Structure of the polypeptide crotamine from the Brazilian rattlesnake Crotalus durissus terrificus.

The crystal structure of the myotoxic, cell-penetrating, basic polypeptide crotamine isolated from the venom of Crotalus durissus terrificus has been determined by single-wavelength anomalous dispersion techniques and refined at 1.7 Šresolution. The structure reveals distinct cationic and hydrophobic surface regions that are located on opposite sides of the molecule. This surface-charge distribution indicates its possible mode of interaction with negatively charged phospholipids and other molecular targets to account for its diverse pharmacological activities. Although the sequence identity between crotamine and human ß-defensins is low, the three-dimensional structures of these functionally related peptides are similar. Since crotamine is a leading member of a large family of myotoxic peptides, its structure will provide a basis for the design of novel cell-penetrating molecules.

Purification, crystallization and preliminary X-ray diffraction analysis of crotamine, a myotoxic polypeptide from the Brazilian snake Crotalus durissus terrificus.

Crotamine, a highly basic myotoxic polypeptide (molecular mass 4881 Da) isolated from the venom of the Brazilian rattlesnake Crotalus durissus terrificus, causes skeletal muscle contraction and spasms, affects the functioning of voltage-sensitive sodium channels by inducing sodium influx and possesses antitumour activity, suggesting potential pharmaceutical applications. Crotamine was purified from C. durissus terrificus venom; the crystals diffracted to 1.9 Å resolution and belonged to the orthorhombic space group I2(1)2(1)2(1) or I222, with unit-cell parameters a = 67.75, b = 74.4, c = 81.01 Å. The self-rotation function indicated that the asymmetric unit contained three molecules. However, structure determination by molecular replacement using NMR-determined coordinates was unsuccessful and a search for potential derivatives has been initiated.

Pseudechis australis venomics: adaptation for a defense against microbial pathogens and recruitment of body transferrin.

The venom composition of Pseudechis australis, a widely distributed in Australia reptile, was analyzed by 2-DE and mass spectrometric analysis. In total, 102 protein spots were identified as venom toxins. The gel is dominated by horizontal trains of spots with identical or very similar molecular masses but differing in the pI values. This suggests possible post-translational modifications of toxins, changing their electrostatic charge. The results demonstrate a highly specialized biosynthesis of toxins destroying the hemostasis (P-III metalloproteases, SVMPs), antimicrobial proteins (L-amino acid oxidases, LAAOs, and transferrin-like proteins, TFLPs), and myotoxins (phospholipase A(2)s, PLA(2)s). The three transferrin isoforms of the Australian P. australis (Elapidae snake) venom are highly homologous to the body transferrin of the African Lamprophis fuliginosus (Colubridae), an indication for the recruitment of body transferrin. The venomic composition suggests an adaptation for a defense against microbial pathogens from the prey. Transferrins have not previously been reported as components of elapid or other snake venoms. Ecto-5'-nucleotidases (5'-NTDs), nerve growth factors (VNGFs), and a serine proteinase inhibitor (SPI) were also identified. The venom composition and enzymatic activities explain the clinical manifestation of the king brown snakebite. The results can be used for medical, scientific, and biotechnological purposes.

Three-dimensional modelling of honeybee venom allergenic proteases: relation to allergenicity.

Api SI and Api SII are serine proteases of the honeybee venom containing allergenic determinants. Each protease consists of two structural modules: an N-terminal CUB (Api SI) or a clip domain (Api SII) and a C-terminal serine protease-like (SPL) domain. Both domains are connected with a linker peptide. The knowledge about the structure and function of Api SI and Api SII is limited mainly to their amino acid sequences. We constructed 3-D models of the two proteases using their amino acid sequences and crystallographic coordinates of related proteins. The models of the SPL domains were built using the structure of the prophenoloxidase-activating factor (PPAF)-II as a template. For modelling of the Api SI CUB domain the coordinates of porcine spermadhesin PSP-I were used. The models revealed the catalytic and substrate-binding sites and the negatively charged residue responsible for the trypsin-like activity. IgE-binding and antigenic sites in the two allergens were predicted using the models and programs based on the structure of known epitopes. Api SI and Api SII show structural and functional similarity to the members of the PPAF-II family. Most probably, they are part of the defence system of Apis mellifera.

The venomics of Bothrops alternatus is a pool of acidic proteins with predominant hemorrhagic and coagulopathic activities.

