Skip to main content
SpringerLink
Log in

Molecular Structure and Mechanisms of Action of Botulinum Neurotoxins

  • Chapter
  • First Online:
Botulinum Toxin Treatment in Surgery, Dentistry, and Veterinary Medicine

  • 443 Accesses

Abstract

Botulinum neurotoxins (BoNTs) are a growing family of bacterial protein toxins that cause a generalized flaccid paralysis of botulism by inactivating neurotransmitter release at peripheral nerve terminals. They are the most potent toxins known thanks to the marvel of their protein design, which underlines their mechanism of action. Their unique biological properties have led them to become also highly effective and successful therapeutic agents for the treatment of a variety of human syndromes. This chapter reports the progress on our understanding of BoNTs, highlighting the different steps of their molecular mechanism of action as key aspects to explain their extreme toxicity but also their unique pharmacological properties.

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

Access this chapter

Log in via an institution
eBook
USD 16.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 109.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 159.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Rossetto O, Pirazzini M, Montecucco C. Botulinum neurotoxins: genetic, structural and mechanistic insights. Nat Rev Microbiol. 2014;12:535–49.

    Article  CAS  PubMed  Google Scholar 

  2. Dong M, Masuyer G, Stenmark P. Botulinum and tetanus neurotoxins. Annu Rev Biochem. 2019;88:811–37.

    Article  CAS  PubMed  Google Scholar 

  3. Peck MW, Smith TJ, Anniballi F, Austin JW, Bano L, Bradshaw M, Cuervo P, Cheng LW, Derman Y, Dorner BG, Fisher A, Hill KK, Kalb SR, Korkeala H, Lindstrom M, Lista F, Luquez C, Mazuet C, Pirazzini M, Popoff MR, Rossetto O, Rummel A, Sesardic D, Singh BR, Stringer SC. Historical perspectives and guidelines for botulinum neurotoxin subtype nomenclature. Toxins. 2017;9(1):38.

    Article  PubMed Central  CAS  Google Scholar 

  4. Montecucco C, Rasotto MB. On botulinum neurotoxin variability. MBio. 2015;6:e02131–14.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  5. Doxey AC, Mansfield MJ, Montecucco C. Discovery of novel bacterial toxins by genomics and computational biology. Toxicon. 2018;147:2–12.

    Article  CAS  PubMed  Google Scholar 

  6. Doxey AC, Mansfield MJ, Lobb B. Exploring the evolution of virulence factors through bioinformatic data mining. mSystems. 2019;21(3):4. pii: e00162-19.

    Google Scholar 

  7. Pirazzini M, Rossetto O, Eleopra R, Montecucco C. Botulinum neurotoxins: biology, pharmacology, and toxicology. Pharmacol Rev. 2017;69(2):200–35.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Gu S, Rumpel S, Zhou J, Strotmeier J, Bigalke H, Perry K, Shoemaker CB, Rummel A, Jin R. Botulinum neurotoxin is shielded by NTNHA in an interlocked complex. Science. 2012;335(6071):977–98.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Simpson LL. The life history of a botulinum toxin molecule. Toxicon. 2013;68:40–59.

    Article  CAS  PubMed  Google Scholar 

  10. Fujinaga Y, Sugawara Y, Matsumura T. Uptake of botulinum neurotoxin in the intestine. Curr Top Microbiol Immunol. 2013;364:45–59.

    CAS  PubMed  Google Scholar 

  11. Lam KH, Jin R. Architecture of the botulinum neurotoxin complex: a molecular machine for protection and delivery. Curr Opin Struct Biol. 2015;31:89–95.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Lacy DB, Tepp W, Cohen AC, DasGupta BR, Stevens RC. Crystal structure of botulinum neurotoxin type A and implications for toxicity. Nat Struct Biol. 1998;5(10):898–902.

    Article  CAS  PubMed  Google Scholar 

  13. Swaminathan S, Eswaramoorthy S. Structural analysis of the catalytic and binding sites of Clostridium botulinum neurotoxin B. Nat Struct Biol. 2000;7:693–9.

