Synergistic effect of Pb(2+) and phosphatidylinositol 4,5-bisphosphate on C2 domain-membrane interactions.

Ca2+-responsive C2 domains are peripheral membrane modules that target their host proteins to anionic membranes upon binding Ca2+ ions. Several C2 domain-containing proteins, such as protein kinase C isoenzymes (PKCs), have been identified as molecular targets of Pb2+, a known environmental toxin. We demonstrated previously that the C2 domain from PKCα (C2α) binds Pb2+ with high affinity and undergoes membrane insertion in the Pb2+-complexed form. The objective of this work was to determine the effect of phosphatidylinositol 4,5-bisphosphate (PIP2) on the C2α–Pb2+ interactions. Using nuclear magnetic resonance (NMR) experiments, we show that Pb2+ and PIP2 synergistically enhance each other’s affinity for C2α. Moreover, the affinity of C2α for PIP2 increases upon progressive saturation of the metal-binding sites. Combining the NMR data with the results of protein-to-membrane Forster resonance energy transfer and vesicle sedimentation experiments, we demonstrate that PIP2 can influence two aspects of C2α–Pb...