The venom proteome of Bothrops alternatus, a venomous snake widespread in South America, was analyzed by 2-D electrophoresis followed by mass spectrometric analysis and determination of enzymatic activities. The venomic composition revealed that metallo- and serine proteinases play primary roles in the pathogenesis of the envenomation by this pitviper. The identified 100 venom components with molecular masses from 10 to 100 kDa belong to six protein families: metalloproteinases, serine/thrombin-like proteinases, phospholipases A(2), L-amino acid oxidases, disintegrins and thrombin inhibitors. Metalloproteinases predominate and belong exclusively to the P-III class including the most potent hemorrhagic toxins. They represent 50% of all identified proteins. Two isoforms were identified: homologous to jararhagin, a hemorrhagic toxin, and to beritractivase, a nonhemorrhagic and pro-coagulant metalloproteinase. The B. alternatus venom is a rich source of proteins influencing the blood coagulation system with a potential for medical application. The isoelectric points of the components are distributed in the acidic pH range (the pI values are between 4 and 7) and no basic proteins were detected.

Snake venomic of Crotalus durissus terrificus--correlation with pharmacological activities.

The snake venomic of Crotalus durissus terrificus was analyzed by 2-D and 1-D electrophoresis and subsequent MS/MS and enzymatic assays. The venomic of the South American rattlesnake comprises toxins from seven protein families: phospholipases A(2), serine proteinases, ecto-5'-nucleotidases, metalloproteinases, nerve growth factors, phosphodiesterases, and glutaminyl cyclase. The venom toxin composition correlates with the clinical manifestation of the crotalinae snake bites and explains pathological effects of the venom such as neurotoxicity, systemic myonecrosis, hemostatic disorders, myoglobinuria, and acute renal failure. The vast majority of toxins are potentially involved in neurotoxicity, myotoxicity, and coagulopathy. The predominant venom components are neurotoxic phospholipases A(2) and serine proteinases. The venom is a rich source of 5'-nucleotidases (7.8% of the identified toxins) inducing hemostatic disorders. Analysis of the venom protein composition provided a catalogue for secreted toxins. The venomic composition of Crotalus d. terrificus and venom gland transcriptome of the synonymous subspecies Crotalus d. collilineatus show differences in the occurrence of protein families and in the abundance of toxins. Some of the venom components identified by the proteomic analysis were not reported in the transcriptome of the Crotalus d. collilineatus venom gland. Enzymatic activities of the Crotalus d. terrificus venom were determined and correlated with the proteomic composition.

3-D Model of the bee venom acid phosphatase: insights into allergenicity.

The acid phosphatase Api m 3 is the major allergen of the honeybee venom. Except for the amino acid sequence, no other structural information for the enzyme is available. We applied homology modeling to assign the three-dimensional structure of Api m 3. The structure of the homodimeric human prostatic acid phosphatase was used to model the Api m 3 tertiary structure. IgE epitopes and antigenic sites were predicted using programs based on the structure of known epitopes and analysis of the 3-D model. The model of Api m 3 revealed an active site similar to those of the histidine-type acid phosphatases with conservation of the catalytically important residues. The observed substitutions in the phosphate ion binding site suggest differences in the substrate specificity in comparison to other acid phosphatases. The analysis of the Api m 3 three-dimensional model revealed a very likely mechanism of enzyme action.

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.

Isolation, crystallization and preliminary X-ray diffraction analysis of L-amino-acid oxidase from Vipera ammodytes ammodytes venom.

L-Amino-acid oxidase from the venom of Vipera ammodytes ammodytes, the most venomous snake in Europe, was isolated and crystallized using the sitting-drop vapour-diffusion method. The solution conditions under which the protein sample was monodisperse were optimized using dynamic light scattering prior to crystallization. The crystals belonged to space group C2, with unit-cell parameters a = 198.37, b = 96.38, c = 109.11 A, beta = 92.56 degrees . Initial diffraction data were collected to 2.6 A resolution. The calculated Matthews coefficient is approximately 2.6 A(3) Da(-1) assuming the presence of four molecules in the asymmetric unit.

Protein Profile Analysis of Two Australian Snake Venoms by One- Dimensional Gel Electrophoresis and MS/MS Experiments.