    Article  CAS  PubMed  Google Scholar 

  14. Kumaran D, Eswaramoorthy S, Furey W, Navaza J, Sax M, Swaminathan S. Domain organization in Clostridium botulinum neurotoxin type E is unique: its implication in faster translocation. J Mol Biol. 2009;386:233–45.

    Article  CAS  PubMed  Google Scholar 

  15. Swaminathan S. Molecular structures and functional relationships in clostridial neurotoxins. FEBS J. 2011;278(23):4467–85.

    Article  CAS  PubMed  Google Scholar 

  16. Pantano S, Montecucco C. The blockade of the neurotransmitter release apparatus by botulinum neurotoxins. Cell Mol Life Sci. 2014;71(5):793–811.

    Article  CAS  PubMed  Google Scholar 

  17. Montecucco C. How do tetanus and botulinum toxins bind to neuronal membranes? Trends Biochem Sci. 1986;11:314–7.

    Article  CAS  Google Scholar 

  18. Rummel A. Double receptor anchorage of botulinum neurotoxins accounts for their exquisite neurospecificity. Curr Top Microbiol Immunol. 2013;364:61–90.

    CAS  PubMed  Google Scholar 

  19. Rummel A. Two feet on the membrane: uptake of clostridial neurotoxins. Curr Top Microbiol Immunol. 2017;406:1–37.

    CAS  PubMed  Google Scholar 

  20. Hamark C, Berntsson RP, Masuyer G, Henriksson LM, Gustafsson R, Stenmark P, Widmalm G. Glycans confer specificity to the recognition of ganglioside receptors by botulinum neurotoxin a. J Am Chem Soc. 2017;139:218–30.

    Article  CAS  PubMed  Google Scholar 

  21. Rummel A, Eichner T, Weil T, Karnath T, Gutcaits A, Mahrhold S, Sandhoff K, Proia RL, Acharya KR, Bigalke H, Binz T. Identification of the protein receptor binding site of botulinum neurotoxins B and G proves the double-receptor concept. Proc Natl Acad Sci U S A. 2007;104:359–64.

    Article  CAS  PubMed  Google Scholar 

  22. Peng L, Berntsson RP, Tepp WH, Pitkin RM, Johnson EA, Stenmark P, Dong M. Botulinum neurotoxin D-C uses synaptotagmin I and II as receptors, and human synaptotagmin II is not an effective receptor for type B, D-C and G toxins. J Cell Sci. 2012;125:3233e3242.

    Article  CAS  Google Scholar 

  23. Strotmeier J, Willjes G, Binz T, Rummel A. Human synaptotagmin-II is not a high affinity receptor for botulinum neurotoxin B and G: increased therapeutic dosage and immunogenicity. FEBS Lett. 2012;586:310–3.

    Article  CAS  PubMed  Google Scholar 

  24. Tao L, Peng L, Berntsson RP, Liu SM, Park S, Yu F, Boone C, Palan S, Beard M, Chabrier PE, Stenmark P, Krupp J, Dong M. Engineered botulinum neurotoxin B with improved efficacy for targeting human receptors. Nat Commun. 2017;8(1):53.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  25. Pang ZP, Melicoff E, Padgett D, Liu Y, Teich AF, Dickey BF, Lin W, Adachi R, Sudhof TC. Synaptotagmin-2 is essential for survival and contributes to Ca2+ triggering of neurotransmitter release in central and neuromuscular synapses. J Neurosci. 2006;26:13493–504.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Li JY, Jahn R, Dahlstrom A. Synaptotagmin I is present mainly in autonomic and sensory neurons of the rat peripheral nervous system. Neuroscience. 1994;63:837–50.

    Article  CAS  PubMed  Google Scholar 

  27. Kranz G, Paul A, Voller B, Posch M, Windischberger C, Auff E, Sycha T. Long-term efficacy and respective potencies of botulinum toxin A and B: a randomized, double-blind study. Br J Dermatol. 2011;164:176–81.

