A Phospholipid Substrate Molecule Residing in the Membrane Surface Mediates Opening of the Lid Region in Group IVA Cytosolic Phospholipase A2

Abstract

The 85-kDa GIVA 3 phospholipase A 2 (GIVA PLA 2 ) is a member of the superfamily of phospholipase A 2 enzymes (1, 2) that cleave fatty acids from the sn-2 position of membrane phospholipids. This enzyme was initially isolated from human platelets, and it is specific for phospholipids containing arachidonic acid in the sn-2 position (3, 4). The release of arachidonic acid is the critical first step in the biosynthesis of eicosanoids, which are potent mediators of inflammation and pain (5). There are a number of enzymes and routes by which arachidonic acid can be released from phospholipids, but experiments with GIVA PLA 2 knock-out mice have demonstrated its importance in many inflammatory processes (6 -8), thereby confirming the key role of the GIVA enzyme.The enzyme is composed of two domains, a Ca 2ϩ binding C2 domain and a ␣/␤-hydrolase domain that contains the catalytic site (9). Crystal structures of both the intact enzyme (PDB 1CJY) (10) and the C2 domain alone (11) (PDB 1RLW) have been solved. For the enzyme to be active, it must be sequestered to a phospholipid interface. Binding Ca 2ϩ to the C2 domain accomplishes this as does the binding of two different lipid mediators, e.g. ceramide 1 phosphate (12, 13) and phosphatidylinositol-(4,5) bis-phosphate (14, 15).The Ca 2ϩ binding C2 domain is a conserved domain that is present on many different lipid-binding proteins (16). Ca 2ϩ binding to this domain sequesters the protein to the lipid surface. Extensive studies have been carried out on the C2 domain using a variety of techniques to determine how Ca 2ϩ binding accomplishes lipid surface binding. These studies have included x-ray reflectivity, site-directed mutagenesis, NMR, EPR, and computational methods (17). These studies have shown (18 -23) that lipid binding entails the penetration of Ca 2ϩ binding loops one and three, composed of amino acids 35-39 and 96 -98, into the interface. However, these studies only deal with C2 binding to the membrane, not the intact cPLA 2 enzyme. How the presence of the ␣/␤-hydrolase domain affects surface binding has not been determined in detail due to the difficulties of working with such a large protein.Unlike the C2 domain, the binding of the catalytic domain of the enzyme to the lipid surface has not been extensively studied. Numerous studies using site-directed mutagenesis of the intact protein have localized amino acids that are important for lipid * This work was supported, in whole or in part, by National Institutes of Health Grants GM20501 (to E. A. D.) and CA099835, CA118595, GM037684, AI0220221, and AI022160 (to V. L. W.). This work was also supported by Discovery Grant UC10591 from the University of California Industry.-University Cooperative Research Program (to V. L. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 858-534-7390; E-mail: edennis@ucsd.edu.3 The abbreviations used...

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