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.

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.

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.

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)

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)

Crystallization and preliminary X-ray diffraction studies of BmooPLA(2)-I, a platelet-aggregation inhibitor and hypotensive phospholipase A(2) from Bothrops moojeni venom

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

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.

Initiating structural studies of Lys49-PLA2 homologues complexed with an anionic detergent, a fatty acid and a natural lipid

Lys49-Phospholipase A(2) (Lys49-PLA(2) - EC 3.1.1.4) homologues damage membranes by a Ca2+-independent mechanism which does not involve catalytic activity. Both MjTX-II from Bothrops moojeni and BthTX-I from Bothrops jararacussu are dimeric in solution and in the crystalline states, and a model for the Ca2+-independent membrane damaging mechanism has been suggested in which flexibility at the dimer interface region pert-nits quaternary structural transitions between open and closed membrane bound dimer conformations which results in the perturbation of membrane phospholipids and disruption of the bilayer structure [1]. With the aim of gaining insights into the structural determinants involved in protein/lipid association, we report here the crystallization and preliminary X-ray analysis of the (i) MjTX-II/SDS complex at a resolution of 2.78Angstrom, (ii) MjTX-II/STE complex at a resolution of 1.8 Angstrom and (W) BthTX-I/DMPC complex at 2.72Angstrom. These complexes were crystallized by the hanging drop vapour-diffusion technique in (i) HEPES buffer (pH 7.5) 1.8M ammonium sulfate with 2% (w/v) polyethyleneglycol 400, in (ii) 0.6-0.8 M sodium citrate as the precipitant (pH 6.0-6.5) and in (iii) sodium citrate buffer (pH 5.8) and PEG 4000 and 20% isopropanol, respectively. Single crystals of these complexes have been obtained and X-ray diffraction data have been collected at room temperature using a R-AXIS IV imaging plate system and graphite monochromated Cu Kalpha X-ray radiation generated by a Rigaku RU300 rotating anode generator for (i) and (W) and using using a Synchrotron Radiation Source (Laboratorio Nacional de Luz Sincrotron, LNLS, Campinas, Brazil) for (ii).

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.

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.

Interactions of mast cell degranulating peptides with model membranes: A comparative biophysical study

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

New Insight into the Mechanism of Action of Wasp Mastoparan Peptides: Lytic Activity and Clustering Observed with Giant Vesicles

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Crystallization and preliminary X-ray diffraction analysis of a class II phospholipase D from Loxosceles intermedia venom

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

Avaliação do desempenho dos reagentes do tempo de tromboplastina parcial ativada utilizados para detectar o anticoagulante lúpico

INTRODUÇÃO: O anticoagulante lúpico é uma imunoglobulina pertencente à família dos anticorpos antifosfolípides. A sua ação in vitro é interferir nos testes de coagulação dependentes de fosfolípides. O tempo de tromboplastina parcial ativada (TTPA) é um teste utilizado como screening na pesquisa do anticoagulante lúpico. Os reagentes utilizados neste teste apresentam grandes variações quanto à sensibilidade. OBJETIVO: Avaliar o desempenho dos reagentes do TTPA e detectar a presença do anticoagulante lúpico através de diferentes testes da coagulação. MATERIAL E MÉTODO: A pesquisa do anticoagulante lúpico foi realizada em 50 amostras plasmáticas de pacientes do sexo feminino através dos testes do TTPA, do tempo de coagulação do caulim (TCC), do tempo de tromboplastina parcial ativada diluída (TTPAd) e do tempo do veneno da víbora de Russel diluído (TVVRd). Três cefalinas comerciais foram avaliadas pelos testes do TTPA e do TTPAd. Na comparação entre os reagentes estudados foi aplicado o cálculo do intervalo de confiança (95%). RESULTADOS: Os três reagentes avaliados apresentaram boa concordância e os métodos utilizados responderam bem à pesquisa do anticoagulante lúpico. DISCUSSÃO E CONCLUSÃO: As três cefalinas comerciais avaliadas podem ser utilizadas na rotina laboratorial para a pesquisa do anticoagulante lúpico.