Crystallization and preliminary diffraction data of a platelet-aggregation inhibitor from the venom of Agkistrodon piscivorus piscivorus (North American water moccasin)

Applaggin (Agkistrodon piscivorus piscivorus platelet-aggregation inhibitor) is a potent inhibitor of blood platelet aggregation derived from the venom of the North American water moccasin, the protein consists of 71 amino acids, is rich in cysteines, contains the sequence-recognition site of adhesion proteins at positions 50-52 (Arg-Gly-Asp) and shares high sequence homology with other snake-venom disintegrins such as echistatin, kistrin and trigramin, Single crystals of applaggin have been grown and X-ray diffraction data have been collected to a resolution of 3.2 Angstrom. The crystals belong to space group P4(1)2(1)2 (or its enantiomorph), with unit-cell dimensions a = b = 63.35, c = 74.18 Angstrom and two molecules per asymmetric unit. Molecular replacement using models constructed from the NMR structures of echistatin and kistrin has not been successful in producing a trial structure for applaggin.

Crystallization and preliminary X-ray diffraction analysis of a Lys49-PLA(2) homologue from Cerrophidion godmani venom.

Lys49-phospholipase A(2) (Lys49-PLA(2)) homologues damage membranes by a Ca2+-independent mechanism which does not involve catalytic activity. The myotoxic Lys-49 phospholipase myotoxin II from Cerrophidion (Bothrops) godmani has been crystallized, and X-ray diffraction data were collected to 2.8 Angstrom resolution. Preliminary analysis reveals the presence of one molecule in the asymmetric unit.

Amino acid sequence of a myotoxic Lys49-phospholipase A(2) homologue from the venom of Cerrophidion (Bothrops) godmani

The complete amino acid sequence of myotoxin II (godMT-II), a myotoxic phospholipase A( 2 )(PLA(2)) homologue from the venom of the Central American crotaline snake Cerrophidion (Bothrops) godmani, was determined by direct protein sequencing methods. GodMT-II is a class II PLA, showing a Lys instead of Asp at position 49. An additional substitution in the calcium binding loop region (Asn instead of Tyr at position 28) suggests the lack of enzymatic activity observed in this toxin is due to loss of its ability to bind the co-factor Ca2+, since the residues involved in forming the catalytic network of PLA(2)s (His-48, Tyr-52 and Asp-99) an conserved in godMT-II. This myotoxin shows highest sequence homology with other Lys-49 PLA(2)s from Bothrops, Agkistrodon and Trimeresurus species, suggesting that they constitute a conserved family of proteins, yet in contrast presents lower homology with Bothrops asper myotoxin III, a catalytically-active PLA(2). The C-terminal region of godMT-II, which is rich in cationic and hydrophobic residues, shares high sequence homology to the corresponding region in the myotoxin II from B. asper, which has been proposed to play an important role in the Ca2+-independent membrane damaging activity. (C) 1998 Elsevier B.V. B.V. All rights reserved.

Structure of a Lys49 phospholipase A(2) homologue isolated from the venom of Bothrops nummifer (jumping viper)

Lys49-Phospholipase A(2) (Lys49-PLA(2)) homologues damage membranes by a Ca2+-independent mechanism which does not involve catalytic activity, We have solved the structure of myotoxin-I, a Lys49-PLA(2) homologue isolated from the venom of Bothrops nummifer (jumping viper) at 2.4 Angstrom resolution using molecular replacement techniques. The final model has been refined to a final R-factor of 18.4% (R-free = 23.2%), and shows excellent geometry, the myotoxin-I from Bothrops nummifer is dimeric in the crystalline state as has been observed for other Lys49-PLA(2) homologues. In addition, a continuous electron density in the active site and substrate binding channel could be successfully modeled as a fatty-acid molecule. (C) 1999 Elsevier B.V. Ltd, All rights reserved.

Crystal structure of the platelet activator convulxin, a disulfide-linked alpha(4)beta(4) cyclic tetramer from the venom of Crotalus durissus terrificus

Convulxin (CVX), a C-type lectin, isolated from the venom of the South American rattlesnake Crotalus durissus terrificus, causes cardiovascular and respiratory disturbances and is a potent platelet activator which hinds to platelet glycoprotein GPVI. The structure of CVX has been solved at 2.4 Angstrom resolution to a crystallographic residual of 18.6% (R-free =26.4%). CVX is a disulfide linked heterodimer consisting of homologous alpha and beta chains. The heterodimers are additionally linked by disulfide bridges to form cyclic alpha(4)beta(4)heterotetramers. These domains exhibit significant homology to the carbohydrate-binding domains of C-type lectins, to the factor IX-binding protein (IX-bp), and to flavocetin-A (Fl-A) but sequence and Structural differences are observed in both the domains in the putative Ca2+ and carbohydrate binding regions. (C) 2003 Elsevier B.V. All rights reserved.

