Crystal Structure of MIOTOXIN ii FROM Bothrops moojeni BOUND TO a fatty acid AT 1.8 Å resolution

FONTES, M.R.M.(1), SOARES, A.M.(2), GIGLIO, J.R.(3), ARNI R.K.(4)

(1) Departamento de Física Biofísica, Instituto de Biociências UNESP, Botucatu, (2) Departamento de Biotecnologia, UNAERP, Ribeirão Preto, (3) Departamento de Bioquímica, Faculdade de Medicina de Ribeirão Preto, USP, Ribeirão Preto, (4) Departamento de Física, IBILCE, UNESP, São José do Rio Preto.

Phospholipases A2 are common components of bothropic venoms responsible for disruption of the cell membrane integrity via hydrolysis of its phospholipids, culminating with cell death. Theses enzymes have an Asp at position 49 (D49) which is a part of the Ca++ loop which promotes the catalytic activity on phosphatidyl choline from egg yolk. A new class of PLA₂-like proteins has been described which, although devoid of PLA₂ activity on phosphatidyl choline, due to a mutation D49K, is still highly myonecrotic. Their effect being Ca++ independent aren’t still completely understood. In this work we show the X-ray structure determination, refinement process and relate structural characteristics of this toxin co-crystallized (MjTX-II) with fatty acid. The MjTX-II was cDNA cloned and sequenced and pharmacological and toxic activities were tested. The toxin was co-crystallized with a fatty acid and data collection was made at LNLS (Campinas). The crystals belong to the space group P2₁2₁2₁ with approximated cell constants a=61.18 b=88.71 and c=51.09 Å. Data was collected up to 1.8 Å resolution. The structure was solved by Molecular Replacement method and the refinement using the CNS program, the final R-factor was 21.9% and free-R-factor 25.3%. It was observed a strong electronic density for the fatty acid for both monomers in the hydrophobic channel close to active site.  The main contacts toxin-fatty acid are His48, Lys49 and the main chain of residues 28-30.

CORRESPONDENCE TO:

Marcos Roberto de Mattos Fontes, Departamento de Física e Biofísica, IB, UNESP, Caixa Postal 510 , Botucatu, SP, CEP: 18618-000, Brasil, Email: fontes@ibb.unesp.br