The C2 domain is a membrane-targeting domain found in many cellular proteins involved in signal transduction or membrane trafficking. The majority of C2 domains co-ordinate multiple Ca2+ ions and bind the membrane in a Ca2+-dependent manner. To understand the mechanisms by which Ca2+ mediates the membrane binding of C2 domains, we measured the membrane binding of the C2 domains of group IV cytosolic phospholipase A2 (cPLA2) and protein kinase C-α (PKC-α) by surface plasmon resonance and lipid monolayer analyses. Ca2+ ions mainly slow the membrane dissociation of cPLA2-C2, while modulating both membrane association and dissociation rates for PKC-α-C2. Further studies with selected mutants showed that for cPLA2 a Ca2+ ion bound to the C2 domain of cPLA2 induces the intra-domain conformational change that leads to the membrane penetration of the C2 domain whereas the other Ca2+ is not directly involved in membrane binding. For PKC-α, a Ca2+ ion induces the inter-domain conformational changes of the protein and the membrane penetration of non-C2 residues. The other Ca2+ ion of PKC-α-C2 is involved in more complex interactions with the membrane, including both non-specific and specific electrostatic interactions. Together, these studies of isolated C2 domains and their parent proteins allow for the determination of the distinct and specific roles of each Ca2+ ion bound to different C2 domains.

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