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Structural studies with the BthTX-I-BPB, the first non-catalytic PLA2 bound to p-bromophenacyl bromide structure – insights into the lack of its toxic activities.
Marchi-Salvador, D.P.1, Fernandes, C.A.H.1, Amui, S.F.2, Soares, A.M.2, and Fontes, M.R.M.1
1 Dept. de Física e Biofísica, Instituto de Biociências, UNESP, Botucatu, SP – fontes@ibb.unesp.br marchi@ibb.unesp.br
2 Dept. de Análises Clínica, Toxicológicas e Bromatológicas, FCF-USP, Ribeirão Preto, SP.
Phospholipases A2 are among the main components of Bothrops venoms
and consists of a broad range of enzymes defined by their ability to
catalyze specifically the hydrolysis of the center (sn-2) ester bond of substrate phospholipids. BthTX-I is a basic myotoxic PLA2, catalytically inactive, purified from Bothrops jararacussu snake venoms. Alkylation of His48 residue of active site with p-bromophenacyl
bromide (BPB), reduced 45%, 85% and 15% of its myotoxic, cytotoxic and
edema inducing activity, respectively, with no significant change in
its liposome-disrupting activity. These toxic activities reduction and
the relationship with the active site residues is not understand, and
the structure of this complex may contribute to this study. Here, we
report the crystallization experiments, X-ray diffraction results,
structure determination, and preliminary analysis of BthTX-I-BPB.
Crystals of this complex were obtained by hanging-drop vapour-diffusion
method in which the protein solution was equilibrated against a
reservoir solution containing sodium acetate pH 6.0, 2-propanol and
polyethylene glycol 4000, 18ºC after two weak. X-ray diffraction data
of a single BthTX-I-BPB crystal was collected using a Synchrotron
Radiation Source (LNLS-MX1, Campinas, Brazil). Data were processed using the Denzo/Scalepack program at 2.28 Å resolution. The crystals belong to P212121 space group with approximated cell constants a=49.2, b=65.8, c=85.4 Å. The data are 94.0% complete with Rmerge=4.7% and I/sigma(I)=4.81 in the last shell. The volume of the unit cell is compatible with two molecules in the asymmetric unit (Vm=2.5 Å3/Da, 51.8% solvent content) and there is a BPB ligand at the active site (His48) for each monomer. This complex is the first Lys49-PLA2 structure chemically modified by p-bromophenacyl bromide solved until this moment and the reasons to the decrease of myotoxicity, citotoxicity and edema inducing activities by binding of BPB may be understand. This work has received financial support from FAPESP, FUNDUNESP, CNPq and LNLS.
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