An extensively glycosylated archaeal pilus survives extreme conditions

Abstract

Pili on the surface of Sulfolobus islandicus are used for many functions, and serve as receptors for certain archaeal viruses. The cells grow optimally at pH 3 and ~80 °C, exposing these extracellular appendages to a very harsh environment. The pili, when removed from cells, resist digestion by trypsin or pepsin, and survive boiling in sodium dodecyl sulfate or 5 M guanidine hydrochloride. We used electron cryo-microscopy to determine the structure of these filaments at 4.1 Å resolution. An atomic model was built by combining the electron density map with bioinformatics without previous knowledge of the pilin sequence—an approach that should prove useful for assemblies where all of the components are not known. The atomic structure of the pilus was unusual, with almost one-third of the residues being either threonine or serine, and with many hydrophobic surface residues. While the map showed extra density consistent with glycosylation for only three residues, mass measurements suggested extensive glycosylation. We propose that this extensive glycosylation renders these filaments soluble and provides the remarkable structural stability. We also show that the overall fold of the archaeal pilin is remarkably similar to that of archaeal flagellin, establishing common evolutionary origins.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: Cryo-EM of the LAL14/1 pilus.
Fig. 2: De novo atomic model building of LAL14/1 pilin.
Fig. 3: The LAL14/1 pilus contains an unusually high percentage of hydrophobic residues.
Fig. 4: O-linked sugar modifications of the LAL14/1 pilus.
Fig. 5: Comparison of the LAL14/1 pilus with bacterial T4P and the archaeal flagellar filament.

Data availability

The three-dimensional reconstruction has been deposited in the Electron Microscopy Data Bank with accession code EMD-0397. The atomic model has been deposited in the Protein Data Bank with accession code 6NAV. The mass spectrometry data have been deposited in PRIDE with accession code PXD012799.

References

  1. 1.

    Prangishvili, D. et al. The enigmatic archaeal virosphere. Nat. Rev. Microbiol. 15, 724–739 (2017).

    CAS  Article  Google Scholar 

  2. 2.

    Chaudhury, P., Quax, T. E. F. & Albers, S. V. Versatile cell surface structures of archaea. Mol. Microbiol. 107, 298–311 (2018).

    CAS  Article  Google Scholar 

  3. 3.

    DiMaio, F. et al. A virus that infects a hyperthermophile encapsidates A-form DNA. Science 348, 914–917 (2015).

    CAS  Article  Google Scholar 

  4. 4.

    Liu, Y. et al. Structural conservation in a membrane-enveloped filamentous virus infecting a hyperthermophilic acidophile. Nat. Commun. 9, 3360 (2018).

    Article  Google Scholar 

  5. 5.

    Ptchelkine, D. et al. Unique architecture of thermophilic archaeal virus APBV1 and its genome packaging. Nat. Commun. 8, 1436 (2017).

    Article  Google Scholar 

  6. 6.

    Kasson, P. et al. Model for a novel membrane envelope in a filamentous hyperthermophilic virus. eLife 6, e26268 (2017).

    Article  Google Scholar 

  7. 7.

    Veesler, D. et al. Atomic structure of the 75 MDa extremophile Sulfolobus turreted icosahedral virus determined by CryoEM and X-ray crystallography. Proc. Natl Acad. Sci. USA 110, 5504–5509 (2013).

    CAS  Article  Google Scholar 

  8. 8.

    Berry, J. L. & Pelicic, V. Exceptionally widespread nanomachines composed of type IV pilins: the prokaryotic Swiss Army knives. FEMS Microbiol. Rev. 39, 134–154 (2015).

    CAS  Article  Google Scholar 

  9. 9.

    Makarova, K. S., Koonin, E. V. & Albers, S. V. Diversity and evolution of type IV pili systems in archaea. Front. Microbiol. 7, 667 (2016).

    Article  Google Scholar 

  10. 10.

    Pohlschroder, M., Ghosh, A., Tripepi, M. & Albers, S. V. Archaeal type IV pilus-like structures—evolutionarily conserved prokaryotic surface organelles. Curr. Opin. Microbiol. 14, 357–363 (2011).

    CAS  Article  Google Scholar 

  11. 11.

    Pohlschroder, M. & Esquivel, R. N. Archaeal type IV pili and their involvement in biofilm formation. Front. Microbiol. 6, 190 (2015).

    Article  Google Scholar 

  12. 12.

    Albers, S. V. & Jarrell, K. F. The archaellum: an update on the unique archaeal motility structure. Trends Microbiol. 26, 351–362 (2018).

    CAS  Article  Google Scholar 

  13. 13.

