Non-Native Metal Ion Reveals the Role of Electrostatics in Synaptotagmin 1-Membrane Interactions.

C2 domains are independently folded modules that often target their host proteins to anionic membranes in a Ca-dependent manner. In these cases, membrane association is triggered by Ca binding to the negatively charged loop region of the C2 domain. Here, we used a non-native metal ion, Cd, in lieu of Ca 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, Förster resonance energy transfer, and vesicle cosedimentation assays, we demonstrate that, although Cd 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 Cd binding to the loop regions of the C2A and C2B domains and the impaired ability of Cd to directly coordinate the lipids. In contrast, electron paramagnetic resonance experiments revealed that Cd 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 a full complement of divalent metal ions and specific lipid coordination in Cd-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 Ca further augments membrane binding by the formation of metal ion-lipid coordination bonds and additional Ca ion binding to the C2 domain loop regions.