Homodimer Stability Influences the Membrane Damaging Activity of Bothropstoxin-I (PLA₂-Lys49) from the venom of Bothrops jararacussu.

RullerR.^{2, Ferreira T. L.3,} Chioato L.³, de Oliveira A.H.C.², Sá, J.M.³; Aragão, E.A.¹, Ward R. J.¹

1 Depto. de Química da FFCLRP, 2 Depto. de Biologia Molecular e Celular e Bioagentes Patogênicos, 3 Depto. Bioquímica e Imunologia FMRP Ribeirão Preto

Bothropstoxin-I (BthTx-I) is a homodimeric Lys49-PLA2 present in the venom of the snake Bothrops jararacussu, which lacks measurable hydrolytic activity against phospholipid substrates. Nevertheless, BthTx-I damages artificial membranes by a Ca²⁺-independent mechanism in which a transition between “open” and “closed” membrane bound dimer conformations has been suggested to play a role. In order to evaluate this model, 3 key amino acid residues making intermolecular contacts at the homodimer interface have been substituted using site-directed mutagenesis. After expression in E. coli, and purification of the recombinant proteins by cation exchange chromatography, the Ca²⁺-independent membrane damaging activities of 7 mutant proteins were evaluated by measuring the release of entrapped calceín from liposomes. The dimer stability of all 7 mutants was estimated by non-linear fitting of fluorescence anisotropy experiments on denaturation by guanidinium hydrochloride. These results were compared with the miotoxic activities as measured by creatine kinase release from mouse gastrocnemius muscle. The principal findings are that reduction of dimer stability leads to reduction in membrane damage. In addition, a reduced membrane damaging activity is also predicted for the monomeric form of the protein and finally, the myotoxic and the Ca²⁺-independent membrane damaging activities are not correlated.

Support: FAPESP, CNPq, PRP-USP and FAEPA 

Correspondence to: roruller@rbp.fmrp.usp.br