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The myotoxic mechanism for PLA 2 -like toxins has been proposed recently to be initiated by an allosteric change induced by a fatty acid binding to the protein, leading to the alignment of the membrane docking site (MDoS) and membrane disrupting site (MDiS). Previous structural studies performed by us demonstrated that MjTX-II, a PLA 2 -like toxin isolated from Bothrops moojeni , presents a different mode of ligand-interaction caused by natural amino acid substitutions and an insertion. Herein, we present four crystal structures of MjTX-II, in its apo state and complexed with fatty acids of different lengths. Analyses of these structures revealed slightly different oligomeric conformations but with both MDoSs in an arrangement that resembles an active-state PLA 2 -like structure. To explore the structural transitions between apo protein and fatty-acid complexes, we performed Normal Mode Molecular Dynamics simulations, revealing that oligomeric conformations of MjTX-II/fatty acid complexes may be reached in solution by the apo structure. Similar simulations with typical PLA 2 -like structures demonstrated that this transition is not possible without the presence of fatty acids. Thus, we hypothesize that MjTX-II does not require fatty acids to be active, although these ligands may eventually help in its stabilization by the formation of hydrogen bonds. Therefore, these results complement previous findings for MjTX-II and help us understand its particular ligand-binding properties and, more importantly, its particular mechanism of action, with a possible impact on the design of structure-based inhibitors for PLA 2 -like toxins in general. [ABSTRACT FROM AUTHOR]

Snake venoms from the Viperidae and Elapidae families often have several phospholipases A 2 (PLA 2 s), which may display different functions despite having a similar structural scaffold. These proteins are considered an important target for the development of drugs against local myotoxic damage because they are not efficiently neutralized by conventional serum therapy. PLA 2 s from these venoms are generally divided into two classes: (i) catalytic PLA 2 s (or Asp49-PLA 2 s) and (ii) non-catalytic PLA 2 -like toxins (or Lys49-PLA 2 s). In many Viperidae venoms, a subset of the basic Asp49-PLA 2 s displays some functional and structural characteristics of PLA 2 -like proteins and group within the same phylogenetic clade, but their myotoxic mechanism is still largely unknown. In the present study, we have crystallized and solved the structure of myotoxin I (MT-I), a basic myotoxic Asp49-PLA 2 isolated from Bothrops asper venom. The structure presents a dimeric conformation that is compatible with that of previous dimers found for basic myotoxic Asp49-PLA 2 s and Lys49-PLA 2 s and has been confirmed by other biophysical and bioinformatics techniques. This arrangement suggests a possible cooperative action between both monomers to exert myotoxicity via two different sites forming a putative membrane-docking site (MDoS) and a putative membrane disruption site (MDiS). This mechanism would resemble that proposed for Lys49-PLA 2 s, but the sites involved appear to be situated in a different region. Thus, as both sites are close to one another, they form a “myotoxic cluster”, which is also found in two other basic myotoxic Asp49-PLA 2 s from Viperidae venoms. Such arrangement may represent a novel structural strategy for the mechanism of muscle damage exerted by the group of basic, Asp49-PLA 2 s found in viperid snake venoms. [ABSTRACT FROM AUTHOR]

Abstract Background Specific compounds found in vegetal species have been demonstrated to be efficient inhibitors of snake toxins, such as phospholipase A 2 -like (PLA 2 -like) proteins. These particular proteins, present in several species of vipers (Viperidae), induce a severe local myotoxic effect in prey and human victims, and this effect is often not efficiently neutralized by the regular serum therapy. PLA 2 -like proteins have been functionally and structurally studied since the early 1990s; however, a comprehensive molecular mechanism was proposed only recently. Methods Myographic and histological techniques were used to evaluate the inhibitory effect of chicoric acid (CA) against BthTX-I myotoxin. Isothermal titration calorimetry assays were used to measure the affinity between the inhibitor and the toxin. X-ray crystallography was used to reveal details of this interaction. Results CA prevented the blockade of indirectly evoked muscle contraction and inhibited muscle damage induced by BthTX-I. The inhibitor binds to the toxin with the highest affinity measured for a natural compound in calorimetric assays. The crystal structure and molecular dynamics simulations demonstrated that CA binds at the entrance of the hydrophobic channel of the toxin and binds to one of the clusters that participates in membrane disruption. Conclusions CA prevents the myotoxic activity of the toxin, preventing its activation by simultaneous binding with two critical regions. General significance CA is a potential myotoxic inhibitor to other PLA 2 -like proteins and a possible candidate to complement serum therapy. Highlights • Chicoric acid offers a complete protection against myotoxic effect of the toxin. • The inhibitor binds to the toxin with high affinity measured for a natural compound. • The inhibitor blocks the hydrophobic channel access of the toxin. • The inhibitor interacts with the membrane disruption site of the toxin. • The present study supports the current myotoxic mechanism. [ABSTRACT FROM AUTHOR]