Skip to main content
SpringerLink
Log in

Cocrystal structure of synaptobrevin-II bound to botulinum neurotoxin type B at 2.0 Å resolution

  • Article
  • Published:

From Nature Structural Biology

View current issue Submit your manuscript

A Retraction to this article was published on 01 July 2009

Abstract

Botulinum neurotoxin serotype B is a zinc protease that disrupts neurotransmitter release by cleaving synaptobrevin-II (Sb2), one of three SNARE proteins involved in neuronal synaptic vesicle fusion. The three-dimensional crystal structure of the apo botulinum neurotoxin serotype B catalytic domain (BoNT/B-LC) has been determined to 2.2 Å resolution, and the complex of cleaved Sb2 with the catalytic domain (Sb2–BoNT/B-LC) has been determined to 2.0 Å resolution. A comparison of the holotoxin catalytic domain and the isolated BoNT/B-LC structure shows a rearrangement of three active site loops. This rearrangement exposes the BoNT/B active site. The Sb2–BoNT/B-LC structure illustrates two distinct binding regions, which explains the specificity of each botulinum neurotoxin for its synaptic vesicle protein. This observation provides an explanation for the proposed cooperativity between binding of full-length substrate and catalysis and suggest a mechanism of synaptobrevin proteolysis employed by the clostridial neurotoxins.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Figure 1: Structures of BoNT/A holotoxin and BoNT/B-LC.
Figure 2: Stereo view showing the electron density contoured at 1.2σ for the cleaved Sb2 peptides (purple).
Figure 3: Molecular and schematic representations of the interactions formed between the bound Sb2 products and BoNT/B-LC.
Figure 4: The proposed reaction mechanism for synaptobrevin proteolysis by botulinum neurotoxin.
Figure 5: The SNARE complex showing Sb2 residues important for toxin binding.

Similar content being viewed by others

Accession codes

Accessions

Protein Data Bank

References

  1. Simpson, L.L. Botulinum neurotoxin and tetanus toxin. Academic Press, San Diego (1989).

    Google Scholar 

  2. Blasi, J. et al. Botulinum neurotoxin A selectively cleaves the synaptic protein SNAP-25. Nature 365, 160– 163 (1993).

    Article  CAS  Google Scholar 

  3. Schiavo, G., Rossetto, O., Santucci, A., DasGupta, B.R. & Montecucco, C. Botulinum neurotoxins are zinc proteins. J. Biol. Chem. 267, 23479– 23483 (1992).

    CAS  PubMed  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. Krieglstein, K.G., DasGupta, B.R. & Henschen, A.H. Covalent structure of botulinum neurotoxin type A: location of sulfhydryl groups, and disulfide bridges and identification of C-termini of light and heavy chains. J. Prot. Chem. 13, 49– 57 (1994).

    Article  CAS  Google Scholar 

  6. Simpson, L.L. Kinetic studies on the interaction between botulinum toxin type A and the cholinergic neuromuscular junction. J. Pharmacol. Exp. Ther. 212, 16– 21 (1980).

    CAS  PubMed  Google Scholar 

  7. Boquet, P., Duflot, E. & Hauttecoeur, B. Low pH induces a hydrophobic domain in the tetanus toxin molecule. Eur. J. Biochem. 144, 339– 344 (1984).

    Article  CAS  Google Scholar 

  8. Lacy, D.B., Stevens, R.C. Sequence homology and structural analysis of the clostridial neurotoxins. J. Mol. Biol. 291, 1091– 1104 (1999).

    Article  CAS  Google Scholar 

  9. Boquet, P. & Duflot, E. Tetanus toxin fragment forms channels in lipid vesicles at low pH. Proc. Natl. Acad. Sci. USA 79, 7614– 7618 (1982).

    Article  CAS  Google Scholar 

  10. Sudhof, T. The synaptic vesicle cycle: a cascade of protein-protein interactions. Nature 375, 645– 653 (1995).

    Article  CAS  Google Scholar 

  11. Hanson, P.I., Heuser, J.E. & Jahn, R. Neurotransmitter release — four years of SNARE complexes. Curr. Opin. Neurobiol. 7, 310– 315 (1997).

    Article  CAS  Google Scholar 

  12. Sudhof, T.C., De Camilli, P., Niemann, H. & Jahn, R. Membrane fusion machinery: insights from synaptic proteins. Cell 75, 1– 4 (1993).

    Article  CAS  Google Scholar 

  13. Swaminathan, S. & Eswaramoorthy, S. The catalytic and binding sites of Clostridium botulinum neurotoxin B defined by structural analysis. Nature Struct. Biol. 3, 693– 699 (2000).

    Article  Google Scholar 

  14. Kurazono, H. et al. Minimal essential domains specifying toxicity of the light chains of tetanus toxin and botulinum neurotoxin type A. J. Biol. Chem. 267, 14721– 14729 (1992).

    CAS  PubMed  Google Scholar 

  15. Lebeda, F.J. & Olson, M.A. Secondary structural predictions for the clostridial neurotoxins. Proteins 20, 293– 300 (1994).

