Crystallographic identification of Ca2+ and Sr2+ coordination sites in synaptotagmin IC2B domain

Synaptotagmin I has two tandem Ca2+-binding C-2 domains, which are essential for fast synchronous synaptic transmission in the central nervous system. We have solved four crystal structures of the C2B domain, one of them in the cation-free form at 1.50 Angstrom resolution, two in the Ca2+-bound form at 1.04 Angstrom (two bound Ca2+ ions) and 1.65 Angstrom (three bound Ca2+ ions) resolution and one in the Sr2+-bound form at 1.18 Angstrom (one bound Sr2+ ion) resolution. The side chains of four highly conserved aspartic acids (D303, D309, D363, and D365) and two main chain oxygens (M302:O and Y364:O), together with water molecules, are in direct contact with two bound Ca2+ ions (sites 1 and 2). At higher Ca2+ concentrations, the side chain of N333 rotates and cooperates with D309 to generate a third Ca2+ coordination site (site 3). Divalent cation binding sites 1 and 2 in the C2B domain were previously identified from NMR NOE patterns and titration studies, supplemented by site-directed mutation analysis. One difference between the crystal and NMR studies involves D371, which is not involved in coordination with any of the identified Ca2+ sites in the crystal structures, while it is coordinated to Ca in site 2 in the NMR structure. In the presence of Sr, which is also capable of triggering exocytosis, but with lower efficiency, only one cation binding site (site 1) was occupied in the crystallographic structure.