The Pseudechis colletti and Pseudechis butleri venoms were analyzed by 1-D gel electrophoresis, followed by mass spectrometric analysis of tryptic peptides obtained from the protein bands. Both venoms contain highly potent pharmacologically active components, which were assigned to the following protein families: basic and acidic phospholipases A2 (PLA2s), L-amino acid oxidases (LAAOs), P-III metalloproteinases (P-III SVMPs), 5'- nucleotidases (5'-NTDs), cysteine-rich secretory proteins (CRISPs), venom nerve growth factors (VNGFs) and post-synaptic neurotoxins. Considerable predominance of PLA2s over other toxins is a characteristic feature of both venoms. The major differences in the venom compositions are the higher concentration of SVMPs and CRISPs in the P. butleri venom, as well as the presence of post-synaptic neurotoxins. Furthermore, the analysis revealed a high concentration of proteins with myotoxic, coagulopathic and apoptotic activities. PLA2s are responsible for the myotoxic and anticoagulant effects observed in patients after envenomation (4). The other protein families, encountered in the two venoms, probably contribute to the major symptoms described for these venoms. These results explain the observed clinical effects of the black snake envenomation. The analyzed venoms contain group P-III metalloproteinases of medical importance with the potency to be used for diagnostic purposes of von Willebrand factor (vWF) disease, for regulation of vWF in thrombosis and haemostasis, for studying the function of the complement system in host defense and in the pathogenesis of diseases. Comparison of venomic data showed similarities in the major venom components of snakes from the genus Pseudechis, resulting in common clinical effects of envenomation, and demonstrating close relationships between venom toxins of Elapidae snakes.

Catalytic efficiencies of alkaline proteinases from microorganisms.

Catalytic efficiencies of proteinase K and mesentericopeptidase were determined using series of peptide-4-nitroanilide substrates and compared with those of subtilisin DY, savinase and esperase. For each enzyme the subsites S1-S4 were characterized. The data for the enzyme specificities were related to our high resolution X-ray models of the five enzymes and their complexes with peptides. The catalytic efficiencies of the alkaline proteinases are modulated by the hydrophobicity, solvent accessibility, flexibility and electrostatic effects in the substrate binding sites. The longer and nonpolar S1 loop offers more possibilities for hydrophobic interactions and increases the enzyme efficiency. S2 is a small narrow cleft which limits the possibilities for effective substitutions in P2. The wide specificity of S3 is due to its location on the protein surface of all investigated proteinases. The affinity of S4 for aromatic groups depends on the nature of the residues building the hydrophobic cavity.

Crystallization and preliminary X-ray diffraction studies of a toxic phospholipase A2 from the venom of Vipera ammodytes meridionalis complexed to a synthetic inhibitor.

A toxic phospholipase A(2) (PLA(2)) is isolated from the neurotoxic complex Vipoxin, the major lethal component of the venom of Vipera ammodytes meridionalis. The enzyme is complexed to the synthetic inhibitor elaidoylamide and crystallized. The crystals belong to the space group P2(1)2(1)2(1), with unit cell dimensions a=46.57 A, b=82.68 A, c=119.47 A and beta=90 degrees. Initial diffraction data to 3.3 A resolution are collected.

Structure-function relationships in the neurotoxin Vipoxin from the venom of Vipera ammodytes meridionalis.

The neurotoxic complex Vipoxin is the lethal component of the venom of Vipera ammodytes meridionalis, the most toxic snake in Europe. It is a complex between a toxic phospholipase A2 (PLA2) and a non-toxic and catalytically inactive protein, stabilizing the enzyme and reducing the activity and toxicity. Structure-function relationships in this complex were studied by spectroscopic methods. A good correlation between the ionization behaviour and accessible surface area (ASA) of the tyrosyl residues was observed. In the toxic PLA2 subunit phenolic groups participate in H-bonding network that stabilizes the catalytically and pharmacologically active conformation. The tryptophan fluorescence decay of Vipoxin is well fitted by two exponentials with lifetimes of 0.1 (54%) and 2.5 (46%) ns. W20P, W31P and W31I are located in the interface between the two subunits and participate in hydrophobic interactions stabilizing the complex. Dissociation of the complex leads to a transition of the tryptophans from hydrophobic to hydrophilic environment, which influences mainly tau2. The longer lifetime is more sensitive to the polarity of the environment. Circular dichroism measurements demonstrate that the two components of the neurotoxin preserve their secondary structure after dissociation of the complex. The results of the spectroscopic studies are in accordance with a mechanism of blockade of transmission across the neuromuscular junctions of the breathing muscles by interaction of a dissociated toxic PLA2 with a membrane. The loss of toxicity is connected with slight changes in the secondary structure of PLA2. CD studies also show a substantial contribution of disulfide bonds to the stability of the neurotoxic complex and its components.