    Article  CAS  Google Scholar 

  16. Lacy, D.B., Tepp, W., Cohen, A.C., DasGupta, B.R. & Stevens, R.C. Crystal structure of botulinum neurotoxin type A and implications for toxicity. Nature Struct. Biol. 5, 898– 902 (1998).

    Article  CAS  Google Scholar 

  17. Pellizzari, R., et al. Structural determinants of the specificity for synaptic vesicle-associated membrane protein/synaptobrevin of tetanus and botulinum type B and G neurotoxins. J. Biol. Chem. 271, 20353– 20358 (1996).

    Article  CAS  Google Scholar 

  18. Shone, C.C., et al. Proteolytic cleavage of synthetic fragments of vesicle-associated membrane protein, isoform-2 by botulinum type B neurotoxin. Eur. J. Biochem. 217, 965– 971 (1993).

    Article  CAS  Google Scholar 

  19. Yamasaki, S. et al. Cleavage of members of the synaptobrevin/VAMP family by types D and F botulinal neurotoxins and tetanus toxin. J. Biol. Chem. 269, 12764– 12772 (1994).

    CAS  PubMed  Google Scholar 

  20. Binz, T. et al. Proteolysis of SNAP-25 by types E and A botulinal neurotoxins. J. Biol. Chem. 269, 1617– 1620 (1994).

    CAS  PubMed  Google Scholar 

  21. Cornille, F. et al. Cooperative exosite-dependent cleavage of synaptobrevin by tetanus toxin light chain. J. Biol. Chem. 272, 3459– 3464 (1997).

    Article  CAS  Google Scholar 

  22. Hazzard, J., Sudhof, T.C. & Rizo, J. NMR analysis of the structure of synaptobrevin and of its interaction with syntaxin. J. Biomol. NMR. 14, 203– 207 (1999).

    Article  CAS  Google Scholar 

  23. Fasshauer, D., Sutton, R.B., Brünger, A.T. & Jahn, R. Conserved structural features of the synaptic fusion complex: SNARE proteins reclassified as Q- and R-SNAREs. Proc. Natl. Acad. Sci. USA 95, 15781– 15786 (1998).

    Article  CAS  Google Scholar 

  24. Sutton, R.B., Fasshauer, D., Jahn, R. & Brünger, A.T. Crystal structure of a SNARE complex involved in synaptic exocytosis at 2.4 Å resolution. Nature 395, 347– 353 (1998).

    Article  CAS  Google Scholar 

  25. Matthews, B.W. Structural basis of the action of thermolysin and related zinc peptidases. Acc. Chem. Res. 21, 333– 340 (1988).

    Article  CAS  Google Scholar 

  26. Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307– 326 (1997).

    Article  CAS  Google Scholar 

  27. Navaza, J. AMoRe and automated package for molecular replacement. Acta Crystallogr. A 50, 157– 163 (1994).

    Article  Google Scholar 

  28. Jones, T. A., Zou, J. Y., Cowan, S. W. & Kjeldgaard, M. Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr. A 47, 110– 119 (1991).

    Article  Google Scholar 

  29. Collaborative Computational Project Number 4. CCP4 Suite: programs for crystallography. Acta Crystallogr. D 50, 760– 763 (1994).

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

    Article  Google Scholar 

  31. Kraulis, P.J. MOLSCRIPT: a program to produce both detailed and schematic plots of protein structures. J. Appl. Crystallogr. 24, 946– 950 (1991).

    Article  Google Scholar 

  32. Merritt, E.A. & Bacon, J.A. Raster3D: photorealistic molecular graphic. Methods Enzymol. 277, 505– 524 (1997).

    Article  CAS  Google Scholar 

  33. Esnouf, R.M. Further additions to MolScript version 1.4, including reading and contouring of electron-density maps. Acta. Crystallogr. D 55, 938– 940 (1999).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We dedicate this paper to H. Niemann, who provided the original light chain constructs to us, and who recently passed away. We thank P. Kuhn and the SSRL staff for their assistance at beamlines 9-1 and 9-2 at the Stanford Synchrotron Radiation Laboratory and we thank M. Adler and J. Nicholson for the synaptobrevin peptide used in the co-crystal structure. We also greatly appreciate the neurotoxin provided by B. DasGupta, B. Tepp, E. Johnson, and M. Goodenough used in previous structural studies and their helpful discussions. Financial support for this research was provided by the U.S. Army and the NN20 program of the U.S. Department of Energy.

Author information

Authors and Affiliations

  1. Department of Chemistry and Molecular Biology, The Scripps Research Institute, La Jolla, 92037, California, USA

    Michael A. Hanson & Raymond C. Stevens

Authors
  1. Michael A. Hanson
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Raymond C. Stevens
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Raymond C. Stevens.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Hanson, M., Stevens, R. Cocrystal structure of synaptobrevin-II bound to botulinum neurotoxin type B at 2.0 Å resolution. Nat Struct Mol Biol 7, 687–692 (2000). https://doi.org/10.1038/77997

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/77997

  • Springer Nature America, Inc.

This article is cited by

Search

Navigation