Structural and functional characterization of suramin-bound MjTX-I from Bothrops moojeni suggests a particular myotoxic mechanism.

Local myonecrosis is the main event resulting from snakebite envenomation by the Bothrops genus and, frequently, it is not efficiently neutralized by antivenom administration. Proteases, phospholipases A2 (PLA2) and PLA2-like toxins are found in venom related to muscle damage. Functional sites responsible for PLA2-like toxins activity have been proposed recently; they consist of a membrane docking-site and a membrane rupture-site. Herein, a combination of functional, biophysical and crystallographic techniques was used to characterize the interaction between suramin and MjTX-I (a PLA2-like toxin from Bothrops moojeni venom). Functional in vitro neuromuscular assays were performed to study the biological effects of the protein-ligand interaction, demonstrating that suramin neutralizes the myotoxic effect of MjTX-I. Calorimetric assays showed two different binding events: (i) inhibitor-protein interactions and (ii) toxin oligomerization processes. These hypotheses were also corroborated with dynamic light and small angle X-ray scattering assays. The crystal structure of the MjTX-I/suramin showed a totally different interaction mode compared to other PLA2-like/suramin complexes. Thus, we suggested a novel myotoxic mechanism for MjTX-I that may be inhibited by suramin. These results can further contribute to the search for inhibitors that will efficiently counteract local myonecrosis in order to be used as an adjuvant of conventional serum therapy.

Structural evidence for a fatty acid-independent myotoxic mechanism for a phospholipase A2-like toxin.

The myotoxic mechanism for PLA2-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 PLA2-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 PLA2-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 PLA2-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 PLA2-like toxins in general.

Crystal structure of a phospholipase A2 from Bothrops asper venom: Insights into a new putative "myotoxic cluster".

Snake venoms from the Viperidae and Elapidae families often have several phospholipases A2 (PLA2s), 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. PLA2s from these venoms are generally divided into two classes: (i) catalytic PLA2s (or Asp49-PLA2s) and (ii) non-catalytic PLA2-like toxins (or Lys49-PLA2s). In many Viperidae venoms, a subset of the basic Asp49-PLA2s displays some functional and structural characteristics of PLA2-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-PLA2 isolated from Bothrops asper venom. The structure presents a dimeric conformation that is compatible with that of previous dimers found for basic myotoxic Asp49-PLA2s and Lys49-PLA2s 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-PLA2s, 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-PLA2s from Viperidae venoms. Such arrangement may represent a novel structural strategy for the mechanism of muscle damage exerted by the group of basic, Asp49-PLA2s found in viperid snake venoms.

Structural and functional evidence for membrane docking and disruption sites on phospholipase A2-like proteins revealed by complexation with the inhibitor suramin.

Local myonecrosis resulting from snakebite envenomation is not efficiently neutralized by regular antivenom administration. This limitation is considered to be a significant health problem by the World Health Organization. Phospholipase A2-like (PLA2-like) proteins are among the most important proteins related to the muscle damage resulting from several snake venoms. However, despite their conserved tertiary structure compared with PLA2s, their biological mechanism remains incompletely understood. Different oligomeric conformations and binding sites have been identified or proposed, leading to contradictory data in the literature. In the last few years, a comprehensive hypothesis has been proposed based on fatty-acid binding, allosteric changes and the presence of two different interaction sites. In the present study, a combination of techniques were used to fully understand the structural-functional characteristics of the interaction between suramin and MjTX-II (a PLA2-like toxin). In vitro neuromuscular studies were performed to characterize the biological effects of the protein-ligand interaction and demonstrated that suramin neutralizes the myotoxic activity of MjTX-II. The high-resolution structure of the complex identified the toxin-ligand interaction sites. Calorimetric assays showed two different binding events between the protein and the inhibitor. It is demonstrated for the first time that the inhibitor binds to the surface of the toxin, obstructing the sites involved in membrane docking and disruption according to the proposed myotoxic mechanism. Furthermore, higher-order oligomeric formation by interaction with interfacial suramins was observed, which may also aid the inhibitory process. These results further substantiate the current myotoxic mechanism and shed light on the search for efficient inhibitors of the local myonecrosis phenomenon.