Contribution of disulfide bonds and calcium to Molluscan hemocyanin stability.

Disulfide bonds and calcium ions contribute significantly to the stability of the hemocyanin from the mollusc Rapana thomasiana grosse (gastropod). An extremely powerful protective effect of Ca2+ at a concentration of 100 mM (100% protection) against the destructive effect of reductants like dithiothreitol was observed. This is important for the practical application of molluscan hemocyanins in experimental biochemistry, immunology and medicine. The reduction of the disulfide bonds in the Rapana hemocyanin leads to a 20% decrease of the a-helical structure. The S-S bonds contribute significantly to the free energy of stabilization in water increasing delta G(D)H2O by 6.9 kJ mol (-1) The data are related to the X-ray model of the Rapana hemocyanin functional unit RtH2e. The results of this study can be of common validity for related respiratory proteins because the cysteine residues are conserved in all sequences of molluscan hemocyanins published so far.

Elapid snake venom analyses show the specificity of the peptide composition at the level of genera Naja and Notechis.

Elapid snake venom is a highly valuable, but till now mainly unexplored, source of pharmacologically important peptides. We analyzed the peptide fractions with molecular masses up to 10 kDa of two elapid snake venoms-that of the African cobra, N. m. mossambica (genus Naja), and the Peninsula tiger snake, N. scutatus, from Kangaroo Island (genus Notechis). A combination of chromatographic methods was used to isolate the peptides, which were characterized by combining complimentary mass spectrometric techniques. Comparative analysis of the peptide compositions of two venoms showed specificity at the genus level. Three-finger (3-F) cytotoxins, bradykinin-potentiating peptides (BPPs) and a bradykinin inhibitor were isolated from the Naja venom. 3-F neurotoxins, Kunitz/basic pancreatic trypsin inhibitor (BPTI)-type inhibitors and a natriuretic peptide were identified in the N. venom. The inhibiting activity of the peptides was confirmed in vitro with a selected array of proteases. Cytotoxin 1 (P01467) from the Naja venom might be involved in the disturbance of cellular processes by inhibiting the cell 20S-proteasome. A high degree of similarity between BPPs from elapid and viperid snake venoms was observed, suggesting that these molecules play a key role in snake venoms and also indicating that these peptides were recruited into the snake venom prior to the evolutionary divergence of the snakes.

Crystal structure of a dimeric Ser49- PLA2-like myotoxic component of the Vipera ammodytes meridionalis venomics reveals determinants of myotoxicity and membrane damaging activity.

Myotoxicity and membrane damage play a central role in the life-threatening effects of the viper envenomation. Myotoxins are an important part of the viper venomics. A Ser49 PLA2-like myotoxin from the venom of Vipera ammodytes meridionalis, the most venomous snake in Europe, was crystallized and its three-dimensional structure determined. The toxin is devoid of phospholipolytic activity. The structure demonstrates a formation of dimers. In the dimers functionally important peptide segments, located on the protein surface, point in the same direction which can strengthen the pharmacological effect. This supports the hypothesis about the physiological importance of the toxin oligomerization for the myotoxicity and membrane damage. The crystallographic model revealed that the structural determinants of myotoxicity (a positively charged C-terminal region and a hydrophobic knuckle) are fully exposed on the protein surface and accessible for interactions with target membranes. Distortion of the catalytic site region explains the absence of enzymatic activity. The structure reveals anion-binding sites which can be considered as possible sites of interactions of the toxin with a negatively charged membrane surface. The high structural similarity of the Ser49 myotoxin and Asp49 PLA2 from the same venom suggests an evolutionary relationship: probably, the Ser49 myotoxin is a product of evolution of the catalytically active phospholipase A2. The first toxin lost the enzymatic activity which is not necessary for the myotoxicity but preserved the cytotoxicity and membrane damaging activity as important components of the venom toxicity.

The structure of a native l-amino acid oxidase, the major component of the Vipera ammodytes ammodytes venomic, reveals dynamic active site and quaternary structure stabilization by divalent ions.

