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

Abstract

During Ca 2+ activation, the Ca 2+ -binding sites of C2 domains typically bind multiple Ca 2+ 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 Ca 2+ activation and positive cooperativity for the C2 domain of cytosolic phospholipase A 2 , which binds two Ca 2+ ions in sites I and II, separated by only 4.1 Å. First, each of the five coordinating side chains in the Ca 2+ -binding cleft is individually mutated and the effect on Ca 2+ -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 Ca 2+ 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 Ca 2+ 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 Ca 2+ binding, enabling the second Ca 2+ ion to bind with higher affinity than the first as required for positive cooperativity.The C2 domain is a ubiquitous membrane-targeting protein module found in a diverse array of signal transducing proteins that regulate key cellular processes at membrane surfaces (reviewed in refs 1-10). These processes include the generation of lipid-derived second messengers, vesicular targetting and fusion, GTPase regulation, protein phosphorylation, pore formation by cytolytic T cells, and ubiquitin-mediated protein degradation. The majority of C2 domains are activated by cytoplasmic Ca 2+ signals and dock to specific membrane-associated targets such as phospholipids or, less commonly, to membrane-bound proteins.The structures of representative Ca 2+ -regulated C2 domains, including the C2A domain of synaptotagmin I (Syt-IA), 1 the C2 domain of cytosolic phospholipase A 2 (α-isoform, cPLA 2 α), and the C2 domain of protein kinase C (PKC), have been determined by X-ray † Support provided by NIH Grants GM R01-063235 (to J.J.F.) and GM R01-054651 (to H.L.S./E.J.), and by a DOE/GTL Grant (to E.J.).

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