Ca2+ Activation of the cPLA2 C2 Domain: Ordered Binding of Two Ca2+ Ions with Positive Cooperativity†

During Ca2+ activation, the Ca2+-binding sites of C2 domains typically bind multiple Ca2+ ions in close proximity. These binding events exhibit positive cooperativity, despite the strong charge repulsion between the adjacent divalent cations. Using both experimental and computational approaches, the present study probes the detailed mechanisms of Ca2+ activation and positive cooperativity for the C2 domain of cytosolic phospholipase A2, which binds two Ca2+ ions in sites I and II, separated by only 4.1 A. First, each of the five coordinating side chains in the Ca2+-binding cleft is individually mutated and the effect on Ca2+-binding affinity and cooperativity is measured. The results identify Asp 43 as the major contributor to Ca2+ affinity, while the two coordinating side chains that provide bridging coordination to both Ca2+ ions, Asp 43 and Asp 40, are observed to make the largest contributions to positive cooperativity. Electrostatic calculations reveal that Asp 43 possesses the highest pseudo-pKa of ...