A Structure-Function Study of the C2 Domain of Cytosolic Phospholipase A2

Abstract

The C2 domain of cytosolic phospholipase A 2 (cPLA 2) is involved in the Ca 2؉ -dependent membrane binding of this protein. To identify protein residues in the C2 do-main of cPLA 2 essential for its Ca 2؉ and membrane bind-ing, we selectively mutated Ca 2؉ ligands and putative membrane-binding residues of cPLA 2 and measured the effects of mutations on its enzyme activity, membrane binding affinity, and monolayer penetration. The muta-tions of five Ca 2؉ ligands (D40N, D43N, N65A, D93N, N95A) show differential effects on the membrane bind-ing and activation of cPLA 2 , indicating that two calcium ions bound to the C2 domain have differential roles. The mutations of hydrophobic residues (F35A, M38A, L39A, Y96A, Y97A, M98A) in the calcium binding loops show that the membrane binding of cPLA 2 is largely driven by hydrophobic interactions resulting from the penetra-tion of these residues into the hydrophobic core of the membrane. Leu 39 and Val 97 are fully inserted into the membrane, whereas Phe 35 and Tyr 96 are partially in-serted. Finally, the mutations of four cationic residues in a ␤-strand (R57E/K58E/R59E/R61E) have modest and negligible effects on the binding of cPLA 2 to zwitterionic and anionic membranes, respectively, indicating that they are not directly involved in membrane binding. In conjunction with our previous study on the C2 domain of protein kinase C-␣ (Medkova, M., and Cho, W. (1998) J. Biol. Chem. 273, 17544 –17552), these results demon-strate that C2 domains are not only a membrane docking unit but also a module that triggers membrane penetra-tion of protein and that individual Ca 2؉ ions bound to the calcium binding loops play differential roles in the membrane binding and activation of their parent proteins. Phospholipases A 2 (PLA 2 s 1 ; EC 3.1.1.4) are a large family of lipolytic enzymes that catalyze the hydrolysis of the fatty acid ester at the sn-2-position of phospholipids (1, 2). Among various mammalian PLA 2 s, 85-kDa cytosolic PLA 2 (cPLA 2) can selec-tively liberate arachidonic acid from membrane phospholipids, which can be converted to potent inflammatory lipid mediators, prostaglandins, thromboxanes, leukotrienes, and lipoxins, col-lectively known as eicosanoids (3). Recent genetic studies showed that the deletion of the cPLA 2 gene results in loss of lipid mediator biosynthesis (4, 5). cPLA 2 is therefore an attrac-tive target for developing specific inhibitors that can be used as novel anti-inflammatory drugs. cPLA 2 is translocated to endo-plasmic reticulum membranes and nuclear envelopes in re-sponse to a rise in intracellular Ca 2ϩ (6, 7). This membrane translocation of cPLA 2 is mediated by its amino-terminal C2 domain, which contains calcium and membrane binding sites (8, 9). However, the mechanism by which the C2 domain of cPLA 2 drives its Ca 2ϩ -dependent membrane targeting is not fully understood. Our recent structure-function study of the C2 domain of protein kinase C-␣ showed that the C2 domain not only brings the protein to the membrane surface but also trig-gers the membrane penetration of protein (10). Also, the study revealed that individual Ca 2ϩ ions bound to the C2 domain play differential roles in membrane binding and activation. Tertiary structures of the C2 domains of protein kinase C (11) and cPLA 2 (12, 13), despite their overall similarity, have no-ticeable differences; i.e. they have different ␤-strand connectiv-ity (14), and, more importantly, the C2 domain of cPLA 2 con-tains two clusters of exposed hydrophobic residues in the calcium binding loops (see Fig. 1A) (12). Consistent with this unique structure, both cPLA 2 (15) and its isolated C2 domain (16) have been shown to be able to penetrate into the mem-brane. In this study, we performed an extensive structure-function analysis of the C2 domain of cPLA 2 to assess the roles of six hydrophobic residues and five Ca 2ϩ ligands located in the calcium binding loops in the membrane binding and activation of cPLA 2 . We also determined the role of a cluster of cationic residues in a ␤-strand. In conjunction with our previous study on protein kinase C-␣, this study reveals interesting similari-ties and differences between the two C2 domains.