    Article  CAS  PubMed  Google Scholar 

  28. Yao G, Zhang S, Mahrhold S, Lam KH, Stern D, Bagramyan K, Perry K, Kalkum M, Rummel A, Dong M, Jin R. N-linked glycosylation of SV2 is required for binding and uptake of botulinum neurotoxin A. Nat Struct Mol Biol. 2016;23:656–62.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Montecucco C, Zanotti G. Botulinum neurotoxin A1 likes it double sweet. Nat Struct Mol Biol. 2016;23:619–21.

    Article  CAS  PubMed  Google Scholar 

  30. Stern D, Weisemann J, Le Blanc A, von Berg L, Mahrhold S, Piesker J, Laue M, Luppa PB, Dorner MB, Dorner BG, Rummel A. A lipid-binding loop of botulinum neurotoxin serotypes B, DC and G is an essential feature to confer their exquisite potency. PLoS Pathog. 2018;14(5):e1007048.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  31. Muraro L, Tosatto S, Motterlini L, Rossetto O, Montecucco C. The N-terminal half of the receptor domain of botulinum neurotoxin A binds to microdomains of the plasma membrane. Biochem Biophys Res Commun. 2009;380(1):76–80.

    Article  CAS  PubMed  Google Scholar 

  32. Zhang S, Berntsson RP, Tepp WH, Tao L, Johnson EA, Stenmark P, Dong M. Structural basis for the unique ganglioside and cell membrane recognition mechanism of botulinum neurotoxin DC. Nat Commun. 2017;8(1):1637.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  33. Montecucco C, Rossetto O, Schiavo G. Presynaptic receptor arrays for clostridial neurotoxins. Trends Microbiol. 2004;12:442–6.

    Article  CAS  PubMed  Google Scholar 

  34. Colasante C, Rossetto O, Morbiato L, Pirazzini M, Molgo J, Montecucco C. Botulinum neurotoxin type A is internalized and translocated from small synaptic vesicles at the neuromuscular junction. Mol Neurobiol. 2013;48:120–7.

    Article  CAS  PubMed  Google Scholar 

  35. Harper CB, Papadopulos A, Martin S, Matthews DR, Morgan GP, Nguyen TH, Wang T, Nair D, Choquet D, Meunier FA. Botulinum neurotoxin type-A enters a non-recycling pool of synaptic vesicles. Sci Rep. 2016;6:19654.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Hughes R, Whaler BC. Influence of nerve-ending activity and of drugs on the rate of paralysis of rat diaphragm preparations by cl. botulinum type a toxin. J Physiol. 1962;160:221–33.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Chanaday NL, Cousin MA, Milosevic I, Watanabe S, Morgan JR. The synaptic vesicle cycle revisited: new insights into the modes and mechanisms. J Neurosci. 2019;39(42):8209–16.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Montal M. Botulinum neurotoxin: a marvel of protein design. Annu Rev Biochem. 2010;79:591–617.

    Article  CAS  PubMed  Google Scholar 

  39. Pirazzini M, Azarnia Tehran D, Leka O, Zanetti G, Rossetto O, Montecucco C. On the translocation of botulinum and tetanus neurotoxins across the membrane of acidic intracellular compartments. Biochim Biophys Acta. 2016;1858:467–74.

    Article  CAS  PubMed  Google Scholar 

  40. Fischer A, Montal M. Crucial role of the disulfide bridge between botulinum neurotoxin light and heavy chains in protease translocation across membranes. J Biol Chem. 2007;282:29604–11.

    Article  CAS  PubMed  Google Scholar 

  41. Azarnia Tehran D, Pirazzini M, Leka O, Mattarei A, Lista F, Binz T, Rossetto O, Montecucco C. Hsp90 is involved in the entry of clostridial neurotoxins into the cytosol of nerve terminals. Cell Microbiol. 2017;19(2):e12647.

    Article  CAS  Google Scholar 

  42. Ratts R, Zeng H, Berg EA, Blue C, McComb ME, Costello CE, vanderSpek JC, Murphy JR. The cytosolic entry of diphtheria toxin catalytic domain requires a host cell cytosolic translocation factor complex. J Cell Biol. 2003;160:1139–50.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Pirazzini M, Azarnia Tehran D, Zanetti G, Megighian A, Scorzeto M, Fillo S, Shone CC, Binz T, Rossetto O, Lista F, Montecucco C. Thioredoxin and its reductase are present on synaptic vesicles, and their inhibition prevents the paralysis induced by botulinum neurotoxins. Cell Rep. 2014;8:1870–8.

