Cation charge and size selectivity of the C2 domain of cytosolic phospholipase A2

C2 domains regulate numerous eukaryotic signaling proteins by docking to target membranes upon binding Ca2+. Effective activation of the C2 domain by intracellular Ca2+ signals requires high Ca2+ selectivity to exclude the prevalent physiological metal ions K+, Na+, and Mg2+. The cooperative binding of two Ca2+ ions to the C2 domain of cytosolic phospholipase A(2) (cPLA(2)-alpha) induces docking to phosphatidylcholine (PC) membranes. The ionic charge and size selectivities of this C2 domain were probed with representative mono-, di-, and trivalent spherical metal cations. Physiological concentrations of monovalent cations and Mg2+ failed to bind to the domain and to induce docking to PC membranes. Superphysiological concentrations of Mg2+ did bind but still failed to induce membrane docking. In contrast, Ca2+, Sr2+, and Ba2+ bound to the domain in the low micromolar range, induced electrophoretic mobility shifts in native polyactylamide gels, stabilized the domain against thermal denaturation, and induced docking to PC membranes. In the absence of membranes, the degree of apparent positive cooperativity in binding of Ca2+, Sr2+, and Ba2+ decreased with increasing cation size, suggesting that the C2 domain binds two Ca2+ or Sr2+ ions, but only one Ba2+ ion. These stoichiometries were correlated with the abilities of the ions to drive membrane docking, such that micromolar concentrations of Ca2+ and Sr2+ triggered docking while even millimolar concentrations of Ba2+ yielded poor docking efficiency. The simplest explanation is that two bound divalent cations are required for stable membrane association. The physiological Ca2+ ion triggered membrane docking at 20-fold lower concentrations than Sr2+, due to both the higher Ca2+ affinity of the free domain and the higher affinity of the Ca2+-loaded domain for membranes. Kinetic studies indicated that Ca2+ ions bound to the free domain are retained at least 5-fold longer than Sr2+ ions. Moreover, the Ca2+-loaded domain remained bound to membranes 2-fold longer than the Sr(2+)loaded domain. For both Ca2+ and Sr2+, the two bound metal ions dissociate from the protein-membrane complex in two kinetically resolvable steps. Finally, representative trivalent lanthanide ions bound to the domain with high affinity and positive cooperativity, and induced docking to PC membranes. Overall, the results demonstrate that both cation charge and size constraints contribute to the high Call selectivity of the C2 domain and suggest that formation of a cPLA2-alpha C2 domain-membrane complex requires two bound multivalent metal ions. These features are proposed to stem from the unique structural features of the metal ion-binding site in the C2 domain.