    Wang, F. et al. Cryoelectron microscopy reconstructions of the Pseudomonas aeruginosa and Neisseria gonorrhoeae type IV pili at sub-nanometer resolution. Structure 25, 1423–1435 (2017).

    CAS  Article  Google Scholar 

  14. 14.

    Kolappan, S. et al. Structure of the Neisseria meningitidis type IV pilus. Nat. Commun. 7, 13015 (2016).

    CAS  Article  Google Scholar 

  15. 15.

    Lopez-Castilla, A. et al. Structure of the calcium-dependent type 2 secretion pseudopilus. Nat. Microbiol. 2, 1686–1695 (2017).

    CAS  Article  Google Scholar 

  16. 16.

    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).

    CAS  Article  Google Scholar 

  17. 17.

    Craig, L. et al. Type IV pilin structure and assembly: X-ray and EM analyses of Vibrio cholerae toxin-coregulated pilus and Pseudomonas aeruginosa PAK pilin. Mol. Cell 11, 1139–1150 (2003).

    CAS  Article  Google Scholar 

  18. 18.

    Hartung, S. et al. Ultrahigh resolution and full-length pilin structures with insights for filament assembly, pathogenic functions, and vaccine potential. J. Biol. Chem. 286, 44254–44265 (2011).

    CAS  Article  Google Scholar 

  19. 19.

    Reardon, P. N. & Mueller, K. T. Structure of the type IVa major pilin from the electrically conductive bacterial nanowires of Geobacter sulfurreducens. J. Biol. Chem. 288, 29260–29266 (2013).

    CAS  Article  Google Scholar 

  20. 20.

    Szabo, Z. et al. Identification of diverse archaeal proteins with class III signal peptides cleaved by distinct archaeal prepilin peptidases. J. Bacteriol. 189, 772–778 (2007).

    CAS  Article  Google Scholar 

  21. 21.

    Albers, S. V., Szabo, Z. & Driessen, A. J. Archaeal homolog of bacterial type IV prepilin signal peptidases with broad substrate specificity. J. Bacteriol. 185, 3918–3925 (2003).

    CAS  Article  Google Scholar 

  22. 22.

    Strom, M. S., Nunn, D. N. & Lory, S. A single bifunctional enzyme, PilD, catalyzes cleavage and N-methylation of proteins belonging to the type IV pilin family. Proc. Natl Acad. Sci. USA 90, 2404–2408 (1993).

    CAS  Article  Google Scholar 

  23. 23.

    Jaubert, C. et al. Genomics and genetics of Sulfolobus islandicus LAL14/1, a model hyperthermophilic archaeon. Open Biol. 3, 130010 (2013).

    Article  Google Scholar 

  24. 24.

    Quemin, E. R. et al. First insights into the entry process of hyperthermophilic archaeal viruses. J. Virol. 87, 13379–13385 (2013).

    CAS  Article  Google Scholar 

  25. 25.

    DiMaio, F. et al. Atomic-accuracy models from 4.5-A cryo-electron microscopy data with density-guided iterative local refinement. Nat. Methods 12, 361–365 (2015).

    CAS  Article  Google Scholar 

  26. 26.

    Braun, T. et al. Archaeal flagellin combines a bacterial type IV pilin domain with an Ig-like domain. Proc. Natl Acad. Sci. USA 113, 10352–10357 (2016).

    CAS  Article  Google Scholar 

  27. 27.

    Yu, X. et al. Filaments from Ignicoccus hospitalis show diversity of packing in proteins containing N-terminal type IV pilin helices. J. Mol. Biol. 422, 274–281 (2012).

    CAS  Article  Google Scholar 

  28. 28.

    Poweleit, N. et al. CryoEM structure of the Methanospirillum hungatei archaellum reveals structural features distinct from the bacterial flagellum and type IV pili. Nat. Microbiol. 2, 16222 (2016).

    CAS  Article  Google Scholar 

  29. 29.

    Daum, B. et al. Structure and in situ organisation of the Pyrococcus furiosus archaellum machinery. eLife 6, e27470 (2017).

    Article  Google Scholar 

  30. 30.

    Bayley, D. P. & Jarrell, K. F. Further evidence to suggest that archaeal flagella are related to bacterial type IV pili. J. Mol. Evol. 46, 370–373 (1998).

    CAS  PubMed  Google Scholar 

  31. 31.

    Ng, S. Y., Chaban, B. & Jarrell, K. F. Archaeal flagella, bacterial flagella and type IV pili: a comparison of genes and posttranslational modifications. J. Mol. Microbiol. Biotechnol. 11, 167–191 (2006).

    CAS  Article  Google Scholar 

  32. 32.

    Faguy, D. M., Jarrell, K. F., Kuzio, J. & Kalmokoff, M. L. Molecular analysis of archael flagellins: similarity to the type IV pilin-transport superfamily widespread in bacteria. Can. J. Microbiol. 40, 67–71 (1994).

