Figure 5.

Ca²⁺ binding by C₂A is an essential component of the electrostatic switch. The crystal structure of the core complex [PDB file 1SFC, containing syntaxin (red), SNAP-25 (green), and VAMP/synaptobrevin (blue)], the NMR structures of the C2A (PDB file 1BYN) and C₂B (PDB file 1K5W) domains of synaptotagmin (yellow), and Ca²⁺ (green spheres “+”) are shown to scale using PyMOL. The membranes, the transmembrane domains, and the link between C2A and C₂B were added in Adobe Photoshop. A) Cross section of a docked vesicle showing two SNARE complexes (SNAREs) and their associated synaptotagmin molecules (syt). B) One syt/SNARE complex viewed from the site of vesicle/presynaptic membrane apposition. A Ca²⁺-independent docking/priming interaction between the C₂B polylysine motif (yellow, space-filled residues) and SNAP-25 [green, space-filled residues, (Rickman et al., 2004; Loewen et al., 2006b)] holds the C₂B Ca²⁺-binding site immediately adjacent to the presynaptic membrane with the C2A Ca²⁺-binding site further removed. In the absence of Ca²⁺, the conserved aspartate residues (red residues: syt^C2A-D2, space-filled; the rest as sticks) within the pockets create a high concentration of negative charge (cluster of “−”s) resulting in electrostatic repulsion of the presynaptic membrane that prevents any membrane interactions by the tips of the C2 domains. C) Upon Ca²⁺ binding, the electrostatic repulsion of the pockets is neutralized thereby initiating the electrostatic switch: a strong attraction of the negatively-charged membrane by the bound Ca²⁺ (green spheres “+”) and the basic residues at the tips of Ca²⁺-binding pockets (blue stick residues “+”). Insertion of the hydrophobic residues (grey stick residues) at the tips of the C2 domains into the core of the presynaptic membrane then triggers fusion by promoting a local Ca²⁺-dependent positive curvature of the plasma membrane (Martens et al., 2007; Hui et al., 2009; Paddock et al., 2011). The C₂ domain interactions with the membrane likely pull the synaptic vesicle (upper grey membrane) toward the presynaptic membrane (lower grey membrane). The Ca²⁺-induced increase in positive charge at the end of the C₂B domain also likely increases the strength of the electrostatic interaction between the C₂B polylysine motif and the SNARE complex, resulting in simultaneous binding of the SNARE complex and the presynaptic membrane (Davis et al., 1999; Bhalla et al., 2006; Loewen et al., 2006a; Dai et al., 2007). D) Membrane penetration by multiple synaptotagmins (large grey ovals, arrows) would pull the plasma membrane toward the vesicle in a ring around the SNARE transmembrane domains (small grey circles, arrowheads) facilitating fusion.