Structural and functional studies with mytoxin II from Bothrops moojeni reveal remarkable similarities and differences compared to other catalytically inactive phospholipases A2-like.

Lys49-phospholipases A2 (Lys49-PLA2s) are proteins found in bothropic snake venoms (Viperidae family) and belong to a class of proteins which presents a phospholipase A2 scaffold but are catalytically inactive. These proteins (also known as PLA2s-like toxins) exert a pronounced local myotoxic effect and are not neutralized by antivenom, being their study relevant in terms of medical and scientific interest. Despite of the several studies reported in the literature for this class of proteins only a partial consensus has been achieved concerning their functional-structural relationships. In this work, we present a comprehensive structural and functional study with the MjTX-II, a dimeric Lys49-PLA2 from Bothrops moojeni venom which includes: (i) high-resolution crystal structure; (ii) dynamic light scattering and bioinformatics studies in order to confirm its biological assembly; (iii) myographic and electrophysiological studies and, (iv) comparative studies with other Lys49-PLA2s. These comparative analyses let us to get important insights into the role of Lys122 amino acid, previously indicated as responsible for Lys49-PLA2s catalytic inactivity and added important elements to establish the correct biological assembly for this class of proteins. Furthermore, we show two unique sequential features of MjTX-II (an amino acid insertion and a mutation) in comparison to all bothropic Lys49-PLA2s that lead to a distinct way of ligand binding at the toxin's hydrophobic channel and also, allowed the presence of an additional ligand molecule in this region. These facts suggest a possible particular mode of binding for long-chain ligands that interacts with MjTX-II hydrophobic channel, a feature that may directly affect the design of structure-based ligands for Lys49-PLA2s.

Structural and functional studies of a bothropic myotoxin complexed to rosmarinic acid: new insights into Lys49-PLA2 inhibition.

Snakebite envenoming is an important public health problem in many tropical and subtropical countries, and is considered a neglected tropical disease by the World Health Organization. Most severe cases are inflicted by species of the families Elapidae and Viperidae, and lead to a number of systemic and local effects in the victim. One of the main problems regarding viperidic accidents is prominent local tissue damage whose pathogenesis is complex and involves the combined actions of a variety of venom components. Phospholipases A2 (PLA2s) are the most abundant muscle-damaging components of these venoms. Herein, we report functional and structural studies of PrTX-I, a Lys49-PLA2 from Bothops pirajai snake venom, and the influence of rosmarinic acid (RA) upon this toxin's activities. RA is a known active component of some plant extracts and has been reported as presenting anti-myotoxic properties related to bothopic envenomation. The myotoxic activity of Lys49-PLA2s is well established in the literature and although no in vivo neurotoxicity has been observed among these toxins, in vitro neuromuscular blockade has been reported for some of these proteins. Our in vitro studies show that RA drastically reduces both the muscle damage and the neuromuscular blockade exerted by PrTX-I on mice neuromuscular preparations (by ∼80% and ∼90%, respectively). These results support the hypothesis that the two effects are closely related and lead us to suggest that they are consequences of the muscle membrane-destabilizing activity of the Lys49-PLA2. Although the C-terminal region of these proteins has been reported to comprise the myotoxic site, we demonstrate by X-ray crystallographic studies that RA interacts with PrTX-I in a different region. Consequently, a new mode of Lys49-PLA2 inhibition is proposed. Comparison of our results with others in the literature suggests possible new ways to inhibit bothropic snake venom myotoxins and improve serum therapy.

Structural, functional, and bioinformatics studies reveal a new snake venom homologue phospholipase A2 class.