    Article  CAS  PubMed  Google Scholar 

  44. Zanetti G, Azarnia Tehran D, Pirazzini M, Binz T, Shone CC, Fillo S, Lista F, Rossetto O, Montecucco C. Inhibition of botulinum neurotoxins interchain disulfide bond reduction prevents the peripheral neuroparalysis of botulism. Biochem Pharmacol. 2015;98(3):522–30.

    Article  CAS  PubMed  Google Scholar 

  45. Rossetto O, Pirazzini M, Lista F, Montecucco C. The role of the single interchains disulfide bond in tetanus and botulinum neurotoxins and the development of antitetanus and antibotulism drugs. Cell Microbiol. 2019;21(11):e13037.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Jahn R, Scheller RH. SNAREs--engines for membrane fusion. Nat Rev. 2006;7(9):631–43.

    Article  CAS  Google Scholar 

  47. Sudhof TC, Rothman JE. Membrane fusion: grappling with SNARE and SM proteins. Science. 2009;323(5913):474–7.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  48. Zhang S, Masuyer G, Zhang J, Shen Y, Lundin D, et al. Identification and characterization of a novel botulinum neurotoxin. Nat Commun. 2017;8:14130.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Zhang S, Lebreton F, Mansfield MJ, Miyashita SI, Zhang J, et al. Identification of a botulinum neurotoxin-like toxin in a commensal strain of Enterococcus faecium. Cell Host Microbe. 2018;23:169–76.e6.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Sudhof TC. The molecular machinery of neurotransmitter release (Nobel lecture). Angew Chem Int Ed Engl. 2014;53:12696–717.

    Article  PubMed  CAS  Google Scholar 

  51. Rossetto O, Schiavo G, Montecucco C, Poulain B, Deloye F, Lozzi L, Shone CC. SNARE motif and neurotoxins. Nature. 1994;372:415–6.

    Article  CAS  PubMed  Google Scholar 

  52. Binz T. Clostridial neurotoxin light chains: devices for SNARE cleavage mediated blockade of neurotransmission. Curr Top Microbiol Immunol. 2013;364:139–57.

    CAS  PubMed  Google Scholar 

  53. Chen S. Clostridial neurotoxins: mode of substrate recognition and novel therapy development. Curr Protein Pept Sci. 2014;15:490–503.

    Article  CAS  PubMed  Google Scholar 

  54. Dressler D. Clinical applications of botulinum toxin. Curr Opin Microbiol. 2012;15:325–36.

    Article  PubMed  Google Scholar 

  55. Johnson EA, Botulism MC. Handb Clin Neurol. 2008;91:333–68.

    Article  PubMed  Google Scholar 

  56. Mazzocchio R, Caleo M. More than at the neuromuscular synapse: actions of botulinum neurotoxin a in the central nervous system. Neuroscientist. 2015;21:44–61.

    Article  PubMed  CAS  Google Scholar 

  57. Antonucci F, Rossi C, Gianfranceschi L, Rossetto O, Caleo M. Longdistance retrograde effects of botulinum neurotoxin A. J Neurosci. 2008;28:3689–96.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Restani L, Giribaldi F, Manich M, Bercsenyi K, Menendez G, Rossetto O, Caleo M, Schiavo G. Botulinum neurotoxins A and E undergo retrograde axonal transport in primary motor neurons. PLoS Pathog. 2012;8:e1003087.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Matak I, Bach-Rojecky L, Filipovic B, Lackovic Z. Behavioral and immunohistochemical evidence for central antinociceptive activity of botulinum toxin A. Neuroscience. 2011;186:201–7.