    CAS  Article  Google Scholar 

  33. 33.

    Song, Y. et al. High-resolution comparative modeling with RosettaCM. Structure 21, 1735–1742 (2013).

    CAS  Article  Google Scholar 

  34. 34.

    Henche, A. L. et al. Structure and function of the adhesive type IV pilus of Sulfolobus acidocaldarius. Environ. Microbiol. 14, 3188–3202 (2012).

    CAS  Article  Google Scholar 

  35. 35.

    Kryndushkin, D., Pripuzova, N., Burnett, B. & Shewmaker, F. Non-targeted identification of prions and amyloid-forming proteins from yeast and mammalian cells. J. Biol. Chem. 288, 27100–27111 (2013).

    CAS  Article  Google Scholar 

  36. 36.

    Nelson, R. et al. Structure of the cross-β spine of amyloid-like fibrils. Nature 435, 773–778 (2005).

    CAS  Article  Google Scholar 

  37. 37.

    Gromiha, M. M. & Selvaraj, S. Comparison between long-range interactions and contact order in determining the folding rate of two-state proteins: application of long-range order to folding rate prediction. J. Mol. Biol. 310, 27–32 (2001).

    CAS  Article  Google Scholar 

  38. 38.

    Broom, A. et al. Designed protein reveals structural determinants of extreme kinetic stability. Proc. Natl Acad. Sci. USA 112, 14605–14610 (2015).

    CAS  Article  Google Scholar 

  39. 39.

    Stranges, P. B. & Kuhlman, B. A comparison of successful and failed protein interface designs highlights the challenges of designing buried hydrogen bonds. Protein Sci. 22, 74–82 (2013).

    CAS  Article  Google Scholar 

  40. 40.

    Pohlschroder, M., Pfeiffer, F., Schulze, S. & Halim, M. F. A. Archaeal cell surface biogenesis. FEMS Microbiol. Rev. 42, 694–717 (2018).

    CAS  Article  Google Scholar 

  41. 41.

    Mann, M. & Jensen, O. N. Proteomic analysis of post-translational modifications. Nat. Biotechnol. 21, 255–261 (2003).

    CAS  Article  Google Scholar 

  42. 42.

    Wall, J. S. & Hainfeld, J. F. Mass mapping with the scanning transmission electron microscope. Annu. Rev. Biophys. Biophys. Chem. 15, 355–376 (1986).

    CAS  Article  Google Scholar 

  43. 43.

    Sojar, H. T. & Bahl, O. P. Chemical deglycosylation of glycoproteins. Methods Enzymol. 138, 341–350 (1987).

    CAS  Article  Google Scholar 

  44. 44.

    Ellen, A. F. et al. Proteomic analysis of secreted membrane vesicles of archaeal Sulfolobus species reveals the presence of endosome sorting complex components. Extremophiles 13, 67–79 (2009).

    CAS  Article  Google Scholar 

  45. 45.

    Altschul, S. F. & Lipman, D. J. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389–3402 (1997).

    CAS  Article  Google Scholar 

  46. 46.

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

    CAS  Article  Google Scholar 

  47. 47.

    Esquivel, R. N., Schulze, S., Xu, R., Hippler, M. & Pohlschroder, M. Identification of Haloferax volcanii pilin N-glycans with diverse roles in pilus biosynthesis, adhesion, and microcolony formation. J. Biol. Chem. 291, 10602–10614 (2016).

    CAS  Article  Google Scholar 

  48. 48.

    Gault, J. et al. Neisseria meningitidis type IV pili composed of sequence invariable pilins are masked by multisite glycosylation. PLoS Pathog. 11, e1005162 (2015).

    Article  Google Scholar 

  49. 49.

    Mer, G., Hietter, H. & Lefevre, J. F. Stabilization of proteins by glycosylation examined by NMR analysis of a fucosylated proteinase inhibitor. Nat. Struct. Biol. 3, 45–53 (1996).

    CAS  Article  Google Scholar 

  50. 50.

    Shental-Bechor, D. & Levy, Y. Effect of glycosylation on protein folding: a close look at thermodynamic stabilization. Proc. Natl Acad. Sci. USA 105, 8256–8261 (2008).

    CAS  Article  Google Scholar 

  51. 51.

    Sola, R. J. & Griebenow, K. Effects of glycosylation on the stability of protein pharmaceuticals. J. Pharm. Sci. 98, 1223–1245 (2009).

    CAS  Article  Google Scholar 

  52. 52.

    Zillig, W. et al. Screening for Sulfolobales, their plasmids and their viruses in Icelandic solfataras. Syst. Appl. Microbiol. 16, 609–628 (1993).

