Microhabitat use by species of the genera Bothrops and Crotalus (Viperidae) in semi-extensive captivity

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

The golden lancehead Bothrops insularis (Serpentes: Viperidae) relies on two seasonally plentiful bird species visiting its island habitat

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Refolding and purification of bothropstoxin-1, a Lys49 - Phospholipase A(2) homologue, expressed as inclusion bodies in Escherichia coli

Hydrolysis of phospholipids by Group II phospholipase A(2) enzymes involves a nucleophilic attack on the sn-2 ester bond by the His48 residue and stabilization of the reaction intermediate by a Ca2+ ion cofactor bound to the Asp49 residue in the protein active site region, Bothropstoxin-I (BthTX-I) is a PLA, variant present in the venom of the snake Bothrops jararacussu which shows a Asp49 to Lys substitution and which lacks hydrolytic activity yet damages artificial membranes by a noncatalytic Ca2+-independent mechanism. In order to better characterize this unusual mechanism of membrane damage, we have established an expression system for BthTX-I in Escherichia coli. The DNA-coding sequence for BthTX-I was subcloned into the vector pET11-d, and the BthTX-I was expressed as inclusion bodies in E, coli BL21(DE3). The native BthTX-I contains seven disulfide bonds, and a straightforward protocol has been developed to refold the recombinant protein at high protein concentration in the presence of surfactants using a size-exclusion chromatography matrix. After refolding, recovery yields of 2.5% (corresponding to 4-5 mg of refolded recombinant BthTX-I per liter of bacterial culture) were routinely obtained. After refolding, identical fluorescent and circular dichroism spectra were obtained for the recombinant BthTX-I compared to those of the native protein. Furthermore, the native and refolded recombinant protein demonstrated identical membrane-damaging properties as evaluated by measuring the release of an entrapped fluorescent marker from liposomes, (C) 2001 Academic Press.

Crystallization of bothrombin, a fibrinogen-converting serine protease isolated from the venom of Bothrops jararaca

Bothrombin, a snake-venom serine protease, specifically cleaves fibrinogen, releasing fibrinopeptide A to form non-crosslinked soft clots, aggregates platelets in the presence of exogeneous fibrinogen and activates blood coagulation factor VIII. Bothrombin shares high sequence homology with other snake-venom proteases such as batroxobin (94% identity), but only 30 and 34% identity with human alpha-thrombin and trypsin, respectively. Single crystals of bothrombin have been obtained and X-ray diffraction data have been collected at the Laboratorio Nacional de Luz Sincrotron to a resolution of 2.8 Angstrom. The crystals belong to the space group P2(1)2(1)2(1), with unit-cell parameters a = 94.81, b = 115.68, c = 155.97 Angstrom.

Insights into metal ion binding in phospholipases A(2): ultra high-resolution crystal structures of an acidic phospholipase A(2) in the Ca2+ free and bound states

The electrophile Ca2+ is an essential multifunctional co-factor in the phospholipase A(2) mediated hydrolysis of phospholipids. Crystal structures of an acidic phospholipase A(2) from the venom of Bothrops jararacussu have been determined both in the Ca2+ free and bound states at 0.97 and 1.60 angstrom resolutions, respectively. In the Ca2+ bound state, the Ca2+ ion is penta-coordinated by a distorted pyramidal cage of oxygen and nitrogen atoms that is significantly different to that observed in structures of other Group I/II phospholipases A(2). In the absence of Ca2+, a water molecule occupies the position of the Ca2+ ion and the side chain of Asp49 and the calcium-binding loop adopts a different conformation. (c) 2005 Elsevier SAS. All rights reserved.

