Lipid binding ridge on loops 2 and 3 of the C2A domain of synaptotagmin I as revealed by NMR spectroscopy

The C(2)A domain of synaptotagmin I, which binds Ca2+ and anionic phospholipids, serves as a Ca2+ sensor during excitation-secretion coupling. We have used multidimensional NMR to locate the region of C(2)A from rat synaptotagmin I that interacts, in the presence of Ca2+, with phosphatidylserine. Untagged, recombinant C(2)A was double-labeled with C-13 and N-15, and triple-resonance NMR data were collected from C(2)A samples containing either Ca2+ alone or Ca2+ plus 6:0 phosphatidylserine. Phospholipid binding led to changes in chemical shifts of backbone atoms in residues Arg(233) and Phe(234) of loop 3 (a loop that also binds Ca2+) and His(198), Val(205), and Phe(206) of loop 2. These residues lie along a straight line on a surface ridge of the C(2)A domain. The only other residue that exhibited appreciable chemical shift changes upon adding lipid was His(254); however, because His(254) is located on the other side of the molecule from the phospholipid docking site defined by the other residues, its shifts may result from nonspecific interactions. The results show that the "docking ridge" responsible for Ca2+-dependent membrane association is localized on the opposite side of the C(2)A domain from the transmembrane and C2B domains of synaptotagmin.