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 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, Förster resonance energy transfer, and vesicle cosedimentation 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, electron paramagnetic resonance 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 a 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.