Thrombomodulin-independent activation of protein C and specificity of hemostatically active snake venom serine proteinases - Crystal structures of native and inhibited Agkistrodon contortrix contortrix protein C activator

Protein C activation initiated by the thrombin-thrombomodulin complex forms the major physiological anticoagulant pathway. Agkistrodon contortrix contortrix protein C activator, a glycosylated single-chain serine proteinase, activates protein C without relying on thrombomodulin. The crystal structures of native and inhibited Agkistrodon contortrix contortrix protein C activator determined at 1.65 and 1.54 angstrom resolutions, respectively, indicate the pivotal roles played by the positively charged belt and the strategic positioning of the three carbohydrate moieties surrounding the catalytic site in protein C recognition, binding, and activation. Structural changes in the benzamidine-inhibited enzyme suggest a probable function in allosteric regulation for the anion-binding site located in the C-terminal extension, which is fully conserved in snake venom serine proteinases, that preferentially binds Cl1- instead of SO42-.

Crystal structure of piratoxin-I: A calcium-independent, myotoxic phospholipase A(2)-homologue from Bothrops pirajai venom

The crystal structure of Piratoxin-I (PrTX-I) a Lys49 homologue isolated from the venom of Bothrops pirajai has been determined and refined at 2.8 Angstrom to a crystallographic residual of 19.7% (R-free = 29.7%). Amino-acid sequence differences between catalytically active phospholipases and PrTX-I in the putative Ca2+-binding loop, specifically the substitutions Tyr28-->Asn, Gly32-->Leu and Asp49-->Lys, result in an altered conformation of this loop, the analysis of the position of the E-amino group of Lys49 in the PrTX-I structure indicates that it fills the site normally occupied by the calcium ion in the catalytically active phospholipases, In contrast to the homologous monomeric Lys49 variant from Agkistrodon piscivorus piscivorus (App), PrTX-I is present as a dimer in the crystalline state, as observed in the structures of myotoxin II from Bothrops asper and Bothropstoxin I from Bothrops jararacussu. The two molecules in the asymmetric unit in the crystal structure of PrTX-I are related by a nearly perfect two-fold symmetry axis, yet the dimeric structure is radically different from the dimeric structure of the phospholipase from Crotalus atrox. In the C. atrox structure the dimer interface occludes the active sites, whereas in the PrTX-I structure they are exposed to solvent, (C) 1998 Elsevier B.V. Ltd. All rights reserved.

Crystallization of the Lys49 PLA(2) homologue, myotoxin II, from the venom of Atropoides nummifer

Myotoxin H, a Lys49 catalytically inactive phospholipase A(2) homologue from Atropoides nummifer venom, was purified, characterized and crystallized. The crystals belongs to the tetragonal system, space group P4(3)2(1)2, with unit cell parameters (a=b=68.66 and c=63.87 Angstrom). Diffraction data were collected to a resolution of 2.32 Angstrom. The crystal structure is currently being determined using molecular replacement techniques. (C) 2004 Elsevier B.V. All rights reserved.

Crystallization and preliminary X-ray crystallographic studies of Protac (R), a commercial protein C activator isolated from Agkistrodon contortrix contortrix venom

The protein C pathway plays an important role in the control and regulation of the blood coagulation cascade and prevents the propagation of the clotting process on the endothelium surface. In physiological systems, protein C activation is catalyzed by thrombin, which requires thrombomodulin as a cofactor. The protein C activator from Agkistrodon contortrix contortrix acts directly on the zymogen of protein C converting it into the active form, independently of thrombomodulin. Suitable crystals of the protein C activator from Agkistrodon contortrix contortrix were obtained from a solution containing 2 M ammonium sulfate as the precipitant and these crystals diffracted to 1.95 angstrom resolution at a synchrotron beamline. The crystalline array belongs to the monoclinic space group C2 with unit cell dimensions a=80.4, b = 63.3 and c = 48.2 angstrom, alpha = gamma = 90.0 degrees and beta = 90.8 degrees. (C) 2005 Elsevier B.V. All rights reserved.

