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Introduction

The tetanus toxin is produced by the bacteria Clostridium tetani. Clostridium bacteria produces distinct neurotoxins that are extremely potent to humans. This bacillus bacteria's spores are widely found in nature, particularly in soil. It enters the body through cuts or abrasion of the skin. The Clostridium bacteria produces two types of neurotoxins. Both Clostridium botulinum and Clostridium tetani form the clostridial neurotoxin family [Krishnamurthy et al., 2005] and have high homology between them. This family is classified as part of the peptidase M27 family of proteins, which are metalloproteases. Metalloproteases bind with a divalent cation, usually zinc, which activates water molecules within the active site to hydrolyze peptide bonds [PDB]. This neurotoxin is catalytically classified as a hydrolase. The active site forms a nucleophilic water, which cleaves a bond of the substrate.

The tetanus toxin effects the central nervous system by inhibiting the release of neurotransmitters, glycine and gamma-aminobutyric acid, into the synaptic cleft of the spinal cord affecting the transmission of nerve impulses throughout the body. This toxin causes tetanus, which is characterized by rigidity, spasms, and paralysis of the voluntary muscles of the body [Krishnamurthy et al., 2005]. Tetanus is often referred to as "lockjaw" because a large majority of the patients experience rigidity of the jaw muscles. The toxin enters the body by way of a cut and into the bloodstream, where it spreads rapidly throughout the body, or by a nerve, which transports the toxin directly to the central nervous system. Tetanus toxin attacks motor nerve cells and hyper-activates them. The overactive nerve impulses cause muscles to go into convulsive spasms.

Although vaccines and treatments have been developed for tetanus, it is still a potent neurological disease around the world.

Structure

The precursor polypeptide of the tetanus toxin is cleaved during post-translational modification into a heavy and light chains. These two chains remain linked by a disulfide bridge. The heavy chain is the C-terminal end of the protein and the light chain is the N-terminal end of the protein. If the two chains are separated, the toxin becomes non-toxic [PDB].

The tetanus toxin is composed of 468 amino acid residues. The secondary structures present are alpha helices, 3^10 helices, and beta sheets. Twenty-seven percent of the secondary structures are , which includes a total of 13 helices (127 residues). The make up 17% of the secondary structures in this neurotoxin. This includes 25 strands consists 81 residues [PDB].

Tetanus toxin has three functional domains: binding, translocation, and catalytic. The heavy chain is responsible for binding the toxin to the specific neural receptors and translocating the catalytic light chain domain into the neural cytosol. The light chain is the zinc-binding domain containing a zinc-binding motif. This metalloprotease activity causes toxicity [Krishnamurthy et al., 2005]. The light chain forms a dimer with about 10% of the protein surface existing between the two monomers. Each monomer binds one . The active sites of the light chain tetanus toxin interact with the solvent region and are embedded inside a cavity centered around a zinc cation and the conserved zinc-dependent motif. Zinc directly coordinates with His232, His236, and Glu270 within the zinc-dependent . Water is another ligand that forms a hydrogen bond with Glu233. The formation of the nucleophilic water molecule and the three other amino acid residues in the tetanus toxin active site form a tetrahedral configuration around the catalytic zinc ion. This interaction between the residues, water, and zinc are essential for the formation of nucleophilic water, which hydrolyzes the peptide bonds of the substrate. There is also a secondary layer important to the functionality of the active site. These residues are in the surrounding structure, approximately 10 Angstroms from the zinc ion, and they include Glu233, His239, Phe274, Arg371, and Tyr374. These residues reinforce the stability and conformation of the active site.

Mechanism of Action

Tetanus toxin binds to the neural cells through gangliosides and a second protein receptor. Once bound, they enter the cytosol via a vesicle membrane. Here, they attack and cleave the proteins that forms the synaptic vesicle fusion apparatus [Krishnamurthy et al., 2005]. The clostridial neurotoxins each have unique binding sites and substrate bond cleavage specificity. Tetanus toxin cleaves vesicle-associated membrane proteins. The VAMP protein is cleaved at the peptide bond Gln76-Phe77 requiring a amino-terminal extension of 22 residues and a peptide of 33-97 residues in length [Kirshnamurthy et al., 2005].

The toxin is produced after the bacterial cell's active, exponential growth phase.

Medical Implications or Possible Applications

Tetanus toxin is still a main concern to public health taking several hundred lives each year. The most commonly used human vaccine is the chemically modified form of the tetanus neurotoxin.

The toxin is most commonly known to affect the muscles of the jaw causing rigidity of the muscles of the jaw and face. This toxin also causes severe spasms in the throat and chest making swallowing and breathing extremely difficult. These are the most common causes of death if tetanus is untreated. Tetanus also causes adverse effects on various muscles throughout the body, notably on the heart, blood pressure, and vital brain centers that cause death later in the disease.