Cation Charge and Size Selectivity of the C2 Domain of Cytosolic Phospholipase A2

The C2 domain is a eukaryotic membrane-targeting protein module present in numerous signal transducing proteins that carry out key cellular functions at membranes (reviewed in refs 1 and 2). These processes include the generation of lipid second messengers, vesicular transport, GTPase regulation, protein phosphorylation, pore formation by cytolytic T cells, and ubiquitin-mediated protein degradation. C2 domains bind a variety of cellular targets, including phospholipids, inositol polyphosphates, and other membrane-associated proteins (1, 2). Many of these C2 domains bind to target membranes in response to the micromolar Ca2+ levels generated during intracellular Ca2+ fluxes. The structures of representative Ca2+-regulated C2 domains, including the C2A domain of synaptotagmin I (Syt-IA),1 the C2 domain of cytosolic phospholipase A2 (α-isoform, cPLA2-α), and the C2 domain of protein kinase C (β-isoform, PKC-β), have been determined by X-ray crystallography and NMR spectroscopy (3–8). These C2 domains are divided into two distinct topological classes (1), both of which are constructed of β-sandwich architecture. At one edge of the β-sandwich, two or more Ca2+ ions bind in an aspartate-lined cleft formed by three interstrand loops. Equilibrium Ca2+ binding measurements have shown that the isolated cPLA2-α C2 domain binds two Ca2+ ions with low micromolar affinity when free in solution, and with even higher affinity when bound to target phosphatidylcholine (PC) membranes (7, 9). Two Ca2+ ions have also been observed in the crystal structure of the domain (6, 8). The binding of the Ca2+ ions is positively cooperative and induces docking of the domain to phosphatidylcholine (PC) membranes (9–11) such that that the C2 domain is activated over the narrow micromolar range of Ca2+ concentrations achieved during intracellular Ca2+ signaling. In contrast, three Ca2+ ions bind to the Syt-IA and PKC-β C2 domains and induce docking to anionic lipids such as phosphatidylserine (PS). It has become clear that C2 domains exhibit different modes of membrane binding and Ca2+ activation parameters adapted for distinct signaling pathways (12). C2 domains regulated by micromolar cytoplasmic Ca2+ signals must possess the ability to selectively bind Ca2+ in the presence of 103–104-fold higher concentrations of K+, Na+, and Mg2+ (13). Several studies have indicated that physiological levels of these background cations do not induce binding of Ca2+-dependent C2 domains to target membranes (11, 14, 15), although a sub-millimolar Mg2+ concentration induces membrane binding by the C2A domain of synaptotagmin III (15). In addition, spherical divalent cations other than Ca2+ induce membrane docking by various C2 domains (14–17), including cPLA2-α (11). In general, the potency of these cations follows this order: Ca2+ > Sr2+ > Ba2+ ≫ Mg2+. This order of potency also reflects the ability of these ions to promote enzymatic activity of cPLA2-α (18). It is not clear, however, whether this cation specificity stems primarily from the metal affinity of the free C2 domain or from the stability of different domain–metal–membrane complexes. In principle, the target membrane could contribute to the observed cation specificity of C2 domains, since (i) target membranes significantly increase the Ca2+ affinity of the cPLA2-α, Syt-IA, and PKC-β C2 domains (12, 19), (ii) the membrane surface contacts the Ca2+-binding loops of the C2 domain when docked to target membranes (reviewed in ref 12), (iii) phospholipid membranes bind divalent cations with selectivity (20), and (iv) phospholipid may complete the coordination shell surrounding the exposed bound Ca2+ by displacing coordinating water molecules as observed for the C2 domain of PKC-α (21). Thus, a complete understanding of the molecular basis of the Ca2+ selectivity of the C2 domain requires the evaluation of metal ion binding by the C2 domain in both its membrane-free and -bound states. This study probed the Ca2+-binding site of the cPLA2-α C2 domain to better define its cation charge and size selectivity. The Ca2+-activated C2 domain is responsible for targeting cPLA2-α to cytoplasmic membranes (22–24), where its Ca2+-independent catalytic domain liberates arachidonic acid from glycerophospholipids to initiate eicosanoid pathways, including inflammation (25–27). When Ca2+ binds, the intrinsic tryptophan fluorescence of the cPLA2-α C2 domain increases due to structural, electrostatic, or dynamic changes that influence the environment of the sole tryptophan (Trp71) partially buried in the domain (9, 12). This fluorescence increase enables evaluation of the equilibrium and kinetic binding parameters of various metal ions for the cPLA2-α C2 domain in the presence and absence of target membranes. The results presented here demonstrate that spherical monovalent cations and Mg2+ are virtually excluded from the site at physiological concentrations, in the absence or presence of target membranes. In contrast, spherical divalent cations (larger than Mg2+) and trivalent cations bind to the site, even in the absence of membranes, and induce membrane docking by the domain. A relationship is observed between the ionic radius and the apparent stoichiometry of divalent cation binding to the isolated C2 domain, as revealed by the Hill coefficient of positive cooperativity. Furthermore, the degree of positive cooperativity is related to the ability of the divalent cation to induce membrane docking, suggesting that the binding of two metal ions is required to induce membrane docking. A model for the formation of a C2 domain–Ca2+–phospholipid complex is discussed.