS  Google Scholar 

  19. Lu, H.M., Motley, S.T. & Lory, S. Interactions of the components of the general secretion pathway: role of Pseudomonas aeruginosa type IV pilin subunits in complex formation and extracellular protein secretion. Mol. Microbiol. 25, 247–259 (1997).

    Article  CAS  Google Scholar 

  20. Douet, V., Loiseau, L., Barras, F. & Py, B. Systematic analysis, by the yeast two-hybrid, of protein interaction between components of the type II secretory machinery of Erwinia chrysanthemi. Res. Microbiol. 155, 71–75 (2004).

    Article  CAS  Google Scholar 

  21. Kuo, W.W., Kuo, H.W., Cheng, C.C., Lai, H.L. & Chen, L.Y. Roles of the minor pseudopilins, XpsH, XpsI and XpsJ, in the formation of XpsG-containing pseudopilus in Xanthomonas campestris pv. campestris. J. Biomed. Sci. 12, 587–599 (2005).

    Article  CAS  Google Scholar 

  22. Hazes, B., Sastry, P.A., Hayakawa, K., Read, R.J. & Irvin, R.T. Crystal structure of Pseudomonas aeruginosa PAK pilin suggests a main-chain-dominated mode of receptor binding. J. Mol. Biol. 299, 1005–1017 (2000).

    Article  CAS  Google Scholar 

  23. Keizer, D.W. et al. Structure of a pilin monomer from Pseudomonas aeruginosa: implications for the assembly of pili. J. Biol. Chem. 276, 24186–24193 (2001).

    Article  CAS  Google Scholar 

  24. Craig, L., Pique, M.E. & Tainer, J.A. Type IV pilus structure and bacterial pathogenicity. Nat. Rev. Microbiol. 2, 363–378 (2004).

    Article  CAS  Google Scholar 

  25. Köhler, R. et al. Structure and assembly of the pseudopilin PulG. Mol. Microbiol. 54, 647–664 (2004).

    Article  Google Scholar 

  26. Craig, L. et al. Type IV pilus structure by cryo-electron microscopy and crystallography: implications for pilus assembly and functions. Mol. Cell 23, 651–662 (2006).

    Article  CAS  Google Scholar 

  27. Robien, M.A., Krumm, B.E., Sandkvist, M. & Hol, W.G. Crystal structure of the extracellular protein secretion NTPase EpsE of Vibrio cholerae. J. Mol. Biol. 333, 657–674 (2003).

    Article  CAS  Google Scholar 

  28. Abendroth, J., Bagdasarian, M., Sandkvist, M. & Hol, W.G. The structure of the cytoplasmic domain of EpsL, an inner membrane component of the type II secretion system of Vibrio cholerae: an unusual member of the actin-like ATPase superfamily. J. Mol. Biol. 344, 619–633 (2004).

    Article  CAS  Google Scholar 

  29. Abendroth, J., Rice, A.E., McLuskey, K., Bagdasarian, M. & Hol, W.G. The crystal structure of the periplasmic domain of the type II secretion system protein EpsM from Vibrio cholerae: the simplest version of the ferredoxin fold. J. Mol. Biol. 338, 585–596 (2004).

    Article  CAS  Google Scholar 

  30. Abendroth, J., Murphy, P., Sandkvist, M., Bagdasarian, M. & Hol, W.G. The X-ray structure of the type II secretion system complex formed by the N-terminal domain of EpsE and the cytoplasmic domain of EpsL of Vibrio cholerae. J. Mol. Biol. 348, 845–855 (2005).

    Article  CAS  Google Scholar 

  31. Korotkov, K.V., Krumm, B., Bagdasarian, M. & Hol, W.G. Structural and functional studies of EpsC, a crucial component of the type 2 secretion system from Vibrio cholerae. J. Mol. Biol. 363, 311–321 (2006).

    Article  CAS  Google Scholar 

  32. Yanez, M.E., Korotkov, K.V., Abendroth, J. & Hol, W.G. Structure of the minor pseudopilin EpsH from the type 2 Secretion system of Vibrio cholerae. J. Mol. Biol. 375, 471–486 (2008).

    Article  CAS  Google Scholar 

  33. Yanez, M.E., Korotkov, K.V., Abendroth, J. & Hol, W.G. The crystal structure of a binary complex of two pseudopilins: EpsI and EpsJ from the type 2 secretion system of Vibrio vulnificus. J. Mol. Biol. 375, 471–486 (2008).

    Article  CAS  Google Scholar 

  34. Pugsley, A.P., Bayan, N. & Sauvonnet, N. Disulfide bond formation in secreton component PulK provides a possible explanation for the role of DsbA in pullulanase secretion. J. Bacteriol. 183, 1312–1319 (2001).

    Article  CAS  Google Scholar 

  35. Durand, E. et al. XcpX controls biogenesis of the Pseudomonas aeruginosa XcpT-containing pseudopilus. J. Biol. Chem. 280, 31378–31389 (2005).

    Article  CAS  Google Scholar 

  36. Reyss, I. & Pugsley, A.P. Five additional genes in the pulC-O operon of the Gram-negative bacterium Klebsiella oxytoca UNF5023 which are required for pullulanase secretion. Mol. Gen. Genet. 222, 176–184 (1990).