Crystallization and preliminary X-ray analysis of bucain, a novel toxin from the Malayan krait Bungarus candidus

Bucain is a three-finger toxin, structurally homologous to snake-venom muscarinic toxins, from the venom of the Malayan krait Bungarus candidus. These proteins have molecular masses of approximately 6000-8000 da and encompass the potent curaremimetic neurotoxins which confer lethality to Elapidae and Hydrophidae venoms. Bucain was crystallized in two crystal forms by the hanging-drop vapour-diffusion technique in 0.1 M sodium citrate pH 5.6, 15% PEG 4000 and 0.15 M ammonium acetate. Form I crystals belong to the monoclinic system space group C2, with unit-cell parameters a = 93.73, b = 49.02, c = 74.09 Angstrom, beta = 111.32degrees, and diffract to a nominal resolution of 1.61 Angstrom. Form II crystals also belong to the space group C2, with unit-cell parameters a = 165.04, b = 49.44, c = 127.60 Angstrom, beta = 125.55degrees, and diffract to a nominal resolution of 2.78 Angstrom. The self-rotation function indicates the presence of four and eight molecules in the crystallographic asymmetric unit of the form I and form II crystals, respectively. Attempts to solve these structures by molecular-replacement methods have not been successful and a heavy-atom derivative search has been initiated.

Purification, characterization and crystallization of Jararacussin-I, a fibrinogen-clotting enzyme isolated from the venom of Bothrops jararacussu

A fibrinogen-clotting enzyme, Jararacussin-I, was purified from the venom of Bothrops jararacussu by a combination of ion exchange chromatography using Resource 15S resin and affinity chromatography using Benzamidine Sepharose 6B resin. Jararacussin-I displays a molecular mass of 28 kDa as estimated by sodium dodecyl sulphate-PAGE and possesses an isoetectric point of 5.0. The coagulant specific activity of the enzyme was determined to be 45.8 NIH U/mg using bovine fibrinogen as the substrate and the esterase specific activity was determined to be 258.7 U/mg. The protease inhibitors, benzamidine and DTT inhibited the esterase specific activity by 72.4 and 69.7%, respectively. The optimal temperature and pH for the degradation of both chains of fibrinogen and esterase specific activity were determined to be 37 degreesC and 7.4-8.0, respectively. The enzyme was inactivated at both 4 and 75 T. Single crystals of Jararacussin-I were obtained and complete three-dimensional X-ray diffraction data was collected at the Brazilian National Synchrotron Source (LNLS) to a resolution of 2.4 Angstrom. (C) 2002 Published by Elsevier B.V. Ltd.

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.

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.

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.

At the interface: Crystal structures of phospholipases A(2)

The protein content of many snake venoms often includes one or more phospholipases A(2) (PLA(2)). In recent years a growing number of venoms from snakes of Agkistrodon, Bothrops and Trimeresurus species have been shown to contain a catalytically inactive PLA(2)-homologue in which the highly conserved aspartic acid at position 49 (Asp49) is substituted by lysine (Lys49). Although demonstrating little or no catalytic activity, these Lys49-PLA(2)s disrupt membranes by a Ca2+-independent mechanism of action. In addition, this family of PLA(2)s demonstrates myotoxic and cytolytic pharmacological activities, however the structural bases underlying these functional properties are poorly understood. Through the application of X-ray crystallography in combination with biophysical and bioinformatics techniques, we are studying structure/function relationships of Lys49-PLA(2)s. We here present results of a systematic X-ray crystallographic and amino acid sequence analysis study of Lys49-PLA(2)s and propose a model to explain the Ca2+ independent membrane damaging activity. (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 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.

Inhibition of myotoxic activity of Bothrops asper myotoxin II by the anti-trypanosomal drug surarnin

Suramin, a synthetic polysulfonated compound, developed initially for the treatment of African trypanosomiasis and onchocerciasis, is currently used for the treatment of several medically relevant disorders. Suramin, heparin, and other polyanions inhibit the myotoxic activity of Lys49 phospholipase A(2) analogues both in vitro and in vivo, and are thus of potential importance as therapeutic agents in the treatment of viperid snake bites. Due to its conformational flexibility around the single bonds that link the central phenyl rings to the secondary amide backbone, the symmetrical suramin molecule binds by an induced-fit mechanism complementing the hydrophobic surfaces of the dimer and adopts a novel conformation that lacks C2 symmetry in the dimeric crystal structure of the suramin-Bothrops asper myotoxin II complex. The simultaneous binding of suramin at the surfaces of the two monomers partially restricts access to the nominal active sites and significantly changes the overall charge of the interfacial recognition face of the protein, resulting in the inhibition of myotoxicity. (c) 2005 Elsevier Ltd. All rights reserved.

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)