Figure 7.

Model of the role played by the conserved basic residues in Ca²⁺-dependent interactions with the anionic presynaptic membrane. The crystal structure of the core complex [PDB file 1SFC, containing syntaxin (red), SNAP-25 (green), and VAMP (vesicle-associated membrane protein)/synaptobrevin (blue)], the nuclear magnetic resonance structures of the C₂A (PDB file 1BYN) and C₂B (PDB file 1K5W) domains of synaptotagmin (yellow), and Ca²⁺ (pink) are shown to scale using PyMOL Molecular Graphics System (DeLano Scientific). The membranes, the transmembrane domains, and the link between C₂A and C₂B were added in Adobe Photoshop. Left, A Ca²⁺-independent priming interaction between the C₂B polylysine motif (yellow, space-filled residues) and SNAP-25 (green, space-filled residues) holds the C₂A and C₂B Ca²⁺ binding sites in close proximity to the presynaptic membrane. We diagrammed the interaction between the C₂B polylysine motif and SNAP-25 within the SNARE complex (Zhang et al., 2002; Rickman et al., 2004, 2006; Dai et al., 2007), because mutation of the C₂B polylysine motif impairs this interaction (Bai et al., 2004) and disrupts synaptotagmin's ability to increase the speed of SNARE-mediated liposome fusion in the absence of any PIP₂ (Loewen et al., 2006). However, an interaction with PIP₂, which is located specifically in the presynaptic membrane, could also serve this purpose (Bai et al., 2004; Araç et al., 2006). In the absence of Ca²⁺, the high concentration of negative charge in the Ca²⁺-binding pockets repulses the negatively charged presynaptic membrane, preventing synaptotagmin's conserved basic residues (blue, space-filled residues) from interacting with the membrane. Right, After Ca²⁺ entry, the negative charge of the Ca²⁺-binding pockets is neutralized by the bound Ca²⁺, which initiates the electrostatic switch: a strong attraction of the negatively charged, phospholipid head groups by the bound Ca²⁺ and the basic residues at the tips of Ca²⁺-binding pockets that draws the synaptic vesicle (SV) toward the presynaptic membrane (PM). Insertion of the hydrophobic residues (gray, stick residues) at the tips of the C₂ domains into the core of the presynaptic membrane (Chapman and Davis, 1998) may help trigger fusion by promoting a local Ca²⁺-dependent buckling of the plasma membrane (Chapman and Davis, 1998; Martens et al., 2007). 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., 2006; Dai et al., 2007).