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 Å. 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 Ca ²⁺ 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-pK_a of the coordinating acidic residues, as well as the highest general cation affinity, due to its relatively buried location within 3.5 Å of seven protein oxygens with full or partial negative charges. These calculations therefore explain the greater importance of Asp 43 in defining the Ca²⁺ affinity. Overall, the experimental and computational results support an activation model in which the first Ca ²⁺ ion binds usually to site I, thereby preordering both bridging side chains Asp 40 and 43, and partially or fully deprotonating the three coordinating Asp residues. This initial binding event prepares the conformation and protonation state of the remaining site for Ca²⁺ binding, enabling the second Ca²⁺ ion to bind with higher affinity than the first as required for positive cooperativity.