Crystallographic identification of Ca ^{2 B domain}

Synaptotagmin I has two tandem Ca²⁺‐binding C₂ 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 Å resolution, two in the Ca²⁺‐bound form at 1.04 Å (two bound Ca²⁺ ions) and 1.65 Å (three bound Ca²⁺ ions) resolution and one in the Sr²⁺‐bound form at 1.18 Å (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 Ca²⁺ in site 2 in the NMR structure. In the presence of Sr²⁺, which is also capable of triggering exocytosis, but with lower efficiency, only one cation binding site (site 1) was occupied in the crystallographic structure.