Biophysics, All India Institute of Medical Sciences^* Department of Biochemistry and Molecular Biology, University of Hamburg c/o DESY, Building 22a, 22603 Hamburg, Germany^**
○Nagendra Singh^* R. Prem Kumar^* Sujata Sharma^* Marcus Perbandt^** Punit Kaur^* Christian Betzel^** Tej P. Singh^*

Inhibition of synthesis of pro-inflammatory eicosanoids by an anti-inflammatory agent indomethacin has been attributed to its action against the enzymes which are involved in the eicosanoids biosynthesis pathway particularly cyclo-oxygenase. In the first step of this pathway, phospholipase A₂ (PLA₂) hydrolyzes acyl group at C2 of phospholipids and produces arachidonic acid which serves as a substrate for cyclooxygenase. Therefore, synthesis of eicosanoids will also be inhibited if the supply of precursor fatty acid is reduced by blocking the action of enzyme PLA₂. It was reported that indomethacin inhibits the action of PLA₂ in an unusual way but its mode of action and the precise site of binding in the hydrophobic channel are not known. Therefore, it is of great interest to analyze the detailed three-dimensional structure of PLA₂ complex with indomethacin. The initial binding studies using SPR indicated the value of 1.3 × 10^-6 M for the dissociation constant (Kd). The crystal structure of the PLA₂ complex with indomethacin was determined at 1.4Å resolution and refined to an R_cryst factor of 18.8% (R_free factor of 20.2%). The final model contains 944 protein atoms, 25 atoms of indomethacin, 235 water molecules and 4 sulfate ions. The structure revealed the site of binding of indomethacin at a different location in the hydrophobic channel than the conventional site occupied by PLA₂ inhibitors. Indomethacin carboxylic group oxygen atom O1 interacts with Asp 49 and His 48 through catalytic water molecule while O2 forms an ionic interaction with Lys 69. In order to be able to form these interactions, indomethacin is oriented parallel to helices H2 and H3. This is a novel site and thus it is of utmost importance for improving the design of PLA₂ inhibitors.