Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

Botulinum neurotoxin B recognizes its protein receptor with high affinity and specificity

Abstract

Botulinum neurotoxins (BoNTs) are produced by Clostridium botulinum and cause the neuroparalytic syndrome of botulism. With a lethal dose of 1 ng kg-1, they pose a biological hazard to humans and a serious potential bioweapon threat1. BoNTs bind with high specificity at neuromuscular junctions and they impair exocytosis of synaptic vesicles containing acetylcholine through specific proteolysis of SNAREs (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors), which constitute part of the synaptic vesicle fusion machinery2,3. The molecular details of the toxin–cell recognition have been elusive. Here we report the structure of a BoNT in complex with its protein receptor: the receptor-binding domain of botulinum neurotoxin serotype B (BoNT/B) bound to the luminal domain of synaptotagmin II, determined at 2.15 Å resolution. On binding, a helix is induced in the luminal domain which binds to a saddle-shaped crevice on a distal tip of BoNT/B. This crevice is adjacent to the non-overlapping ganglioside-binding site of BoNT/B. Synaptotagmin II interacts with BoNT/B with nanomolar affinity, at both neutral and acidic endosomal pH. Biochemical and neuronal ex vivo studies of structure-based mutations indicate high specificity and affinity of the interaction, and high selectivity of BoNT/B among synaptotagmin I and II isoforms. Synergistic binding of both synaptotagmin and ganglioside imposes geometric restrictions on the initiation of BoNT/B translocation after endocytosis. Our results provide the basis for the rational development of preventive vaccines or inhibitors against these neurotoxins.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Structure of the H C B–Syt-II complex.
Figure 2: The H C B–Syt-II complex is stabilized by extensive intermolecular interactions involving two pronounced pockets on the H C B surface.
Figure 3: Site-directed mutagenesis analysis of the toxin-receptor binding site in BoNT/B and in the luminal domains of Syt-I and Syt-II.
Figure 4: Simultaneous binding with membrane-anchored Syt-II and ganglioside imposes geometric restrictions on how BoNT/B binds to the membrane surface.

Similar content being viewed by others

References

  1. Arnon, S. S. et al. Botulinum toxin as a biological weapon: medical and public health management. J. Am. Med. Assoc. 285, 1059–1070 (2001)

    Article  CAS  Google Scholar 

  2. Schiavo, G. et al. Tetanus and botulinum-B neurotoxins block neurotransmitter release by proteolytic cleavage of synaptobrevin. Nature 359, 832–835 (1992)

    Article  ADS  CAS  Google Scholar 

  3. Chen, Y. A., Scales, S. J., Patel, S. M., Doung, Y. C. & Scheller, R. H. SNARE complex formation is triggered by Ca2+ and drives membrane fusion. Cell 97, 165–174 (1999)

    Article  CAS  Google Scholar 

  4. Montecucco, C. & Schiavo, G. Structure and function of tetanus and botulinum neurotoxins. Q. Rev. Biophys. 28, 423–472 (1995)

    Article  CAS  Google Scholar 

  5. Montecucco, C., Rossetto, O. & Schiavo, G. Presynaptic receptor arrays for clostridial neurotoxins. Trends Microbiol. 12, 442–446 (2004)

    Article  CAS  Google Scholar 

  6. Rummel, A., Karnath, T., Henke, T., Bigalke, H. & Binz, T. Synaptotagmins I and II act as nerve cell receptors for botulinum neurotoxin G. J. Biol. Chem. 279, 30865–30870 (2004)

    Article  CAS  Google Scholar 

  7. Nishiki, T. et al. The high-affinity binding of Clostridium botulinum type B neurotoxin to synaptotagmin II associated with gangliosides GT1b/GD1a. FEBS Lett. 378, 253–257 (1996)

    Article  CAS  Google Scholar 

  8. Dong, M. et al. SV2 is the protein receptor for botulinum neurotoxin A. Science 312, 592–596 (2006)

    Article  ADS  CAS  Google Scholar 

  9. Dong, M. et al. Synaptotagmins I and II mediate entry of botulinum neurotoxin B into cells. J. Cell Biol. 162, 1293–1303 (2003)

    Article  CAS  Google Scholar 

  10. Mahrhold, S., Rummel, A., Bigalke, H., Davletov, B. & Binz, T. The synaptic vesicle protein 2C mediates the uptake of botulinum neurotoxin A into phrenic nerves. FEBS Lett. 580, 2011–2014 (2006)

    Article  CAS  Google Scholar 

  11. Pang, Z. P. et al. Synaptotagmin-2 is essential for survival and contributes to Ca+2-triggering of neurotransmitter release in central and neuromuscular synapses. J. Neurosci. (in the press).

