Crystallographic identification of Ca²⁺ and Sr²⁺ coordination sites in synaptotagmin I C₂B 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 C ₂B 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.