Crotacetin, a novel snake venom C-type lectin homolog of convulxin, exhibits an unpredictable antimicrobial activity

Snake venom (sv) C-type lectins encompass a group of hemorrhagic toxins that are capable of interfering with blood stasis. A very well-studied svC-type lectin is the heterodimeric toxin, convulxin (CVX), from the venom of South American rattlesnake Crotalus durissus terrificus. CVX is able to activate platelets and induce their aggregation by acting via p62/GPVI collagen receptor. By using polymerase chain reaction homology screening, we have cloned several cDNA precursors of CVX subunit homologs. One of them, named crotacetin (CTC) beta-subunit, predicts a polypeptide with a topology very similar to the tridimensional conformations of other subunits of CVX-like snake toxins, as determined by computational analysis. Using gel permeation and reverse-phase high-performance liquid chromatography, CTC was purified from C. durissus venoms. CTC can be isolated from the venom of several C. durissus subspecies, but its quantitative predominance is in the venom of C. durissus cascavella. Functional analysis indicates that CTC induces platelet aggregation, and, importantly, exhibits an antimicrobial activity against Gram-positive and -negative bacteria, comparable with CVX.

Amino acid sequence and crystal structure of BaP1, a metalloproteinase from Bothrops asper snake venom that exerts multiple tissue-damaging activities

BaP1 is a 22.7-kD P-I-type zinc-dependent metalloproteinase isolated from the venom of the snake Bothrops asper, a medically relevant species in Central America. This enzyme exerts multiple tissue-damaging activities, including hemorrhage, myonecrosis, dermonecrosis, blistering, and edema. BaP1 is a single chain of 202 amino acids that shows highest sequence identity with metalloproteinases isolated front the venoms of snakes of the subfamily Crotalinae. It has six Cys residues involved in three disulfide bridges (Cys 117-Cys 197, Cys 159-Cys 181, Cys 157-Cys 164). It has the consensus sequence H(142)E(143)XXH(146)XXGXXH(152), as well as the sequence C164I165M166, which characterize the metzincin superfamily of metalloproteinases. The active-site cleft separates a major subdomain (residues 1-152), comprising four a-helices and a five-stranded beta-sheet, from the minor subdomain, which is formed by a single a-helix and several loops. The catalytic zinc ion is coordinated by the N-epsilon2 nitrogen atoms of His 142, His 146, and His 152, in addition to a solvent water molecule, which in turn is bound to Glu 143. Several conserved residues contribute to the formation of the hydrophobic pocket, and Met 166 serves as a hydrophobic base for the active-site groups. Sequence and structural comparisons of hemorrhagic and nonhemorrhagic P-I metalloproteinases from snake venoms revealed differences in several regions. In particular, the loop comprising residues 153 to 176 has marked structural differences between metalloproteinases with very different hemorrhagic activities. Because this region lies in close proximity to the active-site microenvironment, it may influence the interaction of these enzymes with physiologically relevant substrates in the extracellular matrix.

Crystallization and preliminary crystallographic analysis of SMase I, a sphingomyelinase from Loxosceles laeta spider venom

SMase I, a 32 kDa sphingomyelinase found in Loxosceles laeta venom, is responsible for the major pathological effects of spider envenomation. This toxin has been cloned and functionally expressed as a fusion protein containing a 6 x His tag at its N-terminus to yield a 33 kDa protein [Fernandes-Pedrosa et al. (2002), Biochem. Biophys. Res. Commun. 298, 638 - 645]. The recombinant protein possesses all the biological properties ascribed to the whole L. laeta venom, including dermonecrotic and complement-dependent haemolytic activities. Dynamic light-scattering experiments conducted at 291 K demonstrate that the sample possesses a monomodal distribution, with a hydrodynamic radius of 3.57 nm. L. laeta SMase I was crystallized by the hanging-drop vapour-diffusion technique using the sparse-matrix method. Single crystals were obtained using a buffer solution consisting of 0.08 M HEPES and 0.9 M trisodium citrate, which was titrated to pH 7.5 using 0.25 M sodium hydroxide. Complete three-dimensional diffraction data were collected to 1.8 Angstrom at the Laboratorio Nacional de Luz Sincrotron (LNLS, Campinas, Brazil). The crystals belong to the hexagonal system ( space group P6(1) or P6(5)), with unit-cell parameters a = b = 140.6, c = 113.6 Angstrom. A search for heavy-atom derivatives has been initiated and elucidation of the crystal structure is currently in progress.

Antimicrobial activity of an L-amino acid oxidase isolated from Bothrops leucurus snake venom

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Venom peptide analysis of Vipera ammodytes meridionalis (Viperinae) and Bothrops jararacussu (Crotalinae) demonstrates subfamily-specificity of the peptidome in the family Viperidae

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Enzymatic toxins from snake venom: structural characterization and mechanism of catalysis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)