STRUCTURE–BASED DESIGN OF ANTI-INFLAMMATORY AGENTS: CRYSTAL STRUCTURE OF A COMPLEX FORMED BETWEEN COBRA VENOM PHOSPHOLIPASE A2 AND A DESIGNED POTENT PEPTIDE INHIBITOR VAL-ALA-PHE-ARG-SER- (VAFRS) AT 1.9 Å RESOLUTION.

R. K. Singh, J. Makker, P. Vikram, M. Paramsivam, T. Jabeen, S. Sharma, S. Dey, P. Kaur, A. Srinivasan and T. P. Singh

Department of Biophysics, All India Institute of Medical Sciences, New Delhi-110029

India (rajendra84@hotmail.com)

Phospholipase A₂ (PLA₂) is a key enzyme involved in the production of prostaglandins and other related compounds collectively known as eicosanoids. These substances mediate inflammatory response in humans. Among several structure-based designed peptide molecules, a pentapeptide VAFRS showed an inhibition of PLA₂ with a binding constant of 10^-11M. In order to understand the mechanism of binding and evaluate the interactions between PLA₂ and peptide VAFRS, a complex between PLA₂ and VAFRS was prepared. The complex was crystallized using 35% ethanol as a precipitant. The crystals belong to space group P4₁ with a = b = 42.8 Å, and c = 65.9 Å. The crystals diffracted to 1.9 Å resolution. The structure was determined by molecular replacement method and refined to an R-value of 0.182. The peptide was located at the binding site of PLA₂. It formed several hydrogen bonds and van der Waals interactions. The most notable interactions were observed with the Arg of peptide molecule. The side chain of arginine formed multiple interactions with Asp49, His 48, Cys 45, Gly 30, Tyr 28 indicating the significance of its substitution at polar end of the peptide. The VAF part of the peptide was involved in a number of van der Waals interactions in the hydrophobic channel. The prominent conformational differences between the native cobra PLA₂ and the PLA₂ in the complex were restricted to ß-wing and the C- terminal regions of the enzyme. Indeed, the structure provided an excellent model based on the method of structure based drug design.