Membrane-bound orientation and position of the synaptotagmin C2B domain determined by site-directed spin labeling

Site-directed spin labeling is used to determine the orientation and depth of insertion of the second C2 domain from synaptotagmin I (C2B) into membrane vesicles composed of phosphatidylcholine (PC) and phosphatidylserine (PS). EPR line shapes of spin-labeled mutants located with the Ca2+-binding loops of C2B broaden in the presence of Ca2+ and PC/PS vesicles, indicating that these loops undergo a Ca2+-dependent insertion into the membrane interface. Power saturation of the EPR spectra provides a position for each spin-labeled site along the bilayer normal, and these EPR-derived distance constraints, along with a high-resolution structure of the C2B domain, are used to generate a model for the domain orientation and position at the membrane interface. Our data show that the isolated C2B domain from-binding loops 1 and 3 is synaptotagmin 1 penetrates PC/PS membranes, and that the backbone of Ca2+ inserted below the level of a plane defined by the lipid phosphates. The side chains of several loop residues are within the bilayer interior, and both Ca2+-binding sites are positioned near a plane defined by the lipid phosphates. A Tb3+-based fluorescence assay is used to compare the membrane affinity of the C2B domain to that of the first synaptotagmin C2 domain (C2A). Both C2A and C2B bind PC/PS (75:25) membrane vesicles with a micromolar lipid affinity in the presence of metal ion. These results indicate that C2A and C2B have a similar membrane affinity and position when bound to PUPS (75:25) membrane vesicles. EPR spectroscopy indicates that the C2B domain has different interactions with PUPS membranes containing 1 mol % phosphatidylinositol 4,5-bisphosphate.