Independent Folding and Ligand Specificity of the C2 Calciumdependent Lipid Binding Domain of Cytosolic Phospholipase A 2 *

The Ca 2+ -dependent lipid binding domain of the 85-kDa cytosolic phospholipase A 2 (cPLA 2 ) is a homolog of C2 domains present in protein kinase C, synaptotagmin, and numerous other proteins involved in signal transduction. NH 2 -terminal fragments of cPLA 2 spanning the C2 domain were expressed as inclusion bodies in Escherichia coli, extracted with solvent to remove phospholipids, and refolded to yield a domain capable of binding phospholipid vesicles in a Ca 2+ -dependent manner. Unlike other C2 domains characterized to date, the cPLA 2 C2 domain bound preferentially to vesicles comprised of phosphatidylcholine in response to physiological concentrations of Ca 2+. Binding of the cPLA 2 C2 domain to vesicles in the presence of excess Ca 2+ chelator was induced by high concentrations of salts that promote hydrophobic interactions. Despite the selective hydrolysis of arachidonyl-containing phospholipid vesicles by cPLA 2, the cPLA 2 C2 domain did not discriminate among phospholipid vesicles containing saturated or unsaturated sn -2 fatty acyl chains. Moreover, the cPLA 2 C2 domain bound to phospholipid vesicles containing sn -1 and -2 ether linkages and sphingomyelin at Ca 2+ concentrations that caused binding to vesicles containing ester linkages, demonstrating that the carbonyl oxygens of the sn -1 and-2 ester linkage are not critical for binding. These results suggest that the cPLA 2 C2 domain interacts primarily with the headgroup of the phospholipid. The cPLA 2 C2 domain displayed selectivity among group IIA cations, preferring Ca 2+ approximately 50-fold over Sr 2+ and nearly 10,000-fold over Ba 2+ for vesicle binding. No binding to vesicles was observed in the presence of greater than 10 m m Mg 2+. Such strong selectivity for Ca 2+ over Mg 2+ reinforces the view that C2 domains link second messenger Ca 2+ to signal transduction events at the membrane.