Abstract

Many bacteria, including Legionella pneumophila, rely on the type IV secretion system to translocate a repertoire of effector proteins into the hosts for their survival and growth. Type IV coupling protein (T4CP) is a hexameric ATPase that links translocating substrates to the transenvelope secretion conduit. Yet, how a large number of effector proteins are selectively recruited and processed by T4CPs remains enigmatic. DotL, the T4CP of L. pneumophila, contains an ATPase domain and a C-terminal extension whose function is unknown. Unlike T4CPs involved in plasmid DNA translocation, DotL appeared to function by forming a multiprotein complex with four other proteins. Here, we show that the C-terminal extension of DotL interacts with DotN, IcmS, IcmW and an additionally identified subunit LvgA, and that this pentameric assembly binds Legionella effector proteins. We determined the crystal structure of this assembly and built an architecture of the T4CP holocomplex by combining a homology model of the ATPase domain of DotL. The holocomplex is a hexamer of a bipartite structure composed of a membrane-proximal ATPase domain and a membrane-distal substrate-recognition assembly. The presented information demonstrates the architecture and functional dissection of the multiprotein T4CP complexes and provides important insights into their substrate recruitment and processing.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

References

  1. 1.

    , & Microreview: type IV secretion systems: versatility and diversity in function. Cell. Microbiol. 12, 1203–1212 (2010).

  2. 2.

    , , , & Identification of the DotL coupling protein subcomplex of the Legionella Dot/Icm type IV secretion system. Mol. Microbiol. 85, 378–391 (2012).

  3. 3.

    , , & Coupling factors in macromolecular type-IV secretion machineries. Curr. Pharm. Des. 10, 1551–1565 (2004).

  4. 4.

    , & Assembly and mechanisms of bacterial type IV secretion machines. Phil. Trans. R. Soc. Lond. B 367, 1073–1087 (2012).

  5. 5.

    et al. Virb/D4-dependent protein translocation from Agrobacterium into plant cells. Science 290, 979–982 (2000).

  6. 6.

    , , , & Analysis of Vir protein translocation from Agrobacterium tumefaciens using Saccharomyces cerevisiae as a model: evidence for transport of a novel effector protein VirE3. Nucleic Acids Res. 31, 860–868 (2003).

  7. 7.

    , & Energetic components VirD4, VirB11 and VirB4 mediate early DNA transfer reactions required for bacterial type IV secretion. Mol. Microbiol. 54, 1199–1211 (2004).

  8. 8.

    , , & Agrobacterium tumefaciens VirB6 domains direct the ordered export of a DNA substrate through a type IV secretion system. J. Mol. Biol. 341, 961–977 (2004).

  9. 9.

    , & The Legionella pneumophila replication vacuole: making a cosy niche inside host cells. Nat. Rev. Microbiol. 7, 13–24 (2009).

  10. 10.

    & Formation of a pathogen vacuole according to Legionella pneumophila: how to kill one bird with many stones. Cell. Microbiol. 17, 935–950 (2015).

  11. 11.

    & Two distinct defects in intracellular growth complemented by a single genetic locus in Legionella pneumophila. Mol. Microbiol. 7, 7–19 (1993).

  12. 12.

    , , & Identification of a Legionella pneumophila locus required for intracellular multiplication in human macrophages. Proc. Natl Acad. Sci. USA 89, 9607–9611 (1992).

  13. 13.

    , & Host cell killing and bacterial conjugation require overlapping sets of genes within a 22-kb region of the Legionella pneumophila genome. Proc. Natl Acad. Sci. USA 95, 1669–1674 (1998).

  14. 14.

    , , & Conjugative transfer by the virulence system of Legionella pneumophila. Science 279, 873–876 (1998).

  15. 15.

    , & Type IV secretion systems: tools of bacterial horizontal gene transfer and virulence. Cell. Microbiol. 10, 2377–2386 (2008).

  16. 16.

    & Bacterial type IV secretion: conjugation systems adapted to deliver effector molecules to host cells. Trends Microbiol. 8, 354–360 (2000).

  17. 17.

    et al. Identification of the core transmembrane complex of the Legionella Dot/Icm type IV secretion system. Mol. Microbiol. 62, 1278–1291 (2006).

  18. 18.

    et al. The DotL protein, a member of the TraG-coupling protein family, is essential for viability of Legionella pneumophila strain Lp02. J. Bacteriol. 187, 2927–2938 (2005).

  19. 19.

    , , & Conjugative plasmid protein TrwB, an integral membrane type IV secretion system coupling protein—detailed structural features and mapping of the active site cleft. J. Biol. Chem. 277, 7556–7566 (2002).

  20. 20.

    , , & The Legionella IcmSW complex directly interacts with DotL to mediate translocation of adaptor-dependent substrates. PLoS Pathog. 8, e1002910 (2012).

  21. 21.

    & The Legionella pneumophila IcmSW complex interacts with multiple Dot/Icm effectors to facilitate type IV translocation. PLoS Pathog. 3, e188 (2007).

  22. 22.

    & Dali server: conservation mapping in 3D. Nucleic Acids Res. 38, W545–W549 (2010).

