Stepwise visualization of membrane pore formation by suilysin, a bacterial cholesterol-dependent cytolysin

Abstract

Membrane attack complex/perforin/cholesterol-dependent cytolysin (MACPF/CDC) proteins constitute a major superfamily of pore-forming proteins that act as bacterial virulence factors and effectors in immune defence. Upon binding to the membrane, they convert from the soluble monomeric form to oligomeric, membrane-inserted pores. Using real-time atomic force microscopy (AFM), electron microscopy (EM) and atomic structure fitting, we have mapped the structure and assembly pathways of a bacterial CDC in unprecedented detail and accuracy, focussing on suilysin from Streptococcus suis. We show that suilysin assembly is a noncooperative process that is terminated before the protein inserts into the membrane. The resulting ring-shaped pores and kinetically trapped arc-shaped assemblies are all seen to perforate the membrane, as also visible by the ejection of its lipids. Membrane insertion requires a concerted conformational change of the monomeric subunits, with a marked expansion in pore diameter due to large changes in subunit structure and packing.

Article and author information

Author details

  1. Carl Leung

    London Centre for Nanotechnology, University College London, London, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  2. Natalya V Dudkina

    Department of Crystallography, Birkbeck College, London, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  3. Natalya Lukoyanova

    Department of Crystallography, Birkbeck College, London, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  4. Adrian W Hodel

    London Centre for Nanotechnology, University College London, London, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  5. Irene Farabella

    Department of Crystallography, Birkbeck College, London, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  6. Arun P Pandurangan

    Department of Crystallography, Birkbeck College, London, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  7. Nasrin Jahan

    Department of Infection, Immunity, and Inflammation, University of Leicester, Leicester, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  8. Mafalda Pires Damaso

    Department of Infection, Immunity, and Inflammation, University of Leicester, Leicester, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  9. Dino Osmanović

    London Centre for Nanotechnology, University College London, London, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  10. Cyril F Reboul

    Department of Biochemistry and Molecular Biology, Monash University, Melbourne, Australia
    Competing interests
    The authors declare that no competing interests exist.
  11. Michelle A Dunstone

    Department of Biochemistry and Molecular Biology, Monash University, Melbourne, Australia
    Competing interests
    The authors declare that no competing interests exist.
  12. Peter W Andrew

    Department of Infection, Immunity, and Inflammation, University of Leicester, Leicester, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  13. Rana Lonnen

    Department of Infection, Immunity, and Inflammation, University of Leicester, Leicester, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  14. Maya Topf

    Department of Crystallography, Birkbeck College, London, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  15. Helen R Saibil

    Department of Crystallography, Birkbeck College, London, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  16. Bart W Hoogenboom

    London Centre for Nanotechnology, University College London, London, United Kingdom
    For correspondence
    b.hoogenboom@ucl.ac.uk
    Competing interests
    The authors declare that no competing interests exist.

Reviewing Editor

  1. Volker Dötsch, Goethe University, Germany

Version history

  1. Received: August 5, 2014
  2. Accepted: November 24, 2014
  3. Accepted Manuscript published: December 2, 2014 (version 1)
  4. Version of Record published: December 24, 2014 (version 2)

Copyright

© 2014, Leung et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 5,928
    Page views
  • 858
    Downloads
  • 129
    Citations

Article citation count generated by polling the highest count across the following sources: Scopus, Crossref, PubMed Central.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Carl Leung
  2. Natalya V Dudkina
  3. Natalya Lukoyanova
  4. Adrian W Hodel
  5. Irene Farabella
  6. Arun P Pandurangan
  7. Nasrin Jahan
  8. Mafalda Pires Damaso
  9. Dino Osmanović
  10. Cyril F Reboul
  11. Michelle A Dunstone
  12. Peter W Andrew
  13. Rana Lonnen
  14. Maya Topf
  15. Helen R Saibil
  16. Bart W Hoogenboom
(2014)
Stepwise visualization of membrane pore formation by suilysin, a bacterial cholesterol-dependent cytolysin
eLife 3:e04247.
https://doi.org/10.7554/eLife.04247

Share this article

https://doi.org/10.7554/eLife.04247

Further reading

    1. Biochemistry and Chemical Biology
    2. Structural Biology and Molecular Biophysics
    Karolina Honzejkova, Dalibor Kosek ... Tomas Obsil
    Research Article

    Apoptosis signal-regulating kinase 1 (ASK1) is a crucial stress sensor, directing cells toward apoptosis, differentiation, and senescence via the p38 and JNK signaling pathways. ASK1 dysregulation has been associated with cancer and inflammatory, cardiovascular, and neurodegenerative diseases, among others. However, our limited knowledge of the underlying structural mechanism of ASK1 regulation hampers our ability to target this member of the MAP3K protein family towards developing therapeutic interventions for these disorders. Nevertheless, as a multidomain Ser/Thr protein kinase, ASK1 is regulated by a complex mechanism involving dimerization and interactions with several other proteins, including thioredoxin 1 (TRX1). Thus, the present study aims at structurally characterizing ASK1 and its complex with TRX1 using several biophysical techniques. As shown by cryo-EM analysis, in a state close to its active form, ASK1 is a compact and asymmetric dimer, which enables extensive interdomain and interchain interactions. These interactions stabilize the active conformation of the ASK1 kinase domain. In turn, TRX1 functions as a negative allosteric effector of ASK1, modifying the structure of the TRX1-binding domain and changing its interaction with the tetratricopeptide repeats domain. Consequently, TRX1 reduces access to the activation segment of the kinase domain. Overall, our findings not only clarify the role of ASK1 dimerization and inter-domain contacts but also provide key mechanistic insights into its regulation, thereby highlighting the potential of ASK1 protein-protein interactions as targets for anti-inflammatory therapy.