Structural analysis of a group III Glu62-phospholipase A2 from the scorpion, Mesobuthus tamulus: targeting and reversible inhibition by native peptides

Abstract

Group III phospholipase A₂ enzyme transcript from the Mesobuthus tamulus (Indian red scorpion) codes for three distinct products that include a large enzymatic subunit, a pentameric peptide and a small non-enzymatic subunit. The structures of these two subunits were modeled based on their sequence identity to bee venom PLA₂ and the partial sequence of MU2 adaptin subunit of AP2 clathrin adaptor, respectively. The enzymatic subunit comprises of three helices, the calcium binding loop and a substrate binding hydrophobic channel where the structure is stabilized by four disulfide bonds. The active site of the enzyme shows a catalytic histidine residue. Interestingly, there is a conservative mutation of the conserved aspartic acid, a classical participant of catalysis in this enzyme family, to glutamic acid. However, the side chain oxygen atoms of this glutamate are oriented away from the catalytic histidine implicating the non-participation of this residue in stabilizing the tautomeric conformation of the histidine. The acidic non-enzymatic subunit comprises of extensive hydrophobic residues with a conformation of an anti-parallel β-sheets making it ideal for tissue specific targeting. The native pentapeptide with the sequence Alanine-Arginine-Serine-Alanine-Arginine was docked to the enzymatic subunit. The peptide ligand occupies the hydrophobic cavity and makes a plethora of interactions with the residues in the channel, including a hydrogen bond with the crucial catalytic histidine and coordinate bond with the calcium ion. This ligand has a binding constant (K_D) of 1.5 μM. This makes the ligand a potential reversible inhibitor, ideal to prevent the enzyme from interacting with non-specific molecules enroute to the target. The enzyme-ligand complex also provides a model to understand the stereochemistry required for the design of more potent drug molecules against such enzyme drug targets.