Crystallization and preliminary X-ray diffraction analysis of a myotoxic Lys49-PLA(2) from Bothrops jararacussu venom complexed with p-bromophenacyl bromide

For the first time, a non-catalytic and myotoxic Lys49-PLA(2) (BthTX-I from Bothrops jararacussu venom) has been crystallized with BPB inhibitor. X-ray diffraction data were collected and electron-density calculations showed that the ligand is bound to the His48 residue. BthTX-I with His48 chemically modified by BPB shows strongly reduced myotoxic and cytotoxic activities. This suggests a biological correlation between the modification of His48, which is associated with catalytic activity of PLA(2)s, and other toxicological activities of Lys49-PLA(2)s.

Preliminary X-ray crystallographic studies of BthTX-II, a myotoxic Asp49-phospholipase A(2) with low catalytic activity from Bothrops jararacussu venom

For the first time, a complete X-ray diffraction data set has been collected from a myotoxic Asp49-phospholipase A(2) (Asp49-PLA(2)) with low catalytic activity (BthTX-II from Bothrops jararacussu venom) and a molecular-replacement solution has been obtained with a dimer in the asymmetric unit. The quaternary structure of BthTX-II resembles the myotoxin Asp49-PLA(2) PrTX-III (piratoxin III from B. pirajai venom) and all non-catalytic and myotoxic dimeric Lys49-PLA(2)s. In contrast, the oligomeric structure of BthTX-II is different from the highly catalytic and non-myotoxic BthA-I (acidic PLA(2) from B. jararacussu). Thus, comparison between these structures should add insight into the catalytic and myotoxic activities of bothropic PLA(2)s.

Crystallization and preliminary X-ray analysis of S-III-SPIII, a phospholipase A2 isolated from the venom of Bothrops jararacussu.

Large single crystals have been obtained of S-III-SPIII, a phospholipase A(2) from the venom of Bothrops jararacussu. The crystals belong to the orthorhombic system space group C222, and diffract X-rays to a resolution of 1.9 Angstrom. Preliminary analysis reveals the presence of one molecule in the crystallographic asymmetric unit. The crystal structure is currently being determined using molecular replacement techniques.

Structural insights for fatty acid binding in a Lys49-phospholipase A(2): crystal structure of myotoxin II from Bothrops molojeni complexed with stearic acid

The crystal structure of dimeric Lys49-phospholipase A2 myotoxin-II from Bothrops moojeni (MjTX-II) co-crystallized with stearic acid (C18H36O2) has been determined at a resolution of 1.8 angstrom. The electron density maps permitted the unambiguous inclusion of six stearic acid molecules in the refinement. Two stearic acid molecules could be located in the substrate-binding cleft of each monomer in positions, which favor the interaction of their carboxyl groups with active site residues. The way of binding of stearic acids to this Lys49-PLA(2)s is analogous to phospholipids and transition state analogues to catalytically active PLA(2)s. Two additional stearic acid molecules were located at the dimer interface region, defining a hitherto unidentified acyl-binding site on the protein surface. The strictly conserved Lys122 for Lys49-PLA(2)s may play a fundamental role for stabilization of legend-protein complex. The comparison of MjTX-II/satiric acid complex with other Lys-PLA(2)s structures whose putative fatty acids were located at their active site is also analysed. Molecular details of the stearic acid/protein interactions provide insights to binding in croup I/II PLA(2)s and to the possible interactions of Lys49-PLA(2)s with target membranes. (c) 2004 Elsevier SAS. All rights reserved.

Crystallization and preliminary X-ray diffraction studies of two myotoxic Lys49-phospholipases A(2) complexed with alpha-tocopherol

BnSP-7 and BnSP-6, two Lys49-phospholipase A(2) isolated from Bothrops neuwiedi pauloensis snake venom, were co-crystallized with a-tocopherol and X-ray diffraction data were collected for both complexes (2.2 and 2.6 angstrom). A new alternative quaternary conformation for these two complexes compared with all other dimeric Lys49-PLA(2) has been observed.