  12. Eswaramoorthy, S., Kumaran, D. & Swaminathan, S. Crystallographic evidence for doxorubicin binding to the receptor-binding site in Clostridium botulinum neurotoxin B. Acta Crystallogr. D Biol. Crystallogr. 57, 1743–1746 (2001)

    Article  CAS  Google Scholar 

  13. Swaminathan, S. & Eswaramoorthy, S. Structural analysis of the catalytic and binding sites of Clostridium botulinum neurotoxin B. Nature Struct. Biol. 7, 693–699 (2000)

    Article  CAS  Google Scholar 

  14. Rummel, A., Mahrhold, S., Bigalke, H. & Binz, T. The HCC-domain of botulinum neurotoxins A and B exhibits a singular ganglioside binding site displaying serotype specific carbohydrate interaction. Mol. Microbiol. 51, 631–643 (2004)

    Article  CAS  Google Scholar 

  15. Jayaraman, S., Eswaramoorthy, S., Ahmed, S. A., Smith, L. A. & Swaminathan, S. N-terminal helix reorients in recombinant C-fragment of Clostridium botulinum type B. Biochem. Biophys. Res. Commun. 330, 97–103 (2005)

    Article  CAS  Google Scholar 

  16. Perozzo, R., Folkers, G. & Scapozza, L. Thermodynamics of protein–ligand interactions: history, presence, and future aspects. J. Recept. Signal Transduct. Res. 24, 1–52 (2004)

    Article  CAS  Google Scholar 

  17. Stites, W. E. Protein–protein interactions: interface structure, binding thermodynamics, and mutational analysis. Chem. Rev. 97, 1233–1250 (1997)

    Article  CAS  Google Scholar 

  18. Habermann, E., Dreyer, F. & Bigalke, H. Tetanus toxin blocks the neuromuscular transmission in vitro like botulinum A toxin. Naunyn Schmiedebergs Arch. Pharmacol. 311, 33–40 (1980)

    Article  CAS  Google Scholar 

  19. Südhof, T. C. Synaptotagmins: why so many? J. Biol. Chem. 277, 7629–7632 (2002)

    Article  Google Scholar 

  20. Rummel, A. et al. Identification of the protein receptor binding site of botulinum neurotoxins B and G proves the double receptor concept. Proc. Natl Acad. Sci. USA (in the press).

  21. Montecucco, C. How do tetanus and botulinum neurotoxins bind to neuronal membranes? Trends Biochem. Sci. 11, 314–317 (1986)

    Article  CAS  Google Scholar 

  22. Kozaki, S., Kamata, Y., Watarai, S., Nishiki, T. & Mochida, S. Ganglioside GT1b as a complementary receptor component for Clostridium botulinum neurotoxins. Microb. Pathog. 25, 91–99 (1998)

    Article  CAS  Google Scholar 

  23. Rummel, A., Bade, S., Alves, J., Bigalke, H. & Binz, T. Two carbohydrate binding sites in the HCC-domain of tetanus neurotoxin are required for toxicity. J. Mol. Biol. 326, 835–847 (2003)

    Article  CAS  Google Scholar 

  24. Koriazova, L. K. & Montal, M. Translocation of botulinum neurotoxin light chain protease through the heavy chain channel. Nature Struct. Biol. 10, 13–18 (2003)

    Article  CAS  Google Scholar 

  25. Hoch, D. H. et al. Channels formed by botulinum, tetanus, and diphtheria toxins in planar lipid bilayers: relevance to translocation of proteins across membranes. Proc. Natl Acad. Sci. USA 82, 1692–1696 (1985)

    Article  ADS  CAS  Google Scholar 

  26. Dolinsky, T. J., Nielsen, J. E., McCammon, J. A. & Baker, N. A. PDB2PQR: an automated pipeline for the setup of Poisson–Boltzmann electrostatics calculations. Nucleic Acids Res. 32, W665–W667 (2004)

    Article  CAS  Google Scholar 

  27. Li, H., Robertson, A. D. & Jensen, J. H. Very fast empirical prediction and rationalization of protein pKa values. Proteins 61, 704–721 (2005)

    Article  CAS  Google Scholar 

  28. McCoy, A. J., Grosse-Kunstleve, R. W., Storoni, L. C. & Read, R. J. Likelihood-enhanced fast translation functions. Acta Crystallogr. D 61, 458–464 (2005)

    Article  Google Scholar 

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

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank P. Adams and J. Zhou for critical reading of the manuscript, and the staff of beamlines 9-1 at the Stanford Synchrotron Radiation Laboratory (SSRL) and beam line 8.2.2 at the Advanced Light Source (ALS) for help during data collection; H. Bigalke for providing the mouse phrenic nerve facility; and T. Henke and C. Knorr for technical assistance. The SSRL is a national user facility operated by Stanford University on behalf of the US Department of Energy (Office of Basic Energy Sciences). The SSRL Structural Molecular Biology Program is supported by the Department of Energy (Office of Biological and Environmental Research), and by the National Institutes of Health (National Center for Research Resources, Biomedical Technology Program), and the National Institute of General Medical Sciences. The ALS is supported by the Office of Energy Research (Office of Basic Energy Sciences, Material Sciences Division) of the US Department of Energy at Lawrence Berkeley National Laboratory. Support by the Department of Defense and Defense Threat Reduction Agency (to A.T.B.) and by a Deutsche Forschungsgemeinschaft grant (to T.B.) is acknowledged. The atomic coordinates and structure factors of the HCB–Syt-II complex are deposited in the Protein Data Bank under accession code 2NM1.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Axel T. Brunger.

Ethics declarations

Competing interests

The atomic coordinates and structure factors of the HCB–Syt-II complex are deposited in the Protein Data Bank under accession code 2NM1. Reprints and permissions information is available at www.nature.com/reprints. The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Materials and Methods, Supplementary Tables 1-2 and Supplementary Figures 1-5 with legends

Rights and permissions

Reprints and permissions

About this article

Cite this article

Jin, R., Rummel, A., Binz, T. et al. Botulinum neurotoxin B recognizes its protein receptor with high affinity and specificity. Nature 444, 1092–1095 (2006). https://doi.org/10.1038/nature05387

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature05387

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

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