Sandbox Reserved 966

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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 bound

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 ^[1] (see Figure 2)

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. ^[2]

1 Structure
- 1.1 Global aspect
- 1.2 Mechanism & Catalytic site
2 Therapeutic applications
3 Related Structures
4 Contributors
5 References

Structure

Global 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^[3]

The light chain of Clostridium botulinum neurotoxin serotype A presents several typical structures.

The polypeptide forms 11 α-helices (we can notice a kink formed by , who may interact with an H from the N of Gly104)
We can also find tree 3-10 helices (, , )
There are several that 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.

The Cys429 involved in the disulfide bond between the HC and the LC can't be shown here due to the condition for getting the crystal structure.

Mechanism & Catalytic site

Figure 2 : Global aspect of C.botulinum neurotoxin serotype A (made using PyMol and modified with Inkscape by Xavier Hartmann)

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^[4]. 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 cleaves a peptide bond of a protein belonging to the SNARE family Wikipedia page (involved in the phenomenon of vesicle fusion) and especially the Gln197 - Arg198 peptide bond of 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 in red), 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.^[5] 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 ^[6].

Figure 3 : Enzymatic mechanism of BoNT/A. Steps are numbered (made using ChemDraw Ultra by Xavier Hartmann)

At first glance the tunnel for the access of the substrate is clearly visible (unfortunately the representation of the surface is bugged, catalytic site can't be displayed in another color)

The enzymatic reaction is shown on Figure 3 and takes place at the . As it is developed on the scheme, a lot of residues and molecules of water are involved.

Therapeutic applications

In the late 1960s, Edward Schantz and a San Francisco ophthalmologist, Alan Scott, started to work on the use of the botulinum toxin in therapeutic process. First, they tried to treat strabismus and in the late 1970s the neurotoxin was used in many therapeutic applications.^[7]

Hyperhidrosis and CHARGE syndrome

We can now treat excessive sweating with botulinum toxin ^[8] thanks to its action on the receptors of the parasympathetic network. Excessive dribbling can be treated in the same way.

Cosmetic

Non-lethal amount of botulinum toxin can be injected locally to paralyse targeted muscles and reduce wrinkles temporally^[9] (5 to 6 months).

Blepharospesm, 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 relaxation ok the muscles.

Cervical dystonia

BoNT/A is used against cervical dystonia. But it can become inefficient after a period of use.

Related Structures

Structure of the C. botulinum neurotoxin serotype A apo-enzyme 3BOK (light chain)
Structure of the C. botulinum neurotoxin serotype A with an inhibitory peptide bound 3BOO (light chain)
Crystal structure of botulinum neurotoxin serotype A 3BTA (whole toxin)

Contributors

Xavier Hartmann and Rémi Pelletier

References

↑ Rossetto O, Pirazzini M, Montecucco C. Botulinum neurotoxins: genetic, structural and mechanistic insights. Nat Rev Microbiol. 2014 Aug;12(8):535-49. doi: 10.1038/nrmicro3295. Epub 2014 Jun, 30. PMID:24975322 doi:http://dx.doi.org/10.1038/nrmicro3295
↑ Silvaggi NR, Wilson D, Tzipori S, Allen KN. Catalytic features of the botulinum neurotoxin a light chain revealed by high resolution structure of an inhibitory peptide complex. Biochemistry. 2008 May 27;47(21):5736-45. Epub 2008 May 6. PMID:18457419 doi:10.1021/bi8001067
↑ http://www.rcsb.org/pdb/explore/remediatedSequence.do?structureId=3BON&bionumber=1
↑ Rummel A. Double receptor anchorage of botulinum neurotoxins accounts for their exquisite neurospecificity. Curr Top Microbiol Immunol. 2013;364:61-90. doi: 10.1007/978-3-642-33570-9_4. PMID:23239349 doi:http://dx.doi.org/10.1007/978-3-642-33570-9_4
↑ Silvaggi NR, Wilson D, Tzipori S, Allen KN. Catalytic features of the botulinum neurotoxin a light chain revealed by high resolution structure of an inhibitory peptide complex. Biochemistry. 2008 May 27;47(21):5736-45. Epub 2008 May 6. PMID:18457419 doi:10.1021/bi8001067
↑ Breidenbach MA, Brunger AT. Substrate recognition strategy for botulinum neurotoxin serotype A. Nature. 2004 Dec 16;432(7019):925-9. Epub 2004 Dec 12. PMID:15592454 doi:http://dx.doi.org/10.1038/nature03123
↑ http://rms.medhyg.ch/numero-200-page-870.htm
↑ http://rms.medhyg.ch/numero-200-page-870.htm
↑ http://rms.medhyg.ch/numero-200-page-870.htm

