The pre-synaptic fusion machinery

Abstract

Here, we review recent insights into the neuronal presynaptic fusion machinery that releases neurotransmitter molecules into the synaptic cleft upon stimulation. The structure of the pre-fusion state of the SNARE/complexin-1/synaptotagmin-1 synaptic protein complex suggests a new model for the initiation of fast Ca²⁺-triggered membrane fusion. Functional studies have revealed roles of the essential factors Munc18 and Munc13, demonstrating that a part of their function involves the proper assembly of synaptic protein complexes. Near-atomic resolution structures of the NSF/αSNAP/SNARE complex provide first glimpses of the molecular machinery that disassembles the SNARE complex during the synaptic vesicle cycle. These structures show how this machinery captures the SNARE substrate and provide clues as to a possible processing mechanism.

Figure 1

Synaptic fusion complex. (a) Crystal structure of the SNARE/complexin-1/synaptotagmin-1 complex [11••], PDB ID 5W5C (red: syntaxin-1A, green: SNAP-25A, blue: synaptobrevin-2, light blue: central helix of complexin-1, orange: C2B domains of synaptotagmin-1, gray: C2A domains of synaptotagmin-1. (b) Superposition of representative members of the C2 domain superfamily 2.60.40.150 generated by CATH [89] (http://www.cathdb.info) with the structure of the SNARE/complex-1/synaptotagmin-1 complex (red: syntaxin, green: SNAP-25, blue: synaptobrevin, gold: synaptotagmins, purple: Doc2 and Rabphilin, gray: 40 representative structures out of 346 C2 domains with known structure). (c) Release of inhibition model of Ca²⁺-triggering. Note that it is unknown as to which membrane the Ca²⁺-bound synaptotagmin-1 C2 domains might interact with. For illustration purposes, we placed it at the synaptic vesicle membrane, although the presence of PIP₂ increases the binding affinity between Ca²⁺-bound synaptotagmin-1 and the membrane, so it also possible, or even likely, that all synaptotagmin-1 C2 domains localize to the plasma membrane upon Ca²⁺-binding.

Figure 2

Munc18 and Munc13 are chaperones of SNARE complex assembly. (a) Crystal structure of the Munc18/syntaxin-1A complex [38,39] (PDB ID: 3C98) (red: syntaxin-1, gray: Munc18). (b) Interactions between SNAREs and Munc18. Composite model based on the crystal structures of the Vps33/Nyv1 and Vps33/Vam3 complexes [56••] (PDB IDs 5BUZ and 5BV0) (gray: Vps33, red: Vam3, blue: Nyv1). (c) Crystal structure of the C1C2BMUN fragment of Munc13 [60••] (PDB ID 5UE8).

Figure 3

SNARE complex disassembly machinery. (a) EM structure of NSF/αSNAP/SNARE complex at ~3.9 Å resolution [78••] (PDB ID 6MDM). The N, D1, and D2 domains of NSF are colored salmon, light blue, and light purple, respectively. Nucleotides in the D1 and D2 rings are colored yellow. The two αSNAP molecules are shown in gold. The SNARE proteins syntaxin-1a, synaptobrevin-2, and SNAP-25A are colored blue, red, and green, respectively. (b) Close-up view of the interaction between the N-terminal residues of SNAP-25A and the pore of the D1 ATPase ring of NSF. An amino acid essential for SNARE complex disassembly (Y294) is found at the apex of the pore loop of each ATPase subunit; five of six of these tyrosines intercalate between the side chains of the SNAP25 N-terminus, seemingly locking it in place.

Figure 4

Synaptic vesicle cycle. Superimposed on the cycle are structures of the neuronal SNARE complex [3] (PDB ID 1SFC), the SNARE/complexin-1/synaptotagmin-1 complex [11••] (PDB ID 5W5C), the NSF/αSNAP/SNARE complex [78••] (PDB ID 6MDM), the Munc18/syntaxin-1A complex [38,39] (PDB ID: 3C98), and the C1C2BMUN fragment of Munc13 [60••] (PDB ID 5UE8).