Figure 9. Model of the role of Ca2+-dependent penetration of the anionic presynaptic membrane by synaptotagmin. The crystal structure of the core complex [Protein Data Bank (PDB) file 1SFC, containing syntaxin (red), SNAP-25 (synapse associated protein of 25 kD) (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 “+”) were rendered using PyMOL Molecular Graphics System (DeLano Scientific). The synaptic vesicle (SV), the presynaptic membrane (PM), the transmembrane domains, and the link between C2A and C2B were added in Adobe Photoshop. A, Docked synaptic vesicle with two synaptotagmin/SNARE complexes shown. B, syt/SNARE complex viewed end on from the site of synaptic vesicle/presynaptic membrane apposition (SNARE complex transmembrane domains in front of plane of section). Ca2+-independent priming between the C2B polylysine motif (yellow, space-filled residues) and SNAP-25 [green, space-filled residues (Zhang et al., 2002; Rickman et al., 2004; Loewen et al., 2006b)] holds the C2B Ca2+-binding site immediately adjacent to the SNARE complex and the C2A Ca2+-binding site nearby. The negative charge of the Ca2+-binding pockets (cluster of − symbols) prevents interactions between the tips of the C2 domains and the presynaptic membrane attributable to electrostatic repulsion. C, Ca2+ binding neutralizes the negative charge of the pockets resulting in a strong attraction of the negatively charged, phospholipid head groups of the presynaptic membrane by the bound Ca2+ (pink spheres +) and the basic residues at the tips of Ca2+-binding pockets (blue, space-filled residues +). Insertion of the hydrophobic residues at the tips of the C2 domains (gray, space-filled residues) into the core of the presynaptic membrane and then triggers fusion by promoting a local Ca2+-dependent buckling of the plasma membrane (Martens et al., 2007; Hui et al., 2009). D, syt/SNARE complexes viewed from the presynaptic membrane. Multiple syt:SNARE complexes (colored as in A–C) mediate fusion (Jahn and Scheller, 2006) of a single synaptic vesicle (gray ring, not to scale). Ca2+-dependent membrane penetration by synaptotagmin (large gray ovals, arrows) pulls the presynaptic membrane toward the vesicle in the vicinity of the transmembrane regions of the SNARE complexes (small gray circles, arrowheads).