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.

Crystal structures of BnSP-7 and BnSP-6, two Lys49-phospholipases A(2): quaternary structure and inhibition mechanism insights

Phospholipases A(2) are components of Bothrops venoms responsible for disruption of cell membrane integrity via hydrolysis of its phospholipids. A class of PLA(2)-like proteins has been described which despite PLA(2) activity on artificial substrate, due to a D49K mutation, is still highly myonecrotic. This work reports the X-ray structure determination of two Lys49-PLA(2)s from Bothrops neuwiedi pauloensis (BnSP-7 and BnSP-6) and, for the first time, the comparison of eight dimeric Lys49-PLA2s. This comparison reveals that there are not just two (open and closed) but at least six different conformations. The binding of fatty acid observed in three recent Lys49-PLA(2) structures seems to be independent of their quaternary conformation. Cys29 polarization by Lys122 is not significant for BnSP-7 and BnSP-6 or other structures not bound by fatty acids. These structures may be in an active state when nothing is bound to them and the Lys122/Cys29 interactions are weak or absent. (C) 2003 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.

Identification of continuous interaction sites in PLA(2)-based protein complexes by peptide arrays

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Comparison between apo and complexed structures of bothropstoxin-I reveals the role of Lys122 and Ca2+-binding loop region for the catalytically inactive Lys49-PLA(2)s

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

Comparative structural studies on Lys49-phospholipases A(2) from Bothrops genus reveal their myotoxic site

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

The Intriguing Phospholipases A(2) Homologues: Relevant Structural Features on Myotoxicity and Catalytic Inactivity

Phospholipases A(2) homologues are found in the venom of Crotalinae snakes, being their main action related to myonecrosis induction. Although many studies on these toxins had already been performed, their mechanism of action remains unclear. Here, important aspects about these toxins are reviewed, including their correct biological assembly and how essential is the natural substitution D49K for their catalytic inactivity.

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.

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.

Interfacial surface charge and free accessibility to the PLA(2)-active site-like region are essential requirements for the activity of Lys49 PLA(2) homologues

Lys49 phospholipase A(2) homologues are highly myotoxic and cause extensive tissue damage but do not display hydrolytic activity towards natural phospholipids. The binding of heparin, heparin derivatives and polyanionic compounds such as suramin result in partial inhibition (up to 60%) of the myotoxic effects due to a change in the overall charge of the interfacial surface. In vivo experiments demonstrate that polyethylene glycol inhibits more than 90% of the myotoxic effects without exhibiting secondary toxic effects. The crystal structure of bothropstoxin-I complexed with polyethylene glycol reveals that this inhibition is due to steric hindrance of the access to the PLA(2)-active site-like region. These two inhibitory pathways indicate the roles of the overall surface charge and free accessibility to the PLA2-active site-like region in the functioning of Lys49 phospholipases A(2) homologues. Molecular dynamics simulations, small angle X-ray scattering and structural analysis indicate that the oligomeric states both in solution and in the crystalline states of Lys49 phospholipases A2 are principally mediated by hydrophobic contacts formed between the interfacial surfaces. These results provide the framework for the potential application of both clinically approved drugs for the treatment of Viperidae snakebites. (c) 2006 Elsevier Ltd. All rights reserved.

Crystal structure of a dimeric Ser49 PLA(2)-like myotoxic component of the Vipera ammodytes meridionalis venomics reveals determinants of myotoxicity and membrane damaging activity

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Dissociation of enzymatic and pharmacological properties of piratoxins-I and -III, two myotoxic phospholipases A(2) from Bothrops pirajai snake venom

Piratoxins (PrTX) I and III are phospholipases A(2) (PLA(2)s) or PLA(2) homologue myotoxins isolated from Bothrops pirajai snake venom, which also induce myonecrosis, bactericidal activity against Escherichia coli, disruption of artificial membranes, and edema. PrTX-III is a catalytically active hemolytic and anticoagulant Asp49 PLA(2), while PrTX-I is a Lys49 PLA, homologue, which is catalytically inactive on artificial substrates, but promotes blockade of neuromuscular transmission. Chemical modifications of His, Lys, Tyr, and Trp residues of PrTX-I and PrTX-III were performed, together with cleavage of the N-terminal octapeptide by CNBr and inhibition by heparin and EDTA. The lethality, bactericidal activity, myotoxicity, neuromuscular effect, edema inducing effect, catalytic and anticoagulant activities, and the liposome-disruptive activity of the modified toxins were evaluated. A complex pattern of functional differences between the modified and native toxins was observed. However, in general, chemical modifications that significantly affected the diverse pharmacological effects of the toxins did not influence catalytic or membrane disrupting activities. Analysis of structural changes by circular dichroism spectroscopy demonstrated significant changes in the secondary structure only in the case of N-terminal octapeptide cleavage. These data indicate that PrTX-I and PrTX-III possess regions other than the catalytic site, which determine their toxic and pharmacological activities. (C) 2001 Academic Press.

Calcium-Independent Membrane Damage by Venom Phospholipases A(2)

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Isolation and biochemical characterization of a gamma-type phospholipase A2 inhibitor from Crotalus durissus collilineatus snake serum

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Structural bases for a complete myotoxic mechanism: Crystal structures of two non-catalytic phospholipases A(2)-like from Bothrops brazili venom

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