Snake venom phospholipase A(2) inhibitors: Medicinal chemistry and therapeutic potential

Phospholipases A(2) (PLA(2)s) are commonly found in snake venoms from Viperidae, Hydrophidae and Elaphidae families and have been extensively studied due to their pharmacological and physiopathological effects in living organisms. This article reports a review on natural and artificial inhibitors of enzymatic, toxic and pharmacological effects induced by snake venom PLA(2)s. These inhibitors act on PLA(2)S through different mechanisms, most of them still not completely understood, including binding to specific domains, denaturation, modification of specific amino acid residues and others. Several substances have been evaluated regarding their effects against snake venoms and isolated toxins, including plant extracts and compounds from marine animals, mammals and snakes serum plasma, in addition to poly or monoclonal antibodies and several synthetic molecules. Research involving these inhibitors may be useful to understand the mechanism of action of PLA(2)s and their role in envenomations caused by snake bite. Furthermore, the biotechnological potential of PLA(2) inhibitors may provide therapeutic molecular models with antiophidian activity to supplement the conventional serum therapy against these multifunctional enzymes.

Crystal structure of an acidic platelet aggregation inhibitor and hypotensive phospholipase A(2) in the monomeric and dimeric states: insights into its oligomeric state

Phospholipases A(2) belong to the superfamily of proteins which hydrolyzes the sn-2 acyl groups of membrane phospholipids to release arachidonic acid and lysophospholipids. An acidic phospholipase A(2) isolated from Bothrops juraracussu snake venom presents a high catalytic, platelet aggregation inhibition and hypotensive activities. This protein was crystallized in two oligomeric states: monomeric and dimeric. The crystal structures were solved at 1.79 and 1.90 Angstrom resolution, respectively, for the two states. It was identified a Na+ ion at the center of Ca2+-binding site of the monomeric form. A novel dimeric conformation with the active sites exposed to the solvent was observed. Conformational states of the molecule may be due to the physicochemical conditions used in the crystallization experiments. We suggest dimeric state is one found in vivo. (C) 2004 Elsevier B.V. All rights reserved.

Crystallization and preliminary X-ray diffraction studies of BnSP-6, a myotoxic phospholipase A2 from Bothrops neuwiedi venom

BnSP-6 (myotoxin I) is a phospholipase A2 homologue isolated from Bothrops neuwiedi pauloensis venom. Crystals of BnSP-6 were obtained which diffracted X-rays to 2.5 Angstrom resolution using a synchrotron radiation source at room temperature and belong to space group P3(1)21. The unit cell dimensions are a=b=57.7, c=131.1 Angstrom. The structure was solved by molecular replacement using the coordinates of bothropstoxin I from B. jararacussu venom. There are two molecules in the asymmetric unit.

Molecular approaches for structural characterization of Bothrops L-amino acid oxidases with antiprotozoal activity: cDNA cloning, comparative sequence analysis, and molecular modeling

Two L-amino acid oxidases (LAAOs) were identified by random sequencing of cDNA libraries from the venom glands of Bothrops moojeni (BmooLAAO) and Bothrops jararacussu (Bjussu LAAO). Phylogenetic analysis involving other SV-LAAOs showed sequence identities within the range 83-87% being closely related to those from Agkistrodon and Trimeresurus. Molecular modeling experiments indicated the FAD-binding, substrate-binding, and helical domains of Bmoo and Bjussu LAAOs. The RMS deviations obtained by the superposition of those domains and that from Calloselasma rhodostoma LAAO crystal structure confirm the high degree of structural similarity between these enzymes. Purified BjussuLAAO-I and BmooLAAO-I exhibited antiprotozoal activities which were demonstrated to be hydrogen-peroxide mediated. This is the first report on the isolation and identification of cDNAs encoding LAAOs from Bothrops venom. The findings here reported contribute to the overall structural elucidation of SV-LAAOs and will advance the understanding on their mode of action. (c) 2006 Elsevier B.V. All rights reserved.

Influence of Quaternary Conformation on the Biological Activities of the Asp49-phospholipases A(2)s 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.

Structural, functional, and bioinformatics studies reveal a new snake venom homologue phospholipase A(2) class

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

Crystal structure of a phospholipase A(2) homolog complexed with p-bromophenacyl bromide reveals important structural changes associated with the inhibition of myotoxic activity

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

Crystallization and preliminary X-ray crystallographic studies of a Lys49-phospholipase A(2) homologue from Bothrops pirajai venom complexed with rosmarinic acid

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

Anti-hemorrhagic Activity of Four Brazilian Vegetable Species Against Bothrops jararaca Venom

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