INDEPENDENT FOLDING AND LIGAND SPECIFICITY OF THE C2 CALCIUM-DEPENDENT LIPID-BINDING DOMAIN OF CYTOSOLIC PHOSPHOLIPASE A(2)

The Ca2+-dependent lipid binding domain of the 85-kDa cytosolic phosph olipase A(2), (cPLA(2)) is a homolog of C2 domains present in protein kinase C, synaptotagmin, and numerous other proteins involved in signa l transduction, NH2-terminal fragments of cPLA(2), spanning the C2 dom ain were expressed as inclusion bodies in Escherichia coli, extracted with solvent to remove phospholipids, and refolded to yield a domain c apable of binding phospholipid vesicles in a Ca2+-dependent manner. Un like other C2 domains characterized to date, the cPLA(2), C2 domain bo und preferentially to vesicles comprised of phosphatidylcholine in res ponse to physiological concentrations of Ca2+. Binding of the cPLA(2), C2 domain to vesicles in the presence of excess Ca2+ chelator was ind uced by high concentrations of salts that promote hydrophobic interact ions. Despite the selective hydrolysis of arachidonyl-containing phosp holipid vesicles by cPLA(2), the cPLA(2), C2 domain did not discrimina te among phospholipid vesicles containing saturated or unsaturated sn- 2 fatty acyl chains. Moreover, the cPLA(2), C2 domain bound to phospho lipid vesicles containing sn-l and -2 ether linkages and sphingomyelin at Ca2+ concentrations that caused binding to vesicles containing est er linkages, demonstrating that the carbonyl oxygens of the sn-l and-a ester linkage are not critical for binding, These results suggest tha t 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 near ly 10,000-fold over Ba2+ for vesicle binding, No binding to vesicles w as observed in the presence of greater than 10 mM Mg2+. Such strong se lectivity for Ca2+ over Mg2+ reinforces the view that C2 domains link second messenger Ca2+ to signal transduction events at the membrane.