Bothrops moojeni MjTX-II Myotoxin
Russell Thomas '26 and Maria Peacock '26
Contents:
I. Introduction
The Bothrops moojeni MjTX-II protein is a sPLA2 (secreted phospholipase A2) homologue found in Bothrops moojeni venom that functions as a myotoxin. This snake PLA2-like toxin induces cell necrosis via interaction with the fatty acids of cell membranes, with these interactions precipitating a full disruption of membrane stability. While one PLA2-like protein within the Bothrops moojeni venom - Asp49-PLA2 - displays cytotoxicity through its catalytic ability, MjTX-II (a Lys49-PLA2) has not been shown to demonstrate that same catalytic ability. Instead of phospholipid hydrolysis, this Lys49-PLA2-like protein binds fatty acid substrates and physically transfigures the phospholipid membrane.
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II. General Structure
MjTX II is a homodimer, with
133 Amino Acid Residues per monomer.
Each monomer has a bundle of 5 Alpha-Helices and 2 Beta-Sheets as well as some regions with no secondary structure
MjTX-II has two distinct sites that contribute to its myotoxicity:
The Membrane Docking site is composed of a Tyrosine, Arginine, and two Lysine residues. These residues' positively charged molecules interact with the negatively charged phosphate groups in cellular membranes and serve as an attachment point.
The Membrane-disrupting site consists of Proline, Alanine, and Leucine residues located near the C-Terminal domain. These hydrophobic residues interact with the hydrophobic tails of the cell membrane, inciting poration.
III. Substrate Binding in the "Active Site"
MjTX-II has a binding site referred to as the active site, although it displays no catalytic ability. Instead, this site binds its targeted substrate: the phospholipid molecules of the membrane. Stearic Acid molecules are crystallized with the toxin by Watanabe et al. (2014) to imitate the substrate-bound conformation.
This active site is nested between two Alpha-Helices, which are held together by a disulfide bond between Cysteine residues 44 and 105. This active site binds two stearic acid molecules per monomer.
The active site is further reinforced by interactions between three residues: Histidine 48, Aspartate 99, and Tyrosine 52, which are clustered at the crux of the linked alpha helices and form a network of hydrogen bonds.
Histidine 48 also participates in a hydrogen bond with the oxygen in the "head" of the stearic acid molecule, which is also hydrogen bonded to the amino group of Glycine 30 and the carbonyl oxygen of Asparagine 28. In addition, the terminal nitrogen of Lysine 49 provides stablizing hydrogen bonds between the Glycine and the Asparagine.
Many hydrophobic residues, from distant regions of the polypeptide chain, are brought together by the protein's secondary structure to form a hydrophobic "entrance channel" to allow the fatty acid substrates to enter into this active site.
IV. Basis for Myotoxicity
This structure for MjTX II was determined using crystallography, and the crystallized dimer asssumes a conformation termed the "Conventional Dimer"" by dos Santos et al. (2009).
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. However, within the aqueous intra- and inter-cellular environment, the MjTX II dimer assumes a different conformation, termed the "Alternative Dimer"
This conformation has both membrane docking sites much closer together.
These membrane docking sites form part of the dimerization surface, and the site is stabilized by hydrogen bonds between the hydroxyl groups of both Tyrosine 109 residues.
Myotoxicity is a direct result of MjTX II's disruption of cell membranes.
The polar "fingers" of the membrane docking site insert themselves between the charged phosphate groups of the phospholipid bilayer, positioning the membrane disrupting sites above the membrane surface.
When a fatty acid enters the hydrophobic entrance channel and binds to the active site, a conformational change occurs wherein the membrane disrupting sites are pushed closer to the membrane surface. These nonpolar residues interact with the nonpolar "tails" of the phospholipids.
The combination of the membrane docking site and membrane disrupting site interacting with the cell membrane causes severe poration, which allows leakage of essential ions and other cytosolic contents, and eventually leads to cell death.
V. References
Chioato, Lucimara, De Oliveira, Arthur H., Ruller, Roberto, Sá, Juliana M., Ward, Richard J. 2002. Distinct sites for myotoxic and membrane-damaging activities in the C-terminal region of a Lys49-phospholipase A2. Biochemical Journal 366:971-976. Dennis, Edward A., Cao, Jian, Hsu, Yuan-Hao, Magrioti, Victoria, Kokotos, George. 2011. Phospholipase A2 Enzymes: Physical Structure, Biological Function, Disease Implication, Chemical Inhibition, and Therapeutic Intervention. Chemical Reviews 111:6130-6185
dos Santos, Juliana I., Soares, Andreimar Martins, Fontes, Marcos R.M. 2009. Comparative structural studies on Lys49-phospholipases A2 from Bothrops genus reveal their myotoxic site. Journal of Structural Biology 167:106-116.
Fernandes, Carlos A.H., Comparetti, Edson J., Borges, Rafael J., Huancahuire-Vega, Salomon, Ponce-Soto, Luis Alberto, Marangoni, Sergio, Soares, Andreimar M., Fontes, Marcos R.M. 2013. Structural bases for a complete myotoxic mechanism: Crystal structures of two non-catalytic phospholipases A2-like from Bothrops brazili venom. Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics 1834:2772-2778.
Fernandes, Carlos A.H., Borges, Rafael J, Lomonte, Bruno, Fontes, Marcos R.M. 2014. A structure-based proposal for a comprehensive myotoxic mechanism of phospholipase A2-like proteins from viperid snake venoms. Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics
1844:2265-2276.
Watanabe, Leandra, Soares, Andreimar M.,Ward, Richard J., Fontes, Marcos R.M., Arni, Raghuvir. K. 2005. Structural insights for fatty acid binding in a Lys49-phospholipase A2: crystal structure of myotoxin II from Bothrops moojeni complexed with stearic acid. Biochimie 87:161-167
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