Crystallographic identification of Ca2+ and Sr2+ coordination sites in synaptotagmin I C2B domain

Abstract

Synaptotagmin I has two tandem Ca(2+)-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 C(2)B domain, one of them in the cation-free form at 1.50 A resolution, two in the Ca(2+)-bound form at 1.04 A (two bound Ca(2+) ions) and 1.65 A (three bound Ca(2+) ions) resolution and one in the Sr(2+)-bound form at 1.18 A (one bound Sr(2+) 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 Ca(2+) ions (sites 1 and 2). At higher Ca(2+) concentrations, the side chain of N333 rotates and cooperates with D309 to generate a third Ca(2+) coordination site (site 3). Divalent cation binding sites 1 and 2 in the C(2)B 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 Ca(2+) sites in the crystal structures, while it is coordinated to Ca(2+) in site 2 in the NMR structure. In the presence of Sr(2+), which is also capable of triggering exocytosis, but with lower efficiency, only one cation binding site (site 1) was occupied in the crystallographic structure.

Figure 1.

Structure of C₂B-B. (A) Front and side view of ribbon diagrams of the structure of C₂B-B with 2 Ca²⁺ ions. β-Strands are labeled from 1 to 8, while helices are labeled H1 and H2. Ca²⁺ ions are labeled Ca1 and Ca2. (B) Front and back view of electrostatic potential surface of C₂B-B. The acidic and basic residues are colored green and orange, respectively.

Figure 2.

Superimposition of crystal structures C₂B-A, C₂B-B, C₂B-C and C₂B-D and 20 C₂B-NMR structures. The Cα traces of C₂B-A, C₂B-B, C₂B-C and C₂B-D are colored yellow, red, blue, and green, respectively. The C₂B-NMR structures are colored gray. The orientations are the same as in Figure 1A ▶.

Figure 3.

Cation-binding sites in (A) superpositioned C₂B-NMR structures and (B) C₂B-B, (C) C₂B-C, and (D) C₂B-D crystal structures. The Ca²⁺ and Sr²⁺ ions are colored orange. The oxygen atoms that coordinate the cations are colored red. Water molecules are represented as small red spheres. Dashed lines indicate pentagonal bipyramidyl coordination to Ca²⁺ sites (B,C) and tetragonal bipyramidyl coordination to Sr²⁺ site (D) in the crystal structures.

Figure 4.

(A) Partial amino acid sequences alignment of Syt homology group. This includes Syt I: synaptotagmin I (GenBank accession no. P21707); Syt II: synaptotagmin II (P29101); Syt III: synaptotagmin III (P40748); Syt IV: synaptotagmin IV (P50232); Syt V: synaptotagmin V (P47861); Syt VI: synaptotagmin VI (AAA87724); Syt VII: synaptotagmin VII (AAA87725), from Rattus norvegicus. The secondary structural elements of Syt I are also indicated. Schematic of Ca²⁺-binding sites 1, 2 and 3 (B) and Sr²⁺ binding site 1 (C). The cation binding sites are indicated by larger pink balls, while their coordination to amino acids and waters are indicated by smaller pink and yellow balls, respectively. The three Ca²⁺ coordination sites in B are of the pentagonal bipyramidyl type, while the single Sr²⁺ coordination site in C is of the tetragonal bipyramidyl type. These coordinations (distances of ≈2.5 Å) are indicated by blue lines. One additional, slightly longer coordination (distances of ≈2.7 Å) was observed for Ca2+ sites 1 and 2 (dashed green lines), D363:OD2 to Ca1 and D365:OD1 to Ca2, respectively (Table 2). These two interactions are from the bidentate aspartate, which is in coordination with the Ca²⁺-binding site (D363:OD1 to Ca1 and D365:OD2 to Ca2), and therefore excluded from the coordination system. Two hydrogen bonds are indicated by dashed blue lines.