A structure-function study of the C2 domain of cytosolic phospholipase A2. Identification of essential calcium ligands and hydrophobic membrane binding residues.

The C2 domain of cytosolic phospholipase A2 (cPLA2) is involved in the Ca2+-dependent membrane binding of this protein. To identify protein residues in the C2 domain of cPLA2 essential for its Ca2+ and membrane binding, we selectively mutated Ca2+ ligands and putative membrane-binding residues of cPLA2 and measured the effects of mutations on its enzyme activity, membrane binding affinity, and monolayer penetration. The mutations of five Ca2+ ligands (D40N, D43N, N65A, D93N, N95A) show differential effects on the membrane binding and activation of cPLA2, 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 cPLA2 is largely driven by hydrophobic interactions resulting from the penetration of these residues into the hydrophobic core of the membrane. Leu39 and Val97 are fully inserted into the membrane, whereas Phe35 and Tyr96 are partially inserted. Finally, the mutations of four cationic residues in a beta-strand (R57E/K58E/R59E/R61E) have modest and negligible effects on the binding of cPLA2 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-alpha (Medkova, M., and Cho, W. (1998) J. Biol. Chem. 273, 17544-17552), these results demonstrate that C2 domains are not only a membrane docking unit but also a module that triggers membrane penetration of protein and that individual Ca2+ ions bound to the calcium binding loops play differential roles in the membrane binding and activation of their parent proteins.