title: Topology of the substrate-binding site of a Lys49-phospholipase A2 influences Ca2+-independent membrane-damaging activity.
abstract: BthTx-I (bothropstoxin-I) is a myotoxic Lys49-PLA2 (phospholipase A2 with Lys49) isolated from Bothrops jararacussu venom, which damages liposome membranes by a Ca2+-independent mechanism. The highly conserved Phe5/Ala102/Phe106 motif in the hydrophobic substrate-binding site of the Asp49-PLA2s is substituted by Leu5/Val102/Leu106 in the Lys49-PLA2s. The Leu5/Val102/Leu106 triad in BthTx-I was sequentially mutated via all single- and double-mutant combinations to the Phe5/Ala102/Phe106 mutant. All mutants were expressed as inclusion bodies in Escherichia coli, and the thermal stability (Tm), together with the myotoxic and Ca2+-independent membrane-damaging activities of the recombinant proteins, were evaluated. The far-UV CD profiles of the native, wild-type recombinant and the L106F (Leu106-->Phe) and L5F/F102A/L106F mutant proteins were identical. The L5F, V102A, L5F/V102A and V102A/L106F mutants showed distorted far-UV CD profiles; however, only the L5F and L5F/V102A mutants showed significant decreases in Tm. Alterations in the far-UV CD spectra correlated with decreased myotoxicity and protein-induced release of a liposome-entrapped marker. However, the V102A/L106F and L5F/V102A/L106F mutants, which presented high myotoxic activities, showed significantly reduced membrane-damaging activity. This demonstrates that the topology of the substrate-binding region of BthTx-I has a direct effect on the Ca2+-independent membrane damage, and implies that substrate binding retains an important role in this process.
authors.affiliation: Departamento de Bioquímica e Immunologia, FMRP-USP, Ribeirão Preto-SP, Brazil.
authors.forename: Juliana Martha
authors.initials: JM
authors.lastname: Sá
authors.forename: Lucimara
authors.initials: L
authors.lastname: Chioato
authors.forename: Tatiana Lopes
authors.initials: TL
authors.lastname: Ferreira
authors.forename: Arthur H C
authors.initials: AH
authors.lastname: De Oliveira
authors.forename: Roberto
authors.initials: R
authors.lastname: Ruller
authors.forename: José César
authors.initials: JC
authors.lastname: Rosa
authors.forename: Lewis J
authors.initials: LJ
authors.lastname: Greene
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Ward
mesh_terms: D000818:Animals; D001665:Binding Sites; D017837:Bothrops / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D002942:Circular Dichroism / Q000379:methods; D003435:Crotalid Venoms / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D018360:Crystallography, X-Ray / Q000379:methods; D048168:Escherichia coli K12 / Q000235:genetics / Q000378:metabolism; D007425:Intracellular Membranes / Q000187:drug effects / Q000378:metabolism*; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism; D008239:Lysine / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D009498:Neurotoxins / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D010741:Phospholipases A / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology; D054467:Phospholipases A2; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D030162:Reptilian Proteins; D012910:Snake Venoms / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D013379:Substrate Specificity
chemical_list: D003435:Crotalid Venoms; D008081:Liposomes; D009498:Neurotoxins; D011994:Recombinant Proteins; D030162:Reptilian Proteins; D012910:Snake Venoms; C013144:myotoxic component of rattlesnake venom; C078097:bothropstoxin; D010741:Phospholipases A; D054467:Phospholipases A2; D008239:Lysine; D002118:Calcium