Synaptotagmin



Synaptotagmins constitute a family of membrane-trafficking proteins that are characterized by an N-terminal transmembrane region (TMR), a variable linker, and two C-terminal C2 domains - C2A and C2B. There are 15 members in the mammalian synaptotagmin family. There are several C2-domain containing protein families that are related to synaptotagmins, including transmembrane (Ferlins, E-Syts, and MCTPs) and soluble (RIMs, Munc13s, synaptotagmin-related proteins and B/K) proteins.

Functions
Based on their brain/endocrine distribution and biochemical properties, in particular C2 domains of certain synaptotagmins bound to calcium, synaptotagmins were proposed to function as calcium sensors in the regulation of neurotransmitter release and hormone secretion. Although synaptotagmins share a similar domain structure and a high degree of homology in the C2 domains, not all synaptotagmins bind to calcium. In fact, only eight out of the fifteen synaptotagmins are capable of calcium binding. The calcium binding synaptotagmins include synaptotagmins 1, 2, 3, 5, 6, 7, 9, and 10. The remaining seven synaptotagmins do not bind to calcium due to the lack of calcium coordinating residues or spatial orientation of the acidic residues (see the section on C2 domains for details).

Calcium-binding Synaptotagmins act as Ca2+ sensors and are involved in both: It was recently shown that synaptotagmin 1 can displace complexin from the SNARE complex in the presence of calcium. This is thought to be one of the last steps in exocytosis.
 * (i) early synaptic vesicle docking to the presynaptic membrane via interaction with β-neurexin or SNAP-25
 * (ii) late steps of Ca2+ evoked synaptic vesicle fusion with the presynaptic membrane.

C-terminal C2-domains
The C2 domain is a widely occurring conserved sequence motif of 130-140 amino acid residues, which was first defined as the second constant sequence in PKC isoforms. The C2 domain was first shown to bind to calcium in synaptotagmin-1. Subsequent atomic structure analysis of synaptotagmin-1 at 1.9 Å resolution indicated that its C2 domains are composed of a stable eight-stranded β-sandwich with flexible loops emerging from the top and bottom. Nuclear magnetic resonance (NMR) studies of synaptotagmin-1 revealed that calcium binds exclusively to the top loops, and the binding pockets are coordinated by five conserved aspartate residues: three calcium ions bind to C2A via D172, D178, D230, D232, S235 and D238, and two calcium ions bind to C2B via D303, D309, D363, 365 and D371. Not all synaptotagmin C2 domains bind to calcium. In fact, based on sequence similarities and subsequent confirmation by biochemical analyses, only eight synaptotagmins bind to calcium, namely, synaptotagmin-1, -2, -3, -5, -6, -7, -9 and -10. The lack of critical residues involved in calcium binding accounts for the majority of failure in calcium-binding by the other synaptotagmins. This includes both C2 domains of synaptotagmin-11, -12, -13, -14 and -15, and C2A domain of synaptotagmin-4 and -8. Synaptotagmin-4 and -11 C2B domains, which possess all five acidic residues in the top loops, however, do not bind to calcium due to spatial orientation of the calcium ligands that fail to form proper calcium binding sites. For calcium-binding synaptotagmins, although amino acid residues in the top loops other than those mentioned above are not directly involved in coordinating calcium binding, they affect calcium binding affinity, such as R233 in synaptotagmin-1. The diversity of sequences and structures flanking the calcium-coordinating amino acid residues renders the eight synaptotagmins bind to calcium at various affinities, covering the full range of calcium requirements for regulated exocytosis.

The C2A domain regulates the fusion step of synaptic vesicle exocytosis. Consistent with this, the kinetics of $$Ca^{2+}$$-dependent phospholipid binding activity of the C2A domain in vitro are compatible with the very fast nature of neurotransmitter release (within 200 μs). The C2A domain was shown to bind negatively charged phospholipids in a $$Ca^{2+}$$-dependent fashion. $$Ca^{2+}$$-binding alters the protein-protein interactions of synaptotagmin such as increasing the affinity of synaptotagmin for syntaxin.

The C2B domain binds to phosphatidyl-inositol-3,4,5-triphosphate (PIP3) in the absence of calcium ions and to phosphatidylinositol bisphosphate (PIP2) in their presence, suggesting that a lipid-interaction switch occurs during depolarization. Ca2+-binding to the C2B domain confers synaptotagmin dimerization involved in the fusion step of synaptic vesicles by $$Ca^{2+}$$-dependent self-clustering via the C2B domain. $$Ca^{2+}$$-independent is the interaction between the C2B domain and SNAP-25, and between the C2B domain and the "synprint" (synaptic protein interaction) motif of the pore-forming subunit of voltage-gated calcium channels. The C2B domain regulates also the recycling step of synaptic vesicles by binding to the clathrin assembly protein, AP-2.