    Article  CAS  PubMed  Google Scholar 

  60. Marinelli S, Vacca V, Ricordy R, Uggenti C, Tata AM, Luvisetto S, Pavone F. The analgesic effect on neuropathic pain of retrogradely transported botulinum neurotoxin A involves Schwann cells and astrocytes. PLoS One. 2012;7:e47977.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Matak I, Riederer P, Lackovic Z. Botulinum toxin’s axonal transport from periphery to the spinal cord. Neurochem Int. 2012;61:236–9.

    Article  CAS  PubMed  Google Scholar 

  62. Matak I, Lackovic Z. Botulinum toxin A, brain and pain. Prog Neurobiol. 2014;119-120:39–59.

    Article  CAS  PubMed  Google Scholar 

  63. Restani L, Antonucci F, Gianfranceschi L, Rossi C, Rossetto O, Caleo M. Evidence for anterograde transport and transcytosis of botulinum neurotoxin A (BoNT/A). J Neurosci. 2011;31:15650–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Caleo M, Spinelli M, Colosimo F, Matak I, Rossetto O, Lackovic Z, Restani L. Transynaptic action of botulinum neurotoxin type A at Central Cholinergic Boutons. J Neurosci. 2018;38(48):10329–37.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Safarpour Y, Jabbari B. Botulinum toxin treatment of pain syndromes –an evidence based review. Toxicon. 2018;147:120–8.

    Article  CAS  PubMed  Google Scholar 

  66. Mittal SO, Jabbari B. Botulinum neurotoxins and cancer-a review of the literature. Toxins. 2020;12(1):32.

    Article  CAS  PubMed Central  Google Scholar 

  67. Shoemaker CB, Oyler GA. Persistence of Botulinum neurotoxin inactivation of nerve function. Curr Top Microbiol Immunol. 2013;364:179–96.

    CAS  PubMed  PubMed Central  Google Scholar 

  68. Tsai YC, Maditz R, Kuo C-l, Fishman PS, Shoemaker CB, Oyler GA, Weissman AM. Targeting botulinum neurotoxin persistence by the ubiquitin-proteasome system. Proc Natl Acad Sci U S A. 2010;107:16554–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  69. Megighian A, Zordan M, Pantano S, Scorzeto M, Rigoni M, Zanini D, Rossetto O, Montecucco C. Evidence for a radial SNARE super-complex mediating neurotransmitter release at the Drosophila neuromuscular junction. J Cell Sci. 2013;126:3134–40.

    Article  CAS  PubMed  Google Scholar 

  70. Rossetto O, Montecucco C. Tables of toxicity of botulinum and tetanus neurotoxins. Toxins. 2019;11(12):pii:E686.

    Article  CAS  Google Scholar 

  71. Chen S, Barbieri JT. Engineering botulinum neurotoxin to extend therapeutic intervention. PNAS. 2009;106:9180–4.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  72. Sikorra S, Litschko C, Müller C, Thiel N, Galli T, Eichner T, Binz T. Identification and characterization of botulinum neurotoxin a substrate binding pockets and their re-engineering for human SNAP-23. J Mol Biol. 2016;428(2Pt A):372–84.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

Work in the author’s laboratory is supported by grants from the University of Padova.

Author information

Authors and Affiliations

  1. Dipartimento di Scienze Biomediche, Università di Padova, Padova, Italy

    Ornella Rossetto & Marco Pirazzini

Authors
  1. Ornella Rossetto
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Marco Pirazzini
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Ornella Rossetto .

Editor information

Editors and Affiliations

  1. Professor Emeritus of Neurology, Yale University School of Medicine, New Haven, CT, USA

    Bahman Jabbari

Rights and permissions

Reprints and permissions

Copyright information

© 2020 Springer Nature Switzerland AG

About this chapter

Check for updates. Verify currency and authenticity via CrossMark

Cite this chapter

Rossetto, O., Pirazzini, M. (2020). Molecular Structure and Mechanisms of Action of Botulinum Neurotoxins. In: Jabbari, B. (eds) Botulinum Toxin Treatment in Surgery, Dentistry, and Veterinary Medicine. Springer, Cham. https://doi.org/10.1007/978-3-030-50691-9_2

Download citation

Publish with us

Policies and ethics

Search

Navigation