    Article  Google Scholar 

  53. 53.

    Li, X. et al. Electron counting and beam-induced motion correction enable near-atomic-resolution single-particle cryo-EM. Nat. Methods 10, 584–590 (2013).

    CAS  Article  Google Scholar 

  54. 54.

    Mindell, J. A. & Grigorieff, N. Accurate determination of local defocus and specimen tilt in electron microscopy. J. Struct. Biol. 142, 334–347 (2003).

    Article  Google Scholar 

  55. 55.

    Tang, G. et al. EMAN2: an extensible image processing suite for electron microscopy. J. Struct. Biol. 157, 38–46 (2007).

    CAS  Article  Google Scholar 

  56. 56.

    Frank, J. et al. SPIDER and WEB: processing and visualization of images in 3D electron microscopy and related fields. J. Struct. Biol. 116, 190–199 (1996).

    CAS  Article  Google Scholar 

  57. 57.

    Scheres, S. H. RELION: implementation of a Bayesian approach to cryo-EM structure determination. J. Struct. Biol. 180, 519–530 (2012).

    CAS  Article  Google Scholar 

  58. 58.

    Afonine, P. V. et al. New tools for the analysis and validation of cryo-EM maps and atomic models. Acta Crystallogr. D Struct. Biol. 74, 814–840 (2018).

    CAS  Article  Google Scholar 

  59. 59.

    Pettersen, E. F. et al. UCSF Chimera—a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).

    CAS  Article  Google Scholar 

  60. 60.

    Krogh, A., Larsson, B., von Heijne, G. & Sonnhammer, E. L. Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. J. Mol. Biol. 305, 567–580 (2001).

    CAS  Article  Google Scholar 

  61. 61.

    Drozdetskiy, A., Cole, C., Procter, J. & Barton, G. J. JPred4: a protein secondary structure prediction server. Nucleic Acids Res. 43, W389–W394 (2015).

    CAS  Article  Google Scholar 

  62. 62.

    Wang, R. Y. et al. De novo protein structure determination from near-atomic-resolution cryo-EM maps. Nat. Methods 12, 335–338 (2015).

    CAS  Article  Google Scholar 

  63. 63.

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

    CAS  Article  Google Scholar 

  64. 64.

    Williams, C. J. et al. MolProbity: more and better reference data for improved all-atom structure validation. Protein Sci. 27, 293–315 (2018).

    CAS  Article  Google Scholar 

  65. 65.

    Pronk, S. et al. GROMACS 4.5: a high-throughput and highly parallel open source molecular simulation toolkit. Bioinformatics 29, 845–854 (2013).

    CAS  Article  Google Scholar 

  66. 66.

    Wall, J. S. & Simon, M. N. Scanning transmission electron microscopy of DNA–protein complexes. Methods Mol. Biol. 148, 589–601 (2001).

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This work was supported by NIH grants GM122510 (to E.H.E.) and GM123089 (to F.D.), as well as l’Agence Nationale de la Recherche project ENVIRA (ANR-17-CE15-0005-01 to M.K.). M.A.B.K. was supported by NIH grant T32 GM080186. The cryo-EM imaging conducted at the Molecular Electron Microscopy Core facility at the University of Virginia was supported by the School of Medicine and built with NIH grant G20-RR31199. The Titan Krios and Falcon II direct electron detectors were obtained with NIH grants S10-RR025067 and S10-OD018149, respectively. We thank V. Conticello for the suggestion of TFMS. We are also grateful to the Ultrastructural BioImaging (UTechS UBI) unit of Institut Pasteur for access to electron microscopes.

Author information

Affiliations

Authors

Contributions

V.C.-K. isolated and purified the pili. J.S.W. performed the STEM analysis. N.S. performed the mass spectrometry. G.A.P.d.O. performed the amyloid assays. F.D. performed the interfacial analysis. F.W. performed the cryo-EM, three-dimensional reconstruction and model building, with assistance from T.O. and E.H.E. M.K. and Z.S. performed the bioinformatics analysis. M.A.B.K. performed the TFMS deglycosylation. D.P., M.K. and E.H.E. designed the project. F.W., D.P., M.K. and E.H.E. wrote the paper.

Corresponding authors

Correspondence to Mart Krupovic or Edward H. Egelman.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary Table 1 and Supplementary Figures 1–10.

Reporting Summary

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Wang, F., Cvirkaite-Krupovic, V., Kreutzberger, M.A.B. et al. An extensively glycosylated archaeal pilus survives extreme conditions. Nat Microbiol 4, 1401–1410 (2019). https://doi.org/10.1038/s41564-019-0458-x

Download citation

Further reading

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing