Skip to main content

Thank you for visiting 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.

Substrate recognition strategy for botulinum neurotoxin serotype A


Clostridal neurotoxins (CNTs) are the causative agents of the neuroparalytic diseases botulism and tetanus1,2. CNTs impair neuronal exocytosis through specific proteolysis of essential proteins called SNAREs3. SNARE assembly into a low-energy ternary complex is believed to catalyse membrane fusion, precipitating neurotransmitter release; this process is attenuated in response to SNARE proteolysis4,5,6,7. Site-specific SNARE hydrolysis is catalysed by the CNT light chains, a unique group of zinc-dependent endopeptidases3. The means by which a CNT properly identifies and cleaves its target SNARE has been a subject of much speculation; it is thought to use one or more regions of enzyme–substrate interaction remote from the active site (exosites)8,9,10. Here we report the first structure of a CNT endopeptidase in complex with its target SNARE at a resolution of 2.1 Å: botulinum neurotoxin serotype A (BoNT/A) protease bound to human SNAP-25. The structure, together with enzyme kinetic data, reveals an array of exosites that determine substrate specificity. Substrate orientation is similar to that of the general zinc-dependent metalloprotease thermolysin11. We observe significant structural changes near the toxin's catalytic pocket upon substrate binding, probably serving to render the protease competent for catalysis. The novel structures of the substrate-recognition exosites could be used for designing inhibitors specific to BoNT/A.

Your institute does not have access to this article

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: The nearly identical active sites of different BoNT light chains.
Figure 2: The interface between sn2 and BoNT/A.
Figure 3: Detailed views of α- and β-exosites.
Figure 4: Kinetic characterization of sn2 mutants.
Figure 5: Wall-eyed stereo view of β-exosite conformational changes.
Figure 6: Exosite-based model of BoNT/A substrate recognition.


  1. Humeau, Y., Doussau, F., Grant, N. J. & Poulain, B. How botulinum and tetanus neurotoxins block neurotransmitter release. Biochimie 82, 427–446 (2000)

    CAS  Article  Google Scholar 

  2. Dolly, J. O., Black, J., Williams, R. S. & Melling, J. Acceptors for botulinum neurotoxin reside on motor nerve terminals and mediate its internalization. Nature 307, 457–460 (1984)

    ADS  CAS  Article  Google Scholar 

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

    ADS  CAS  Article  Google Scholar 

  4. 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)

    CAS  Article  Google Scholar 

  5. Söllner, T. et al. SNAP receptors implicated in vesicle targeting and fusion. Nature 362, 318–324 (1993)

    ADS  Article  Google Scholar 

  6. Pellegrini, L. L., O'Connor, V., Lottspeich, F. & Betz, H. Clostridial neurotoxins compromise the stability of a low energy SNARE complex mediating NSF activation of synaptic vesicle fusion. EMBO J. 14, 4705–4713 (1995)

    CAS  Article  Google Scholar 

  7. Xu, T., Binz, T., Niemann, H. & Neher, E. Multiple kinetic components of exocytosis distinguished by neurotoxin sensitivity. Nature Neurosci. 1, 192–200 (1998)

    CAS  Article  Google Scholar 

  8. Rossetto, O. et al. SNARE motif and neurotoxins. Nature 372, 415–416 (1994)

    ADS  CAS  Article  Google Scholar 

  9. 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)

    CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

  11. Holden, H. M., Tronrud, D. E., Monzingo, A. F., Weaver, L. H. & Matthews, B. W. Slow- and fast-binding inhibitors of thermolysin display different modes of binding: crystallographic analysis of extended phosphonamidate transition-state analogues. Biochemistry 26, 8542–8553 (1987)

    CAS  Article  Google Scholar 

  12. 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)

    CAS  Article  Google Scholar 

  13. Hanson, M. A. & Stevens, R. C. Cocrystal structure of synaptobrevin-II bound to botulinum neurotoxin type B at 2.0 Å resolution. Nature Struct. Biol. 7, 687–692 (2000)

    CAS  Article  Google Scholar 

  14. Agarwal, R., Eswaramoorthy, S., Kumaran, D., Binz, T. & Swaminathan, S. Structural analysis of botulinum neurotoxin type E catalytic domain and its mutant glu212 → gln reveals the pivotal role of the glu212 carboxylate in the catalytic pathway. Biochemistry 43, 6637–6644 (2004)

    CAS  Article  Google Scholar 

  15. Vaidyanathan, V. V. et al. Proteolysis of SNAP-25 isoforms by botulinum neurotoxin types A, C, and E: domains and amino acid residues controlling the formation of enzyme-substrate complexes and cleavage. J. Neurochem. 72, 327–337 (1999)

    CAS  Article  Google Scholar 

  16. Foran, P., Shone, C. C. & Dolly, J. O. Differences in the protease activities of tetanus and botulinum B toxins revealed by the cleavage of vesicle-associated membrane protein and various sized fragments. Biochemistry 33, 15365–15374 (1994)

    CAS  Article  Google Scholar 

  17. Washbourne, P., Pellizzari, R., Baldini, G., Wilson, M. C. & Montecucco, C. Botulinum neurotoxin types A and E require the SNARE motif in SNAP-25 for proteolysis. FEBS Lett. 418, 1–5 (1997)

    CAS  Article  Google Scholar 

  18. Li, L., Binz, T., Niemann, H. & Singh, B. R. Probing the mechanistic role of glutamate residue in the zinc-binding motif of type A botulinum neurotoxin light chain. Biochemistry 39, 2399–2405 (2000)

    CAS  Article  Google Scholar 

  19. Binz, T., Bade, S., Rummel, A., Kollewe, A. & Alves, J. Arg(362) and Tyr(365) of the botulinum neurotoxin type A light chain are involved in transition state stabilization. Biochemistry 41, 1717–1723 (2002)

    CAS  Article  Google Scholar 

  20. Brooijmans, N., Sharp, K. A. & Kuntz, I. D. Stability of macromolecular complexes. Proteins 48, 645–653 (2002)

    CAS  Article  Google Scholar 

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

    ADS  CAS  Article  Google Scholar 

  22. Rupp, B. & Segelke, B. Questions about the structure of the botulinum neurotoxin B light chain in complex with a target peptide. Nature Struct. Biol. 8, 663–664 (2001)

    CAS  Article  Google Scholar 

  23. Sukonpan, C. et al. Synthesis of substrates and inhibitors of botulinum neurotoxin type A metalloprotease. J. Pept. Res. 63, 181–193 (2004)

    CAS  Article  Google Scholar 

  24. Schmidt, J. J. & Bostian, K. A. Proteolysis of synthetic peptides by type A botulinum neurotoxin. J. Protein Chem. 14, 703–708 (1995)

    CAS  Article  Google Scholar 

  25. Fiebig, K. M., Rice, L. M., Pollock, E. & Brunger, A. T. Folding intermediates of SNARE complex assembly. Nature Struct. Biol. 6, 117–123 (1999)

    CAS  Article  Google Scholar 

  26. Segelke, B., Knapp, M., Kadkhodayan, S., Balhorn, R. & Rupp, B. Crystal structure of Clostridium botulinum neurotoxin protease in a product-bound state: evidence for noncanonical zinc protease activity. Proc. Natl Acad. Sci. USA 101, 6888–6893 (2004)

    ADS  CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

  28. Dall'Acqua, W. & Carter, P. Substrate-assisted catalysis: molecular basis and biological significance. Protein Sci. 9, 1–9 (2000)

    CAS  Article  Google Scholar 

  29. Fasshauer, D., Bruns, D., Shen, B., Jahn, R. & Brunger, A. T. A structural change occurs upon binding of syntaxin to SNAP-25. J. Biol. Chem. 272, 4582–4590 (1997)

    CAS  Article  Google Scholar 

  30. Debnath, A. K., Radigan, L. & Jiang, S. Structure-based identification of small molecule antiviral compounds targeted to the gp41 core structure of the human immunodeficiency virus type 1. J. Med. Chem. 42, 3203–3209 (1999)

    CAS  Article  Google Scholar 

Download references


We thank T. Binz and J. Ernst for providing initial BoNT/A and sn2 constructs and P. Adams, T. Fenn, S. Kaiser, Z. Panepucci, P. Strop and W. Weis for technical assistance and critical reading. Portions of this research were carried out at the Stanford Synchrotron Radiation Laboratory, 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. Portions of this research were conducted at the Advanced Light Source which is supported by the Office of Energy Research (Office of Basic Energy Sciences, Materials Sciences Division) of the US Department of Energy at Lawrence Berkeley National Laboratory. This work was supported in part by an NIH grant to A.T.B.

Author information

Authors and Affiliations


Corresponding author

Correspondence to Axel T. Brunger.

Ethics declarations

Competing interests

The authors declare that they have no competing financial interests.

Supplementary information

Supplementary Information

This file contains the Supplementary Methods section of the paper with references, along with Supplementary Tables 1 and 2, and Supplementary Figures 1, 2 and 3. (PDF 428 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Breidenbach, M., Brunger, A. Substrate recognition strategy for botulinum neurotoxin serotype A. Nature 432, 925–929 (2004).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

Further reading


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.


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