Retrieved from "http://52.214.119.220/wiki/index.php/Sandbox_Reserved_966"

@@ Line 20: / Line 20: @@
 The Cys429 involved in the disulfide bond between the HC and the LC can't be shown here due to the condition for getting the crystal structure.
-*The light chain of Clostridium botulinum neurotoxin serotype A has 11 α-helices :
+=== Mechanism & Catalytic site ===
-**Helix 1 : <scene name='60/604485/H1/2'>H1</scene>
-**Helix 2 : <scene name='60/604485/H2/1'>H2</scene> (we can notice the kink formed by <scene name='60/604485/Thr101/1'>Thr101</scene>, who may interact with an H from the N of Gly104)
-**Helix 4 : <scene name='60/604485/H4/1'>H4</scene>
-**Helix 5 : <scene name='60/604485/H5/1'>H5</scene>
-**Helix 6 : <scene name='60/604485/H6/1'>H6</scene>
-**Helix 7 : <scene name='60/604485/H7/1'>H7</scene>
-**Helix 8 : <scene name='60/604485/H8/1'>H8</scene>
-**Helix 9 : <scene name='60/604485/H9/1'>H9</scene>
-**Helix 10 : <scene name='60/604485/H10/1'>H10</scene>
-**Helix 11 : <scene name='60/604485/H11/1'>H11</scene>
-**Helix 12 : <scene name='60/604485/H12/1'>H12</scene>
-* It also has three 3-10 helices :
-**Helix 3 : <scene name='60/604485/H3/2'>H3</scene>
-**Helix 13 : <scene name='60/604485/H13/1'>H13</scene>
-**Helix 14 : <scene name='60/604485/H14/1'>H14</scene>
-* There are several  <scene name='60/604485/Sheets/1'>β sheets </scene> that are anti parallel except <scene name='60/604485/Sheets_parrallel/1'>this one.</scene>
-* An interesting structure is also a typical  <scene name='60/604485/Betaturn/1'>β-turn</scene> : 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.
-The Cys429 involved in the disulfide bond between the HC and the LC can't be shown here due to the condition for getting the crystal structure.
-=== Mechanism ===
 [[Image:BoNT.jpg|thumb|'''Figure 2 :''' Global aspect of C.botulinum neurotoxin serotype A (made using PyMol and modified with Inkscape by Xavier Hartmann) ]]
 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<ref>PMID: 23239349</ref>. ''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.
@@ Line 52: / Line 26: @@
 It is the light chain that carries the metalloprotease activity. Neurotoxin serotype A cleaves a peptide bond of a protein belonging to the SNARE family [http://en.wikipedia.org/wiki/SNARE_(protein) ''Wikipedia page''] (involved in the phenomenon of vesicle fusion) and especially the Gln197 - Arg198 peptide bond of SNAP-25 [http://en.wikipedia.org/wiki/SNAP25 ''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 (SNKTRIDQAN[[QR]]ATKML, cleavage site in red), 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.<ref>PMID: 18457419</ref> 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 <ref>PMID:15592454</ref>.
 [[Image:Mecanismefinal.jpg|thumb|'''Figure 3 :''' Enzymatic mechanism of BoNT/A. Steps are numbered (made using ChemDraw Ultra by Xavier Hartmann) ]]
-===Catalytic site ===
 At first glance the tunnel for the access of the substrate is clearly visible <scene name='60/604485/Catalytic_site_view/1'>view of the catalytic site</scene> ''(unfortunately the representation of the surface is bugged, catalytic site can't be displayed in another color)''