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Cholix Toxin from Vibrio cholerae

Introduction

Cholix toxin (CT) are a class of protein toxin originating from the bacteria Vibrio cholerae. It is the third member of eEF2 specific ADP-ribosyltransferase toxins. The toxin uses ADP-riboyltransferase to inactivate eukaryotic elongation factor 2 by transferring ADP-ribose from NAD+ which inhibits protein synthesis and causes cell death. This protein toxin has been known to cause disease in both plants and animals. Specifically, the toxin can cause the disease Cholera. It enters eukaryotic cells through endocytosis. Once inside, the toxin transfers an ADP-ribose group to an Arg residue of the GTP binding protein G. This then activates adenylate cyclase which leads to an increase amount of cAMP, causing a secretion of Cl-,HCO3-, and water from epithelial cells from the site of colonization. The result is dehydration and loss of electrolytes in infected hosts. Cholix toxins are composed of a receptor binding, translocation, and catalytic domain.

Structure

X-Ray Crystallography

To observe the structure, the Cholix Toxin was crystallized by vapor diffusion against reservoirs containing 23% polyethlene gylcol-10,000, 7.5% ethylene glycol, and 0.1 m HERPES. In addition, the reservoirs were kept at a of pH 7.5 and at a temperature of 19°C. About 40 µL of reservior solution containing 1.25 mM NAD+ solution was added to a 2 µL crystal containing group. The NAD+ was allowed to soak into the crystals for approximately 2-3 minutes. The crystals were then transferred to paratone-N for visualization via X-Ray Diffraction

Protein Structure

The sequence of the Cholix Toxin is 666 amino acids in length. It contains a total of four disulfide bonds which are located between Cys-43:Cys-47, Cys-240:Cys: 257, Cys-310:Cys-332, and Cys-426:Cys-433. The secondary structure of the Cholix Toxin consists of 11 and 4 The Cholix Toxin contains three different domains. Though it is not shown, the active site for this toxin is located at Glu-613.

Molecule

The Cholix Toxin is a bipartite molecule consisting of 1 A subunit which further divides into two peptides: A1 and A2 and 5 B subunits. The A subunit is considered the active subunit while the B subunit serves to bind to GM1 ganglioside on the surfaces of intestinal epithelial cells. Both of these subunits are highly essential for the functionality of this toxin.

Mechanism of Action

Basic Mechanism

1. The Cholix Toxin binds to the GM1 gangliosides of host cell membrane.
2. Toxin gains access inside the cell through endocytosis.
3. CT-GM1 complex forms.
4. CT-GM1 complex travels through the Golgi cisternae and to the Endoplasmic Reticulum (ER).
5. A1 peptide of the toxin will unfold and dissociated from B pentamer.
6. The unfolded A1 is translocated to the cystol and will eventually leave the membrane after translocation.
7. A1 will move by diffusion to attack the adenylate cyclase complex.
8. The B subunit does not move across the membrane, rather it remains bound to the GM1 receptors.
9. The active subunit A1 uses its enzymatic ability to transfer ADP-ribose group to an Arg residue of the α subunit of the GTP binding protein Gs.
10. The Gs-α subunit becomes activated and breaks away from the Gs membrane-bound subunit and goes to attach itself to the catalytic site of the adenylate cyclase, activating this enzyme.
11. "The activation of adenylate cyclase causes increase in cAMP levels which inhibits Na+ and Cl- absorption and increases secretion of Cl- and HCO3- which leads to dehydration and loss of electrolytes in the infected host. The activation of adenylate cyclase also causes pore formation within the intestinal epithelial cell membrane which leads to cell death." ^[1]

ADP-ribosylation of Gs-α subunit of G-Proteins

Diseases

References

1. ↑ Fieldhouse, R., Jorgensen, R., Lugo, M., & Merrill, A. (2012). The 1.8a cholix toxin crystal structure in complex with NAD+ and evidence for a new kinetic model. 1-14.