C2 domains are independently folded modules that often target their host proteins to anionic membranes in a Ca2+-dependent manner. In these cases, membrane association is triggered by Ca2+ binding to the negatively charged loop region of the C2 domain. Here, we used a non-native metal ion, Cd2+, in lieu of Ca2+ to gain insight into the contributions made by long-range Coulombic interactions and direct metal ion-lipid bridging to membrane binding. Using X-ray crystallography, NMR, FRET, and vesicle co-sedimentation assays, we demonstrate that, although Cd2+ binds to the loop region of C2A/B domains of Synaptotagmin 1 with high affinity, long-range Coulombic interactions are too weak to support membrane binding of individual domains. We attribute this behavior to two factors: the stoichiometry of Cd2+ binding to the loop regions of the C2A and C2B domains and the impaired ability of Cd2+ to directly coordinate the lipids. In contrast, EPR experiments revealed that Cd2+ does support membrane binding of the C2 domains in full-length Synaptotagmin 1, where the high local lipid concentrations that result from membrane tethering can partially compensate for lack of full complement of divalent metal ions and specific lipid coordination in Cd2+-complexed C2A/B domains. Our data suggest that long-range Coulombic interactions alone can drive the initial association of C2A/B with anionic membranes, and that Ca2+ further augments membrane binding by the formation of metal ion-lipid coordination bonds and additional Ca2+ ion binding to the C2 domain loop regions.

words]Lead (Pb) is a potent neurotoxin that disrupts synaptic neurotransmission. We report that Synaptotagmin I (SytI), a key regulator of Ca 2+ -evoked neurotransmitter release, has two highaffinity Pb 2+ binding sites that belong to its cytosolic C2A and C2B domains. The crystal structures of Pb 2+ -complexed C2 domains revealed that protein-bound Pb 2+ ions have holodirected coordination geometries and all-oxygen coordination spheres. The on-rate constants of Pb 2+ binding to the C2 domains of SytI are comparable to those of Ca 2+ and are diffusion-limited. In contrast, the off-rate constants are at least two orders of magnitude smaller, indicating that Pb 2+ can serve as both thermodynamic and kinetic trap for the C2 domains. We demonstrate, using NMR spectroscopy, that population of these sites by Pb 2+ ions inhibits further Ca 2+ binding despite the existing coordination vacancies. Our work offers a unique insight into the bioinorganic chemistry of Pb(II) and suggests a mechanism by which low concentrations of Pb 2+ ions can interfere with the Ca 2+ -dependent function of SytI in the cell.