The crystal structure of the major component of the Vipera ammodytes ammodytes venomic, a flavotoxin, member of the l-amino acid oxidase (LAAO) family, has been determined and refined at 2.6 Å resolution. The asymmetric unit consists of four molecules, each bound to oxidized FAD, representing a dimer of dimers. The binding of four Zn(2+) ions stabilizes the enzymatically active quaternary structure and is considered important for the biological activity of LAAO and other flavoproteins. Each monomer consists of three domains with a cofactor bound between the FAD and substrate binding domains, and a solvent exposed glycosylation site which is considered crucial for the toxicity. Comparison of LAAO structures in the absence and presence of a substrate indicates conformational changes in the dynamic active site. The active site H-bond network involving the triad Lys326-Water-N5 of FAD is formed only upon substrate binding, and results in the increased mobility of the isoalloxazine system. Details of the catalytic transformation of amino acid substrates are discussed.

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

Snake venom peptidomes are valuable sources of pharmacologically active compounds. We analyzed the peptidic fractions (peptides with molecular masses < 10,000 Da) of venoms of Vipera ammodytes meridionalis (Viperinae), the most toxic snake in Europe, and Bothrops jararacussu (Crotalinae), an extremely poisonous snake of South America. Liquid chromatography/mass spectrometry (LC/MS), direct infusion electrospray mass spectrometry (ESI-MS) and matrix-assisted desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) were applied to characterize the peptides of both snake venoms. 32 bradykinin-potentiating peptides (BPPs) were identified in the Crotalinae venom and their sequences determined. 3 metalloproteinase inhibitors, 10 BPPs and a Kunitz-type inhibitor were observed in the Viperinae venom peptidome. Variability in the C-terminus of homologous BPPs was observed, which can influence the pharmacological effects. The data obtained so far show a subfamily specificity of the venom peptidome in the Viperidae family: BPPs are the major peptide component of the Crotalinae venom peptidome lacking Kunitz-type inhibitors (with one exception) while the Viperinae venom, in addition to BPPs, can contain peptides of the bovine pancreatic trypsin inhibitor family. We found indications for a post-translational phosphorylation of serine residues in Bothrops jararacussu venom BPP (S[combining low line]QGLPPGPPIP), which could be a regulatory mechanism in their interactions with ACE, and might influence the hypotensive effect. Homology between venom BPPs from Viperidae snakes and venom natriuretic peptide precursors from Elapidae snakes suggests a structural similarity between the respective peptides from the peptidomes of both snake families. The results demonstrate that the venoms of both snakes are rich sources of peptides influencing important physiological systems such as blood pressure regulation and hemostasis. The data can be used for pharmacological and medical applications.

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

Snake venoms are cocktails of enzymes and non-enzymatic proteins used for both the immobilization and digestion of prey. The most common snake venom enzymes include acetylcholinesterases, l-amino acid oxidases, serine proteinases, metalloproteinases and phospholipases A(2) . Higher catalytic efficiency, thermal stability and resistance to proteolysis make these enzymes attractive models for biochemists, enzymologists and structural biologists. Here, we review the structures of these enzymes and describe their structure-based mechanisms of catalysis and inhibition. Some of the enzymes exist as protein complexes in the venom. Thus we also discuss the functional role of non-enzymatic subunits and the pharmacological effects of such protein complexes. The structures of inhibitor-enzyme complexes provide ideal platforms for the design of potent inhibitors which are useful in the development of prototypes and lead compounds with potential therapeutic applications.

Three-dimensional model of the honeybee venom allergen Api m 7: structural and functional insights.

Api m 7 is one of the major protease allergens of the honeybee venom. It consists of a serine protease-like (SPL) and a CUB domain. The knowledge about the structure and function of Api m 7 is limited mainly to its amino acid sequence. Three-dimensional models of the two structural domains were constructed using their amino acid sequences and the crystallographic coordinates of prophenoloxidase-activating factor (PPAF-II) as a template for the SPL domain and the coordinates of porcine spermadhesin PSP-II for the CUB domain. The structural organization of Api m 7 suggests that the CUB domain is involved in interactions with natural substrates while the SPL domain probably activates zymogens. IgE epitopes and antigenic sites were predicted. Api m 7 shows structural and functional similarity to the members of the PPAF-II family. Possible substrates, function and evolution of the enzyme are discussed in the paper.