Calcium-triggered fusion

Overview. Synaptic transmission between pre- and post-synaptic neurons occurs when the pre-synaptic neuron terminal is temporarily depolarized upon an action potential, opening Ca2+channels near the active zones of synapses. Because the extracellular Ca2+concentration is much higher than the cytoplasmic concentration, Ca2+will flow into the cytoplasm. In turn, Ca2+will trigger fusion of neurotransmitter-filled synaptic vesicles with the presynaptic membrane in less than a millisecond. Upon fusion, neurotransmitter molecules are released into the synaptic cleft, and then bind to receptors that are located in the postsynaptic membrane. Many of the key factors of the core synaptic fusion machinery have been identified, including fusogenic SNAREs, the Ca2+-sensor synaptotagmin, the activator/regulator complexin, the assembly factors Munc18 and Munc13, and the disassembly factors NSF and SNAP.

Our crystal structures of the complex of the neuronal SNARE complex and synaptotagmin-1 (Zhou et al., 2015 in the presence of  Ca2+ or Mg2+ revealed pairwise interactions between synaptotagmin-1 and the SNARE complex. Of these, the primary interface (i.e., the one with the largest interface area) is shown in Figure 1a. The interacting residues are conserved for synaptotagmins and SNAREs involved in fast synchronous release. We tested the function of amino acid residues involved in the primary interface in neuronal cultures by mutagenesis. These mutations also greatly reduced the Ca2+-triggered amplitude in a reconstituted fusion. Moreover, the primary interface has been validated by several independent follow-up studies. Our structure suggests that the complex localizes the synaptotagmin C2B domain near the fusion site (Figure 1b) which may in turn bend membranes or it may dissociate upon Ca2+-binding.

Figure 1. (a) The primary interface between the SNARE complex and synaptotagmin-1 (Zhou et al., 2015). Synaptobrevin-2, blue; SNAP-25, green; syntaxin-1A, red; synaptotagmin-1 C2B (the colors indicate the loops involved in Ca2+-binding (gold), the primary SNARE/synaptotagmin-1 interface (green), the tripartite SNARE/complexin-1/synaptotagmin-1 interface (purple, see Figure 3a), and the polybasic region (blue) (PDB IDs 5CCG and 5CCI)). For clarity, only the primary C2B-SNARE interface is shown. (b) Model how the SNARE/synaptagmin-1 complex might induce membrane bending.

Our crystal structure of the tripartite SNARE/complexin-1/synaptotagmin-1 complex (Figure 2a) (Zhou et al., 2017) additionally revealed a tripartite interface between one synaptotagmin-1 C2B domain and both the SNARE complex and complexin-1 (Figure 2a). To test the functional relevance of the interfaces that we observed in the crystal structure of SNARE/complexin-1/synaptotagmin-1 complex, we again performed structure-guided mutagenesis, and then tested these mutations by electrophysiological experiments. We showed that both Ca2+-triggered synaptic release and suppression of spontaneous release are sensitive to mutations of synaptotagmin-1 C2B residues involved in either the SNARE/complexin-1/synaptotagmin-1 tripartite or the SNARE/synaptotagmin-1 primary interfaces.

Figure 2. Crystal structure of the Ca2+-free tripartite complex between the half-zippered SNARE complex (synaptobrevin-2, blue; SNAP-25, green, syntaxin-1A, red), complexin-1 (yellow), and synaptotagmin-1 C2B (gray, green, purple, blue, and gold, same color code as in Figure 2a) (PDB ID 5W5C). Among the most striking structural features of this tripartite interface is the continuation of the complexin-1 central α-helix into the α-helix HA of synaptotagmin-1. (b) Close up view of a point contact between proteoliposomes with neuronal SNAREs, complexin-1, synaptotagmin-1, and Munc13. Shown is a slice of an isosurface representation of a cryo-electron tomogram of a proteoliposome that mimics a synaptic vesicle (top) and a proteoliposome that mimics the plasma membrane (bottom) (Gipson et al., 2017). (c) Model of Ca2+-triggered fusion starting from an inhibited (locked) state (Zhou et al., 2017).

Key insights. The SNARE/complexin-1/synaptotagmin-1 complexes form protein "stalks" that juxtapose the membranes, but keep them far enough away to reduce the chances of membrane fusion. In other words, these complexes actually inhibit fusion and set the stage for synchronous Ca2+-triggering (Brunger et al., 2018).  After release of inhibition by dislocation of the synaptotagmin-1 C2B domain involved in the tripartite interface, the activating properties of synaptotagmin-1, along with full zippering of the SNARE complex, likely assist the fusion process (Figure 2c).