Phospholipases A2 (PLA2s) are enzymes responsible for membrane disruption through Ca(2+) -dependent hydrolysis of phospholipids. Lys49-PLA2s are well-characterized homologue PLA2s that do not show catalytic activity but can exert a pronounced local myotoxic effect. These homologue PLA2s were first believed to present residual catalytic activity but experiments with a recombinant toxin show they are incapable of catalysis. Herein, we present a new homologue Asp49-PLA2 (BthTX-II) that is also able to exert muscle damage. This toxin was isolated in 1992 and characterized as presenting very low catalytic activity. Interestingly, this myotoxic homologue Asp49-PLA2 conserves all the residues responsible for Ca(2+) coordination and of the catalytic network, features thought to be fundamental for PLA2 enzymatic activity. Previous crystallographic studies of apo BthTX-II suggested this toxin could be catalytically inactive since a distortion in the calcium binding loop was observed. In this article, we show BthTX-II is not catalytic based on an in vitro cell viability assay and time-lapse experiments on C2C12 myotube cell cultures, X-ray crystallography and phylogenetic studies. Cell culture experiments show that BthTX-II is devoid of catalytic activity, as already observed for Lys49-PLA2s. Crystallographic studies of the complex BthTX-II/Ca(2+) show that the distortion of the calcium binding loop is still present and impairs ion coordination even though Ca(2+) are found interacting with other regions of the protein. Phylogenetic studies demonstrate that BthTX-II is more phylogenetically related to Lys49-PLA2s than to other Asp49-PLA2s, thus allowing Crotalinae subfamily PLA2s to be classified into two main branches: a catalytic and a myotoxic one.

Crystallization and preliminary X-ray crystallographic studies of a Lys49-phospholipase A2 homologue from Bothrops pirajai venom complexed with rosmarinic acid.

PrTX-I, a noncatalytic and myotoxic Lys49-phospholipase A(2) from Bothrops pirajai venom, was crystallized in the presence of the inhibitor rosmarinic acid (RA). This is the active compound in the methanolic extract of Cordia verbenacea, a plant that is largely used in Brazilian folk medicine. The crystals diffracted X-rays to 1.8 A resolution and the structure was solved by molecular-replacement techniques, showing electron density that corresponds to RA molecules at the entrance to the hydrophobic channel. The crystals belong to space group P2(1)2(1)2(1), indicating conformational changes in the structure after ligand binding: the crystals of all apo Lys49-phospholipase A(2) structures belong to space group P3(1)21, while the crystals of complexed structures belong to space groups P2(1) or P2(1)2(1)2(1).

Influence of quaternary conformation on the biological activities of the Asp49-phospholipases A2s from snake venoms.

One of the main components of snake venoms are the Asp49-phospholipases A(2), also known as svPLA(2)s. The study of these toxins is a matter of great scientific interest due to their wide variety of biological effects. In this work we present strong evidences found in literature and other aspects which strengthen the importance of quaternary assembly for understanding the activities and molecular evolution of svPLA(2)s.

The intriguing phospholipases A2 homologues: relevant structural features on myotoxicity and catalytic inactivity.

Phospholipases A(2) homologues are found in the venom of Crotalinae snakes, being their main action related to myonecrosis induction. Although many studies on these toxins had already been performed, their mechanism of action remains unclear. Here, important aspects about these toxins are reviewed, including their correct biological assembly and how essential is the natural substitution D49K for their catalytic inactivity.

Comparative structural studies on Lys49-phospholipases A(2) from Bothrops genus reveal their myotoxic site.