  23. 23.

    , , & The MPI bioinformatics Toolkit as an integrative platform for advanced protein sequence and structure analysis. Nucleic Acids Res. 44, W410–W415 (2016).

  24. 24.

    , , & lvgA, a novel Legionella pneumophila virulence factor. Infect. Immun. 71, 2394–2403 (2003).

  25. 25.

    & The Legionella pneumophila IcmS–LvgA protein complex is important for Dot/Icm-dependent intracellular growth. Mol. Microbiol. 61, 596–613 (2006).

  26. 26.

    , & Modeling of loops in protein structures. Protein Sci. 9, 1753–1773 (2000).

  27. 27.

    & Comparative protein structure modeling using MODELLER. Curr. Protoc. Bioinformatics 47, 5.6.1–5.6.37 (2014).

  28. 28.

    , & IcmS-dependent translocation of SdeA into macrophages by the Legionella pneumophila type IV secretion system. Mol. Microbiol. 56, 90–103 (2005).

  29. 29.

    et al. Identification of Icm protein complexes that play distinct roles in the biogenesis of an organelle permissive for Legionella pneumophila intracellular growth. Mol. Microbiol. 38, 719–736 (2000).

  30. 30.

    , , , & Molecular characterization of the Dot/Icm-translocated AnkH and AnkJ eukaryotic-like effectors of Legionella pneumophila. Infect. Immun. 78, 1123–1134 (2010).

  31. 31.

    , , & The Legionella IcmS–IcmW protein complex is important for Dot/Icm-mediated protein translocation. Mol. Microbiol. 55, 912–926 (2005).

  32. 32.

    et al. Probing cellular protein complexes using single-molecule pull-down. Nature 473, 484–488 (2011).

  33. 33.

    , & A Legionella pneumophila effector protein encoded in a region of genomic plasticity binds to Dot/Icm-modified vacuoles. PLoS Pathog. 5, e1000278 (2009).

  34. 34.

    et al. The bacterial conjugation protein TrwB resembles ring helicases and F1-ATPase. Nature 409, 637–641 (2001).

  35. 35.

    et al. Structural basis of specific TraD–TraM recognition during F plasmid-mediated bacterial conjugation. Mol. Microbiol. 70, 89–99 (2008).

  36. 36.

    , & Vire2, a type IV secretion substrate, interacts with the VirD4 transfer protein at cell poles of Agrobacterium tumefaciens. Mol. Microbiol. 49, 1699–1713 (2003).

  37. 37.

    et al. Chimeric coupling proteins mediate transfer of heterologous type IV effectors through the Escherichia coli pKM101-encoded conjugation machine. J. Bacteriol. 198, 2701–2718 (2016).

  38. 38.

    et al. The all-alpha domains of coupling proteins from the Agrobacterium tumefaciens VirB/VirD4 and Enterococcus faecalis pCF10-encoded type IV secretion systems confer specificity to binding of cognate DNA substrates. J. Bacteriol. 197, 2335–2349 (2015).

  39. 39.

    , & Bacterial type IV secretion systems: versatile virulence machines. Future Microbiol. 7, 241–257 (2012).

  40. 40.

    & From flagellum assembly to virulence: the extended family of type III export chaperones. Trends Microbiol. 8, 202–204 (2000).

  41. 41.

    , & The various and varying roles of specific chaperones in type III secretion systems. Curr. Opin. Microbiol. 6, 7–14 (2003).

  42. 42.

    & Maintenance of an unfolded polypeptide by a cognate chaperone in bacterial type III secretion. Nature 414, 77–81 (2001).

  43. 43.

    et al. A C-terminal translocation signal required for Dot/lcm-dependent delivery of the Legionella RalF protein to host cells. Proc. Natl Acad. Sci. USA 102, 826–831 (2005).

  44. 44.

    et al. The E block motif is associated with Legionella pneumophila translocated substrates. Cell. Microbiol. 13, 227–245 (2011).

  45. 45.

    et al. Computational modeling and experimental validation of the Legionella and Coxiella virulence-related type-IVB secretion signal. Proc. Natl Acad. Sci. USA 110, E707–E715 (2013).

  46. 46.

    , & Genetic analysis of the Legionella pneumophila DotB ATPase reveals a role in type IV secretion system protein export. Mol. Microbiol. 57, 70–84 (2005).

  47. 47.

    , , , & Electrostatics of nanosystems: application to microtubules and the ribosome. Proc. Natl Acad. Sci. USA 98, 10037–10041 (2001).

  48. 48.

    et al. Mechanistic and structural insights into the proteolytic activation of Vibrio cholerae MARTX toxin. Nat. Chem. Biol. 5, 469–478 (2009).

  49. 49.

    et al. PHENIX: a comprehensive python-based system for macromolecular structure solution. Acta Crystallogr. D 66, 213–221 (2010).

  50. 50.

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

  51. 51.

    & Processing of X-ray diffraction data collected in oscillation mode. Method Enzymol. 276, 307–326 (1997).

  52. 52.

    , , & Features and development of Coot. Acta Crystallogr. D 66, 486–501 (2010).

