Independent folding and ligand specificity of the C2 calcium-dependent lipid binding domain of cytosolic phospholipase A2

The Ca2+-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, NH2-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 Ca2+-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 Ca2+. Binding of the cPLA(2), C2 domain to vesicles in the presence of excess Ca2+ 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-l and -2 ether linkages and sphingomyelin at Ca2+ concentrations that caused binding to vesicles containing ester linkages, demonstrating that the carbonyl oxygens of the sn-l and-a 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 Ca2+ approximately 50-fold over Sr2+ and nearly 10,000-fold over Ba2+ for vesicle binding, No binding to vesicles was observed in the presence of greater than 10 mM Mg2+. Such strong selectivity for Ca2+ over Mg2+ reinforces the view that C2 domains link second messenger Ca2+ to signal transduction events at the membrane.