Phospholipases A(2) (PLA(2)s) are membrane-associated enzymes that hydrolyze phospholipids at the sn-2 position, releasing lysophospholipids and free fatty acids. Phospholipase A(2) homologues (Lys49-PLA(2)s) are highly myotoxic and cause extensive tissue damage despite not showing measurable catalytic activity. They are found in different snake venoms and represent one third of bothropic venom composition. The importance of these toxins during envenomation is related to the pronounced local myotoxic effect they induce since this effect is not neutralized by serum therapy. We present herein three structures of Lys49-PLA(2)s from Bothrops genus snake venom crystallized under the same conditions, two of which were grown in the presence of alpha-tocopherol (vitamin E). Comparative structural analysis of these and other Lys49-PLA(2)s showed two different patterns of oligomeric conformation that are related to the presence or absence of ligands in the hydrophobic channel. This work also confirms the biological dimer indicated by recent studies in which both C-termini are in the dimeric interface. In this configuration, we propose that the myotoxic site of these toxins is composed by the Lys 20, Lys115 and Arg118 residues. For the first time, a residue from the short-helix (Lys20) is suggested as a member of this site and the importance of Tyr119 residue to myotoxicity of bothropic Lys49-PLA(2)s is also discussed. These results support a complete hypothesis for these PLA(2)s myotoxic activity consistent with all findings on bothropic Lys49-PLA(2)s studied up to this moment, including crystallographic, bioinformatics, biochemical and biophysical data.

Structural and functional properties of Bp-LAAO, a new L-amino acid oxidase isolated from Bothrops pauloensis snake venom.

An L-amino acid oxidase (Bp-LAAO) from Bothrops pauloensis snake venom was highly purified using sequential chromatography steps on CM-Sepharose, Phenyl-Sepharose CL-4B, Benzamidine Sepharose and C18 reverse-phase HPLC. Purified Bp-LAAO showed to be a homodimeric acidic glycoprotein with molecular weight around 65kDa under reducing conditions in SDS-PAGE. The best substrates for Bp-LAAO were L-Met, L-Leu, L-Phe and L-Ile and the enzyme showed a strong reduction of its catalytic activity upon L-Met and L-Phe substrates at extreme temperatures. Bp-LAAO showed leishmanicidal, antitumoral and bactericidal activities dose dependently. Bp-LAAO induced platelet aggregation in platelet-rich plasma and this activity was inhibited by catalase. Bp-LAAO-cDNA of 1548bp codified a mature protein with 516 amino acid residues corresponding to a theoretical isoelectric point and molecular weight of 6.3 and 58kDa, respectively. Additionally, structural and phylogenetic studies identified residues under positive selection and their probable location in Bp-LAAO and other snake venom LAAOs (svLAAOs). Structural and functional investigations of these enzymes can contribute to the advancement of toxinology and to the elaboration of novel therapeutic agents.

Preliminary X-ray crystallographic studies of a Lys49-phospholipase A2 homologue from Bothrops pirajai venom complexed with p-bromo-phenacyl bromide and alpha-tocopherol inhibitors.

PrTX-I, a non-catalytic and myotoxic Lys49-PLA(2) from Bothrops pirajai venom has been crystallized alone and in complex with bromophenacyl bromide (BPB), alpha tocopherol and alpha tocopherol acetate inhibitors. These crystals have shown to diffract X-rays between 2.34 and 1.65 A resolution. All complexes crystals are isomorphous and belong to the space group P2(1) whereas native PrTX-I crystals belong to the P3(1)21.

Crystallization and preliminary X-ray diffraction studies of two myotoxic Lys49-phospholipases A2 complexed with alpha-tocopherol.

BnSP-7 and BnSP-6, two Lys49-phospholipase A2 isolated from Bothrops neuwiedi pauloensis snake venom, were co-crystallized with alpha-tocopherol and X-ray diffraction data were collected for both complexes (2.2 and 2.6 A). A new "alternative" quaternary conformation for these two complexes compared with all other dimeric Lys49-PLA2 has been observed.

Crystallization and preliminary X-ray diffraction analysis of an acidic phospholipase A(2) complexed with p-bromophenacyl bromide and alpha-tocopherol inhibitors at 1.9- and 1.45-A resolution.

An acidic phospholipase A(2) (PLA(2)) isolated from Bothrops jararacussu snake venom was crystallized with two inhibitors: alpha-tocopherol (vitamin E) and p-bromophenacyl bromide (BPB). The crystals diffracted at 1.45- and 1.85-A resolution, respectively, for the complexes with alpha-tocopherol and p-bromophenacyl bromide. The crystals are not isomorphous with those of the native protein, suggesting the inhibitors binding was successful and changes in the quaternary structure may have occurred.