  53. 53.

    et al. Crystallography & NMR system: a new software suite for macromolecular structure determination. Acta Crystallogr. D 54, 905–921 (1998).

  54. 54.

    , & Determination of domain structure of proteins from X-ray solution scattering. Biophys. J. 80, 2946–2953 (2001).

  55. 55.

    , & CRYSOL—a program to evaluate X-ray solution scattering of biological macromolecules from atomic coordinates. J. Appl. Crystallogr. 28, 768–773 (1995).

  56. 56.

    & GNOM—a program package for small-angle scattering data-processing. J. Appl. Crystallogr. 24, 537–540 (1991).

  57. 57.

    et al. Accurate FRET measurements within single diffusing biomolecules using alternating-laser excitation. Biophys. J. 88, 2939–2953 (2005).

  58. 58.

    , , , & Dynamic release of bending stress in short dsDNA by formation of a kink and forks. Angew. Chem. Int. Ed. 54, 8943–8947 (2015).

  59. 59.

    , & A practical guide to single-molecule FRET. Nat. Methods 5, 507–516 (2008).

  60. 60.

    , , & Single-molecule pull-down for studying protein interactions. Nat. Protoc. 7, 445–452 (2012).

  61. 61.

    et al. Template based protein structure modeling by global optimization in CASP11. Proteins 84(Suppl. 1), 221–232 (2016).

  62. 62.

    , , , & Multiple sequence alignment by conformational space annealing. Biophys. J. 95, 4813–4819 (2008).

  63. 63.

    , & New optimization method for conformational energy calculations on polypeptides: conformational space annealing. J. Comput. Chem. 18, 1222–1232 (1997).

  64. 64.

    , & Unbiased global optimization of Lennard–Jones clusters for N < or = 201 using the conformational space annealing method. Phys. Rev. Lett. 91, 080201 (2003).

  65. 65.

    et al. All-atom chain-building by optimizing MODELLER energy function using conformational space annealing. Proteins 75, 1010–1023 (2009).

  66. 66.

    , & Improved prediction of protein side-chain conformations with SCWRL4. Proteins 77, 778–795 (2009).

Download references

Acknowledgements

This study made use of Beamlines 4C and 5C at Pohang Accelerator Laboratory, Korea, and was supported by the National Research Foundation of Korea (NRF) (grant no. 2008-00576) and KAIST Future Systems Healthcare Project, Ministry of Science, ICT and Future Planning. J.D.K. was supported by a TJ PARK postdoctoral fellowship from POSCO TJ PARK Foundation. The authors thank the Korea Institute for Advanced Study for providing computing resources (KIAS Center for Advanced Computation Linux Cluster).

Author information

Author notes

    • Mi-Jeong Kwak
    •  & J. Dongun Kim

    These authors contributed equally to this work.

Affiliations

  1. Department of Biological Sciences, KAIST Institute for the Biocentury, Korea Advanced Institute of Science and Technology, Daejeon 34141, Korea

    • Mi-Jeong Kwak
    • , J. Dongun Kim
    • , Hyunmin Kim
    • , Seonghoon Kim
    •  & Byung-Ha Oh
  2. Department of Physics, Pohang University of Science and Technology, Pohang, Kyungbuk 37673, Korea

    • Cheolhee Kim
    •  & Nam Ki Lee
  3. Department of Molecular Microbiology and Immunology, Keck School of Medicine, University of Southern California, 1975 Zonal Avenue, Los Angeles, California 90033, USA

    • James W. Bowman
    •  & Jae U. Jung
  4. Center for Advanced Computation, School of Computational Sciences, Korea Institute for Advanced Study, Seoul 02455, Korea

    • Keehyoung Joo
    •  & Jooyoung Lee
  5. Pohang Accelerator Laboratory, Pohang University of Science and Technology, Pohang, Kyungbuk 37673, Korea

    • Kyeong Sik Jin
    •  & Yeon-Gil Kim

Authors

  1. Search for Mi-Jeong Kwak in:

  2. Search for J. Dongun Kim in:

  3. Search for Hyunmin Kim in:

  4. Search for Cheolhee Kim in:

  5. Search for James W. Bowman in:

  6. Search for Seonghoon Kim in:

  7. Search for Keehyoung Joo in:

  8. Search for Jooyoung Lee in:

  9. Search for Kyeong Sik Jin in:

  10. Search for Yeon-Gil Kim in:

  11. Search for Nam Ki Lee in:

  12. Search for Jae U. Jung in:

  13. Search for Byung-Ha Oh in:

Contributions

M.-J.K., J.D.K., H.K., S.K. and Y.-G.K. performed X-ray crystallography and biochemical experiments. C.K. conducted the ALEX-FRET experiment, J.W.B. the SiMPull, K.J. and J.L. homology modelling, and K.S.J. the SAXS. B.-H.O., M.-J.K., J.D.K., N.K.L. and J.U.J. conceived the experiments and wrote the manuscript. All authors discussed the results.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Byung-Ha Oh.

Supplementary information

PDF files

  1. 1.

    Supplementary Information

    Supplementary Figures 1–7, Supplementary Table 1, Supplementary References.

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nmicrobiol.2017.114

Further reading