C2 domains from different Ca²⁺ signaling pathways display functional and mechanistic diversity

The ubiquitous C2 domain is a conserved Ca²⁺-triggered membrane-docking module that targets numerous signaling proteins to membrane surfaces where they regulate diverse processes critical for cell signaling. In this study, we quantitatively compared the equilibrium and kinetic parameters of C2 domains isolated from three functionally distinct signaling proteins: cytosolic phospholipase A₂-α (cPLA₂-α), protein kinase C-β (PKC-β), and synaptotagmin-IA (Syt-IA). The results show that equilibrium C2 domain docking to mixed phosphatidylcholine and phosphatidylserine membranes occurs at micromolar Ca²⁺ concentrations for the cPLA₂-α C2 domain, but requires 3- and 10-fold higher Ca₂₊ concentrations for the PKC-β and Syt-IA C2 domains ([Ca²⁺]_1/2 = 4.7, 16, 48 μM, respectively). The Ca²⁺-triggered membrane docking reaction proceeds in at least two steps: rapid Ca²⁺ binding followed by slow membrane association. The greater Ca²⁺ sensitivity of the cPLA₂-α domain results from its higher intrinsic Ca²⁺ affinity in the first step compared to the other domains. Assembly and disassembly of the ternary complex in response to rapid Ca²⁺ addition and removal, respectively, require greater than 400 ms for the cPLA₂-α domain, compared to 13 ms for the PKC-β domain and only 6 ms for the Syt-IA domain. Docking of the cPLA₂-α domain to zwitterionic lipids is triggered by the binding of two Ca²⁺ ions and is stabilized via hydrophobic interactions, whereas docking of either the PKC-β or the Syt-IA domain to anionic lipids is triggered by at least three Ca²⁺ ions and is maintained by electrostatic interactions. Thus, despite their sequence and architectural similarity, C2 domains are functionally specialized modules exhibiting equilibrium and kinetic parameters optimized for distinct Ca²⁺ signaling applications. This specialization is provided by the carefully tuned structural and electrostatic parameters of their Ca²⁺- and membrane-binding loops, which yield distinct patterns of Ca²⁺ coordination and contrasting mechanisms of membrane docking.