Roles of catalytic domain residues in interfacial binding and activation of group IV cytosolic phospholipase A2

Abstract

Group IV cytosolic phospholipase A2 (cPLA2) has been shown to play a critical role in eicosanoid biosynthesis. cPLA2 is composed of the C2 domain that mediates the Ca2+-dependent interfacial binding of protein and the catalytic domain. To elucidate the mechanism of interfacial activation of cPLA2, we measured the effects of mutations of selected ionic and hydrophobic residues in the catalytic domain on the enzyme activity and the membrane binding of cPLA2. Mutations of anionic residues located on (Glu419 and Glu420) or near (Asp436, Asp438, Asp439, and Asp440) the active site lid enhanced the affinity for cPLA2 for anionic membranes, implying that the electrostatic repulsion between these residues and the anionic membrane surface might trigger the opening of the active site. This notion is further supported by a biphasic dependence of cPLA2 activity on the anionic lipid composition of the vesicles. Mutations of a cluster of cationic residues (Lys541, Lys543, Lys544, and Arg488), while significantly enhancing the activity of enzyme, abrogated the specific activation effect by phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2). These data, in conjunction with cell activity of cPLA2 and mutants transfected into HEK293 cells, suggest that the cationic residues form a specific binding site for PtdIns(4,5)P2 and that the specific PtdIns(4,5)P2 binding is involved in cellular activation of cPLA2. Also, three hydrophobic residues at the rim of the active site (Ile399, Leu400, and Leu552) were shown to partially penetrate the membrane, thereby promoting membrane binding and activation of cPLA2. Based on these results, we propose an interfacial activation mechanism for cPLA2 which involves the removal of the active site lid by non-specific electrostatic repulsion, the interdomain hinge movement induced by specific PtdIns(4,5)P2 binding, and the partial membrane penetration by catalytic domain hydrophobic residues.