    Article  CAS  Google Scholar 

  37. Helaine, S., Dyer, D.H., Nassif, X., Pelicic, V. & Forest, K.T. 3D structure/function analysis of PilX reveals how minor pilins can modulate the virulence properties of type IV pili. Proc. Natl. Acad. Sci. USA 104, 15888–15893 (2007).

    Article  CAS  Google Scholar 

  38. Holm, L. & Sander, C. Mapping the protein universe. Science 273, 595–603 (1996).

    Article  CAS  Google Scholar 

  39. Hansen, J.K. & Forest, K.T. Type IV pilin structures: insights on shared architecture, fiber assembly, receptor binding and type II secretion. J. Mol. Microbiol. Biotechnol. 11, 192–207 (2006).

    Article  CAS  Google Scholar 

  40. Jones, S. & Thornton, J.M. Principles of protein-protein interactions. Proc. Natl. Acad. Sci. USA 93, 13–20 (1996).

    Article  CAS  Google Scholar 

  41. Jones, S. & Thornton, J.M. Protein-protein interactions: a review of protein dimer structures. Prog. Biophys. Mol. Biol. 63, 31–65 (1995).

    Article  CAS  Google Scholar 

  42. Nunn, D. Bacterial type II protein export and pilus biogenesis: more than just homologies? Trends Cell Biol. 9, 402–408 (1999).

    Article  CAS  Google Scholar 

  43. Vignon, G. et al. Type IV-like pili formed by the type II secreton: specificity, composition, bundling, polar localization, and surface presentation of peptides. J. Bacteriol. 185, 3416–3428 (2003).

    Article  CAS  Google Scholar 

  44. Alm, R.A. & Mattick, J.S. Genes involved in the biogenesis and function of type-4 fimbriae in Pseudomonas aeruginosa. Gene 192, 89–98 (1997).

    Article  CAS  Google Scholar 

  45. Hu, N.T. et al. XpsG, the major pseudopilin in Xanthomonas campestris pv. campestris, forms a pilus-like structure between cytoplasmic and outer membranes. Biochem. J. 365, 205–211 (2002).

    Article  CAS  Google Scholar 

  46. Durand, E. et al. Type II protein secretion in Pseudomonas aeruginosa: the pseudopilus is a multifibrillar and adhesive structure. J. Bacteriol. 185, 2749–2758 (2003).

    Article  CAS  Google Scholar 

  47. van Duyne, G.D., Standaert, R.F., Karplus, P.A., Schreiber, S.L. & Clardy, J. Atomic structures of the human immunophilin FKBP-12 complexes with FK506 and rapamycin. J. Mol. Biol. 229, 105–124 (1993).

    Article  CAS  Google Scholar 

  48. Luft, J.R. et al. A deliberate approach to screening for initial crystallization conditions of biological macromolecules. J. Struct. Biol. 142, 170–179 (2003).

    Article  CAS  Google Scholar 

  49. Terwilliger, T. SOLVE and RESOLVE: automated structure solution, density modification and model building. J. Synchrotron Radiat. 11, 49–52 (2004).

    Article  CAS  Google Scholar 

  50. Bricogne, G., Vonrhein, C., Flensburg, C., Schiltz, M. & Paciorek, W. Generation, representation and flow of phase information in structure determination: recent developments in and around SHARP 2.0. Acta Crystallogr. D Biol. Crystallogr. 59, 2023–2030 (2003).

    Article  CAS  Google Scholar 

  51. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).

    Article  Google Scholar 

  52. McCoy, A.J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).

    Article  CAS  Google Scholar 

  53. Murshudov, G.N., Vagin, A.A. & Dodson, E.J. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D Biol. Crystallogr. 53, 240–255 (1997).

    Article  CAS  Google Scholar 

  54. Painter, J. & Merritt, E.A. Optimal description of a protein structure in terms of multiple groups undergoing TLS motion. Acta Crystallogr. D Biol. Crystallogr. 62, 439–450 (2006).

    Article  Google Scholar 

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Acknowledgements

We thank M. Yanez, S. Turley, J. Bosch and J. Abendroth for help and valuable discussions; S. Moseley from the Department of Microbiology, University of Washington, for providing the ETEC genomic DNA; the Hauptman-Woodward Institute in Buffalo for crystal screening; and the support staff of beamline 9-2 of the Stanford Synchrotron Radiation Laboratory (SSRL) for assistance during data collection. Portions of this research were carried out at the SSRL, supported by the Department of Energy and by the US National Institutes of Health (NIH). This work was supported by grant AI34501 from the NIH and by the Howard Hughes Medical Institute (HHMI).

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  1. Department of Biochemistry, Biomolecular Structure Center, University of Washington, Box 357742, Seattle, 98195, Washington, USA

    Konstantin V Korotkov & Wim G J Hol

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  1. Konstantin V Korotkov
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  2. Wim G J Hol
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K.V.K. cloned, purified, crystallized and determined the GspK–GspI–GspJ structure, and K.V.K. and W.G.J.H. designed the research and wrote the manuscript.

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Correspondence to Wim G J Hol.

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Korotkov, K., Hol, W. Structure of the GspK–GspI–GspJ complex from the enterotoxigenic Escherichia coli type 2 secretion system. Nat Struct Mol Biol 15, 462–468 (2008). https://doi.org/10.1038/nsmb.1426

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