Many membrane-associated enzymes, including those of the phospholipase C (PLC) superfamily, are regulated by specific interactions with lipids. Previously, we have shown that the C2 domain of PLC δ1 is required for phosphatidylserine (PS)-dependent enzyme activation and that activation requires the presence of Ca 2+. To identify the site of interaction and the role of Ca 2+ in the activation mechanism, we mutagenized three highly conserved Ca 2+ binding residues (Asp-653, Asp-706, and Asp-708) to Gly in the C2 domain of PLC δ1. The PS-dependent Ca 2+ binding affinities of the mutant enzymes D653G, D706G, and D708G were reduced by 1 order of magnitude, and the maximal level of Ca 2+ binding was reduced to half of that of the native enzyme. The level of Ca 2+-dependent PS binding was also reduced in the mutant enzymes. Under basal conditions, the Ca 2+ dependence and the maximal level of hydrolysis of phosphatidylinositol 4,5-bisphosphate were not altered in the mutants. However, the Ca 2+-dependent PS stimulation was severely defective. PS reduces the K m of the native enzyme almost 20-fold, but far less for the mutants. Replacing Asp-653, Asp-706, and Asp-708 simultaneously with glycine in the C2 domain of PLC δ1 leads to a complete and selective loss of the stimulation and binding by PS. These results show that D653, D706, and D708 are required for Ca 2+ binding in the C2 domain and demonstrate a mechanism by which C2 domains can mediate regulation of enzyme activity by specific lipid ligands.
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