Sandbox Reserved 966

This Sandbox is Reserved from 15/11/2014, through 15/05/2015 for use in the course "Biomolecule" taught by Bruno Kieffer at the Strasbourg University. This reservation includes Sandbox Reserved 951 through Sandbox Reserved 975.

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Structure of the "Clostridium botulinum" neurotoxin serotype A light chain with Zn2+ cofactor boundStructure of the "Clostridium botulinum" neurotoxin serotype A light chain with Zn2+ cofactor bound

<StructureSection load='3BON' size='340' side='right' caption='BoNT/A-LC' scene='60/604485/General_introduction/2'> The Clostridium botulinum neurotoxin produced by the bacteria Clostridium botulinum (and some strains of Clostridium butyricium and Clostridium baratii) is the most lethal toxin known today. Seven serotypically botulinum neurotoxins (BoNTs) have been found, each of them is categorized into subtypes depending on their amino acid sequence. The protein is initially synthesized as a single polypeptide chain of ≈150 kDa and is then cleaved by a protease to yield the mature toxin, which consists of a light chain (LC which is 50 kDa) and a heavy chain (HC which is 100 kDa). LC and HC are held together by a long peptide belt, non-covalent interactions and a single inter-chain disulphide bond The Clostridium botulinum neurotoxin serotype A light chain (BoNT/A-LC) shown below is composed of 425 residues. It was obtained with high resolution X-ray crystal structure using an inhibitory peptide and the catalytic Zn(II) ion.

StructureStructure

Global aspectGlobal aspect

The Ramachandran diagram (see Figure 1) shows the distribution of the residues in the protein. Amino acids are globally in favored regions.

Figure 1 : Ramachandran diagram of C.botulinum neurotoxin serotype A light chain (made using Python by Rémi Pelletier)

The of BoNT/A-LC

• The light chain of Clostridium botulinum neurotoxin serotype A has 11 α-helices :
  • Helix 1 :
  • Helix 2 :
  • Helix 4 :
  • Helix 5 :
  • Helix 6 :
  • Helix 7 :
  • Helix 8 :
  • Helix 9 :
  • Helix 10 :
  • Helix 11 :
  • Helix 12 :

• It also has three 3-10 helices :
  • Helix 3 :
  • Helix 13 :
  • Helix 14 :

• There are several most of them are anti parallel except

• An interesting structure is also a typical  : indeed the chain makes a sharp reversal by 180° within 4 residues, moreover Cα from the i residue and the Cα from the i+3 residue are separated by less than 7 angstroms.

MechanismMechanism

The neurotoxin inactivates neurotransmitter release owing to its metalloprotease activity. It targets the presynaptic membrane of peripheral nerve terminals using a binding mode based on the use of two independent receptors: a polysialoganglioside (PSG) and a protein receptor in the lumen of synaptic vesicles. Clostridium botulinum neurotoxin is initially synthesized as a single polypeptide chain of ≈150 kDa and is then cleaved by a protease to yield the mature toxin, which consists of a light chain (LC which is 50 kDa) and a heavy chain (HC which is 100 kDa). The heavy chain presents the PSG binding domain and is therefore implied in the entry of the neurotoxin in the nerve cell thanks to vesicles; then the disulfide bond is broken and the light chain is released. It is the light chain that carries the metalloprotease activity. Neurotoxin serotype A targets a protein belonging to the SNARE family Wikipedia page on what a SNARE protein is (involved in the phenomenon of vesicle fusion and especially the protein SNAP-25 Wikipedia page blocking the release of neurotransmitter release inducing paralysis. The BoNT LCs are remarkable among proteases for the extremely long substrates required for efficient proteolysis. With a minimum substrate of 17 amino acids (SNKTRIDQANQRATKML, cleavage site underlined), the BoNT/A protease accepts shorter peptide substrates than any of the other serotypes. In comparison, other microbial metalloproteases have been found to have activity against substrates as short as dipeptides. Structural and biochemical data on the BoNT/ A-LC suggest that most of the specific interactions between the enzyme and the SNAP-25 substrate occur at sites remote from the active site. Indeed, the only amino acid within the cleavage sequence that is required for efficient proteolysis is the P1′ Arg198 .

Figure 2 : Enzymatic mechanism (made using ChemDraw Ultra by Xavier Hartmann

Therapeutic applicationsTherapeutic applications

In the late 1960s, a Edward Schantz and a San Francisco opthalmologist, Alan Scott, start to work on the using of the botulinum toxin in therapeutic process. First, they try to treat strabismus and in the late 1970s the neurotoxin is used in many therapeutic applications.

Hyperhidrosis and CHARGE syndromeHyperhidrosis and CHARGE syndrome

We can now treat excessive sweating with botulinium toxin thanks to its action on the receptors of the parasympathetic network. Excessive dribbling can be treated by the same way.

CosmeticCosmetic

Non-lethal amount of botulinum toxin can be injected localy to paralyse targeted muscles and reduce wrinkles temporaly (5 to 6 months).

Blepharospesm, nystagmus, torticollis and strabismusBlepharospesm, nystagmus, torticollis and strabismus

This pathologic can be treated by the same methode. Every anomalous behaviour of muscle tissue can be fixed thanks to the paralyzing effect of the toxin. It is based on the muscles relaxation.

Cervical dystoniaCervical dystonia

BTX-A is used against cervical dystonia. But it can become inefficient after a periode of use.