Initiating structural studies of Lys49-PLA2 homologues complexed with an anionic detergent, a fatty acid and a natural lipid.
Lys49-Phospholipase A2 (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 permits 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. 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 2.78A, (ii) MjTX-II/STE complex at a resolution of 1.8 A and (iii) BthTX-I/DMPC complex at 2.72A. 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 (iii) and using using a Synchrotron Radiation Source (Laboratório Nacional de Luz Sincrotron, LNLS, Campinas, Brazil) for (ii).
Watanabe
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Department of Physics, IBILCE/UNESP, São José do Rio Preto, SP, CEP 15054-000, Brasil.
Fontes
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Soares
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Giglio
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Arni
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D000818:Animals; D003435:Crotalid Venoms / Q000737:chemistry*; D003460:Crystallization; D004134:Dimyristoylphosphatidylcholine / Q000737:chemistry; D010741:Phospholipases A / Q000737:chemistry*; D054467:Phospholipases A2; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry; D013229:Stearic Acids / Q000737:chemistry; D014961:X-Ray Diffraction / Q000706:statistics & numerical data
D003435:Crotalid Venoms; D013229:Stearic Acids; D012967:Sodium Dodecyl Sulfate; C031183:stearic acid; C114084:myotoxin II, Bothrops moojeni; D010741:Phospholipases A; D054467:Phospholipases A2; D004134:Dimyristoylphosphatidylcholine