CRYSTAL STRUCTURE OF A BOTHROPSTOXIN I, A MYOTOXIN K49 LIKE PHOSPHOLIPASE A2

Mini-Symposium - Abstracts

4 CRYSTAL STRUCTURE OF A BOTHROPSTOXIN I, A MYOTOXIN K49 LIKE PHOSPHOLIPASE A2

M.T.S. GIOTTO , W.F. AZEVEDO , E. HORJALES , G. OLIVA , Y.P. MASCARENHAS ,R.C. GARRATT , A.C.O. CINTRA , J.R. GIGLIO

Institute of Physics of São Carlos, University of São Paulo, São Carlos, SP; Department of Biochemistry, School of Medicine of Ribeirão Preto, University of São Paulo, Ribeirão Preto, SP, Brazil.

Bothropstoxin-I (BthTx-I) is a myotoxin isolated from the Brazilian snake Bothrops jararacussu that is a member of the phospholipase A2 family, but presents no catalytic activity due to a D49K substitution. Protein was provided by Prof. Dr. J.R. Giglio and Dr. A.C.O Cintra from the Department of Biochemistry of Ribeirão Preto and used in crystallization experiments that were performed using the vapor diffusion technique in hanging drops at 18ºC. The BthTx-I crystallized in 0.1 M HEPES, pH ranging from 7.0 to 7.6. The precipitant was (NH4)SO4 in concentrations ranging from 57 to 62%. Data collection was initially performed using the automatic difractometer R-AXIS IIC from the Rigaku Co. at the Laboratory of Crystallography of the Institute of Physics of São Carlos, University of São Paulo.

Subsequently, a second data set was collected at SERC Daresbury Laboratory in England using synchrotron radiation. BthTx-I crystallizes in space group P3|2I with a=b=57.58Å c=131.29Å, =ß=90º and . Processing of the data was performed with the MOSFLM program yielding an Rmerge=6.3% and completeness of 99.6% at 2.1 of resolution. The structure was solved by Molecular Replacement using the package AMoRe with the Agkistrodon piscivorus piscivorus enzyme as search model and refined using the refinement program X-PLOR to a final R=18.7% and Rfree =27.4%. The asymmetric unit contains two monomers that can be chosen so that they present similar interactions to those described for the homologous myotoxin II from B. asper. The interface is, however, surprisingly small when compared to other dimeric structures and is less complementary than expected. A theoretical model for phospholipid building to BthTx-I suggests that no direct interactions between the sn-2 ester bond and K49 would be expected, thus explaining the lack of catalytic activity.

Financial support: FINEP and CNPq

CORRESPONDENCE TO:

Dra. Maria Teresa S. Giotto - Departamento de Física e Informática, Instituto de Física de São Carlos, USP, CEP 13560-970, Caixa Postal 369, São Carlos, SP, Brasil. email: [e-mail]

Publication Dates

• Publication in this collection
  08 Jan 1999
• Date of issue
  1997