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

Ca(2+)-responsive C2 domains are peripheral membrane modules that target their host proteins to anionic membranes upon binding Ca(2+) ions. Several C2 domain-containing proteins, such as protein kinase C isoenzymes (PKCs), have been identified as molecular targets of Pb(2+), a known environmental toxin. We demonstrated previously that the C2 domain from PKCα (C2α) binds Pb(2+) with high affinity and undergoes membrane insertion in the Pb(2+)-complexed form. The objective of this work was to determine the effect of phosphatidylinositol 4,5-bisphosphate (PIP(2)) on the C2α-Pb(2+) interactions. Using nuclear magnetic resonance (NMR) experiments, we show that Pb(2+) and PIP(2) synergistically enhance each other's affinity for C2α. Moreover, the affinity of C2α for PIP(2) increases upon progressive saturation of the metal-binding sites. Combining the NMR data with the results of protein-to-membrane Förster resonance energy transfer and vesicle sedimentation experiments, we demonstrate that PIP(2) can influence two aspects of C2α-Pb(2+)-membrane interactions: the affinity of C2α for Pb(2+) and the association of Pb(2+) with the anionic sites on the membrane. Both factors may contribute to the toxic effect of Pb(2+) resulting from the aberrant modulation of PKCα activity. Finally, we propose a mechanism for Pb(2+) outcompeting Ca(2+) from membrane-bound C2α.