Structural and functional characterization of brazilitoxins II and III (BbTX-II and -III), two myotoxins from the venom of Bothrops brazili snake.
ABSTRACT We report the purification and biochemical/pharmacological characterization of two myotoxic PLA(2) (BbTX-II K49 PLA(2) homologue and BbTX-III PLA(2)) from Bothrops brazili venom. Both were purified by a single chromatographic step on reverse phase HPLC, showing M(r) approximately 14 kDa for both myotoxins, showing high content of hydrophobic and basic amino acids as well as 14 half-cysteine residues. The BbTX-II K49 PLA(2) homologue and BbTX-III PLA(2), had a sequence of 121 amino acid residues. BbTX-II: [amino acid sequence: see text] with pI value 8.73. BbTX-III: [amino acid sequence: see text] with a pI value of 8.46. BbTX-III presented PLA(2) activity in the presence of a synthetic substrate and showed a minimum sigmoidal behavior, reaching its maximal activity at pH 8.0 and 35-45 degrees C. Maximum PLA(2) activity required Ca(2+). In vitro, BbTX-II K49 PLA(2) homologue and BbTX-III PLA(2) caused a blockade of the neuromuscular transmission in young chick biventer cervicis preparations in a similar way to other Bothrops species. In mice, BbTX-II K49 PLA(2) homologue and BbTX-III PLA(2) induces myonecrosis and edema-forming activity. All these biological effects induced by the BbTX-II K49 PLA(2) homologue, occur in the absence of a measurable PLA(2) activity in vitro, further supporting the concept of catalytic independent mechanisms exerted by Lys49 proteins.
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ABSTRACT: Catalytically inactive phospholipase A(2) (PLA(2)) homologues play key roles in the pathogenesis induced by snake envenomation, causing extensive tissue damage via a mechanism still unknown. Although, the amino acid residues directly involved in catalysis are conserved, the substitution of Asp49 by Arg/Lys/Gln or Ser prevents the binding of the essential calcium ion and hence these proteins are incapable of hydrolyzing phospholipids. In this work, the crystal structure of a Lys49-PLA(2) homologue from Bothrops brazili (MTX-II) was solved in two conformational states: (a) native, with Lys49 singly coordinated by the backbone oxygen atom of Val31 and (b) complexed with tetraethylene glycol (TTEG). Interestingly, the TTEG molecule was observed in two different coordination cages depending on the orientation of the nominal calcium-binding loop and of the residue Lys49. These structural observations indicate a direct role for the residue Lys49 in the functioning of a catalytically inactive PLA(2) homologue suggesting a contribution of the active site-like region in the expression of pharmacological effects such as myotoxicity and edema formation. Despite the several crystal structures of Lys49-PLA(2) homologues already determined, their biological assembly remains controversial with two possible conformations. The extended dimer with the hydrophobic channel exposed to the solvent and the compact dimer in which the active site-like region is occluded by the dimeric interface. In the MTX-II crystal packing analysis was found only the extended dimer as a possible stable quaternary arrangement.International journal of biological macromolecules 05/2012; 51(3):209-14. · 2.37 Impact Factor
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ABSTRACT: Bothrops brazili is a snake found in the forests of the Amazonian region whose commercial therapeutic anti-bothropic serum has low efficacy for local myotoxic effects, resulting in an important public health problem in this area. Catalytically inactive phospholipases A2-like (Lys49-PLA2s) are among the main components from Bothrops genus venoms and are capable to cause drastic myonecrosis. Several studies have shown that the C-terminal region of these toxins, which includes a variable combination of positively charged and hydrophobic residues, is responsible for their activity. In this work we describe the crystal structures of two Lys49-PLA2s (BbTX-II and MTX-II) from Bothrops brazili venom and a comprehensive structural comparison with several Lys49-PLA2s. Based on these results, it was identified two independent sites of interaction between protein and membrane which leads to the proposition of a new myotoxic mechanism for bothropic Lys49-PLA2s composed by five different steps. This proposition is able to fully explain the action of these toxins and may be useful to develop efficient inhibitors for complement the conventional antivenom administration.Biochimica et Biophysica Acta 10/2013; · 4.66 Impact Factor
- 01/2012: pages 1-34; , ISBN: 978-953-51-0813-9