Structure of a calcium-independent phospholipase-like myotoxic protein from Bothrops asper venom

Myotoxin II, a myotoxic calcium-independent phospholipase-like protein isolated from the venom of Bothrops asper, possesses no detectable phospholipase activity. The crystal structure has been determined and refined at 2.8 A to an R-factor of 16.5% (F > 3sigma) with excellent stereochemistry. Amino-acid differences between catalytically active phospholipases and myotoxin II in the Ca(2+)-binding region, specifically the substitutions Tyr28-->Asn, Gly32-->Leu and Asp49-->Lys, result in an altered local conformation. The key difference is that the epsilon-amino group of Lys49 fills the site normally occupied by the calcium ion in catalytically active phospholipases. In contrast to the homologous monomeric Lys49 variant from Agkistrodon piscivorus piscivorus, myotoxin II is present as a dimer both in solution and in the crystalline state. The two molecules in the asymmetric unit are related by a nearly perfect twofold axis, yet the dimer is radically different from the dimer formed by the phospholipase from Crotalus atrox. Whereas in C. atrox the dimer interface occludes the active sites, in myotoxin II they are exposed to solvent.UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP

Rapid purification of serine proteinases from Bothrops alternatus and Bothrops moojeni venoms

AbstractEnvenomation by Bothrops species results, among other symptoms, in hemostatic disturbances. These changes can be ascribed to the presence of enzymes, primarily serine proteinases some of which are structurally similar to thrombin and specifically cleave fibrinogen releasing fibrinopeptides. A rapid, three-step, chromatographic procedure was developed to routinely purify serine proteinases from the venoms of Bothrops alternatus and Bothrops moojeni. The serine proteinase from B. alternatus displays an apparent molecular mass of ∼32 kDa whereas the two closely related serine proteinases from B. moojeni display apparent molecular masses of ∼32 kDa and ∼35 kDa in SDS–PAGE gels. The partial sequences indicated that these enzymes share high identity with serine proteinases from the venoms of other Bothrops species. These proteins coagulate plasma and possess fibrinogenolytic activity but lack fibrinolytic activity

Exfoliative Toxins of Staphylococcus aureus

Virulent strains of Staphylococcus aureus secrete exfoliative toxins (ETs) that cause the loss of cell‐cell adhesion in the superficial epidermis. S. aureus ETs are serine proteases, which exhibit exquisite substrate specificity, and their mechanisms of action are extremely complex. To date, four different serotypes of ETs have been identified and three of them (ETA, ETB and ETD) are associated with toxin‐mediated staphylococcal syndromes related to human infections leading to diseases of medical and veterinary importance

Crystallization and preliminary diffraction data of two myotoxins isolated from the venoms of Bothrops asper (Terciopelo) and Bothrops nummifer (jumping viper)

Two myotoxins isolated from B. asper (myotoxin II) and B. nummifer (myotoxin I) snake venoms have been crystallized and their diffraction properties are described. These myotoxins are phospholipase A2 variants which lack enzymatic activity; B. asper myotoxin II is a lysine-49 phospholipase. Crystals were obtained at room temperature by standard hanging-drop vapour diffusion methods. Crystals diffracted to a resolution of 2.8 and 2.3 A, respectively.UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP

Proteome analysis of snake venom toxins: pharmacological insights

Snake venoms are an extremely rich source of pharmacologically active proteins with a considerable clinical and medical potential. To date, this potential has not been fully explored, mainly because of our incomplete knowledge of the venom proteome and the pharmacological properties of its components, in particular those devoid of enzymatic activity. This review summarizes the latest achievements in the determination of snake venom proteome, based primarily on the development of new strategies and techniques. Detailed knowledge of the venom toxin composition and biological properties of the protein constituents should provide the scaffold for the design of new more effective drugs for the treatment of the hemostatic system and heart disorders, inflammation, cancer and consequences of snake bites, as well as new tools for clinical diagnostic and assays of hemostatic parameters

Structure of thrombin complexed with selective non-electrophilic inhibitors having cyclohexyl moieties at P1

The crystal structures of five new non-electrophilic β-strand-templated thrombin active-site inhibitors have been determined bound to the enzyme. Four co-crystallize with hirugen and inhibitor isomorphously to produce thrombin-hirugen crystals (monoclinic, space group C2), while one co-crystallizes in the hexagonal system, space group P65. A 1,4-substituted cyclohexyl moiety is conserved at the P1 position of all the inhibitors, along with a fused hetero-bicyclic five- and six-membered ring that occupies the P2 site. Amino, amidino and aminoimidazole groups are attached to the cyclohexyl ring for recognition at the S1 specificity site, while benzylsulfonyl and diphenyl groups enhance the binding at the S3 subsite. The cyclohexyl groups at the P1 positions of three of the inhibitors appear to be in the energetically favored chair conformation, while the imidazole-substituted cyclohexyl rings are in a boat conformation. Somewhat unexpectedly, the two cyclohexyl-aminoimidazole groups bind differently in the specificity site; the unique binding of one is heretofore unreported. The other inhibitors generally mimic arginyl binding at S1. This group of inhibitors combines the nonelectrophilicity and selectivity of DAPA-like compounds and the more optimal binding features of the S1-S3 sites of thrombin for peptidic molecules, which results in highly potent (binding constants 12 nM-16 pM, one being 1.1 μM) and selective (ranging from 140 to 20 000 times more selective compared with trypsin) inhibitors of thrombin. The binding modes of these novel inhibitors are correlated with their binding constants, as is their selectivity, in order to provide further insight for the design of therapeutic antithrombotic agents that inhibit thrombin directly at the active site

Crystallization and preliminary X-ray diffraction analysis of a novel Arg49 phospholipase A2 homologue from Zhaoermia mangshanensis venom

A single crystal of zhaoermiatoxin with maximum dimensions of 0.2 × 0.2 × 0.5 mm was used for X-ray diffraction data collection to a resolution of 2.05 Å using synchrotron radiation and the diffraction pattern was indexed in the hexagonal space group P64, with unit-cell parameters a = 72.9, b = 72.9, c = 93.9 Å