During neurotransmitter release, the neuronal SNARE proteins synaptobrevin/VAMP, syntaxin, and SNAP-25 form a four-helix bundle, the SNARE complex, that pulls the synaptic vesicle and plasma membranes together possibly causing membrane fusion. Complexin binds tightly to the SNARE complex and is essential for efficient Ca(2+)-evoked neurotransmitter release. A combined X-ray and TROSY-based NMR study now reveals the atomic structure of the complexin/SNARE complex. Complexin binds in an antiparallel alpha-helical conformation to the groove between the synaptobrevin and syntaxin helices. This interaction stabilizes the interface between these two helices, which bears the repulsive forces between the apposed membranes. These results suggest that complexin stabilizes the fully assembled SNARE complex as a key step that enables the exquisitely high speed of Ca(2+)-evoked neurotransmitter release.

␣-Synuclein is a small cytosolic protein of presynaptic nerve terminals composed of seven 11-residue repeats and a hydrophilic tail. ␣-Synuclein misfolding and dysfunction may contribute to the pathogenesis of Parkinson's disease and neurodegenerative dementias, but its normal folding and function are unknown. In solution, ␣-synuclein is natively unstructured but assumes an ␣-helical conformation upon binding to phospholipid membranes. We now show that this conformation of ␣-synuclein consists of two ␣-helical regions that are interrupted by a short break. The structural organization of the ␣-helices of ␣-synuclein was not anticipated by sequence analyses and may be important for its pathogenic role.In recent years, the presynaptic protein ␣-synuclein has attracted much attention because of its involvement in neurodegenerative diseases (1-3). Two independent mutations in human ␣-synuclein cause familial Parkinson's disease, and wild type ␣-synuclein is a major component of Lewy bodies, cytoplasmic inclusion bodies found in Parkinson's disease and in several forms of neurodegenerative dementia. However, independent of its role in neurodegenerative diseases, ␣-synuclein is an interesting protein in its own right. It is an abundant presynaptic protein that may regulate neurotransmitter release and may contribute to synaptic plasticity (4 -6). ␣-Synuclein is the founding member of a protein family that additionally includes ␤-and ␥-synucleins and synoretin (7-9). The sequences of all synucleins are similar, although only ␣-synuclein is implicated in disease. Synucleins are composed of six copies (␤-synuclein) or seven copies (all other synucleins) of an unusual 11-residue imperfect repeat, followed by a variable short hydrophilic tail. Synucleins are soluble, natively unfolded proteins that avidly bind to negatively charged phospholipid membranes and become ␣-helical upon binding (10). Although secondary structure predictions indicate that the synuclein repeats could form an amphipathic structure consistent with lipid binding, the ␣-helical conformation is puzzling because the synuclein repeats are punctuated by central glycine residues. Furthermore, in Lewy bodies ␣-synuclein is thought to be in a ␤-strand aggregate, but aggregation of ␣-synuclein into dimers and multimers is promoted by lipid environments that induce an ␣-helical conformation (11-13). In the present study, we have examined the conformation of ␣-synuclein in lipidic environments to understand the relation of its sequence to its physicochemical properties and to map a potential pathway of misfolding in neurodegenerative disease. EXPERIMENTAL PROCEDURESProduction of ␣-Synuclein-Recombinant ␣-synuclein was expressed in bacteria as GST-fusion proteins with a TEV protease recognition sequence preceding the N-terminal methionine and cleaved with TEV protease (Invitrogen), resulting in a single additional glycine residue at the N terminus. After TEV cleavage, ␣-synuclein was isolated as the only heat-stable component upon boiling for 15 min, purified by i...

Synaptotagmin acts as a Ca(2+) sensor in neurotransmitter release through its two C(2) domains. Ca(2+)-dependent phospholipid binding is key for synaptotagmin function, but it is unclear how this activity cooperates with the SNARE complex involved in release or why Ca(2+) binding to the C(2)B domain is more crucial for release than Ca(2+) binding to the C(2)A domain. Here we show that Ca(2+) induces high-affinity simultaneous binding of synaptotagmin to two membranes, bringing them into close proximity. The synaptotagmin C(2)B domain is sufficient for this ability, which arises from the abundance of basic residues around its surface. We propose a model wherein synaptotagmin cooperates with the SNAREs in bringing the synaptic vesicle and plasma membranes together and accelerates membrane fusion through the highly positive electrostatic potential of its C(2)B domain.

Complexins constitute a family of four synaptic high-affinity SNARE complex binding proteins. They positively regulate a late, post-priming step in Ca2+-triggered synchronous neurotransmitter release, but the underlying molecular mechanisms are unclear. We show here that SNARE complex binding of Complexin I via its central α-helix is necessary but unexpectedly not sufficient for its key function in promoting neurotransmitter release. An accessory α-helix N-terminal of the SNARE complex binding region plays an inhibitory role in fast synaptic exocytosis, while its N-terminally adjacent sequences facilitate Ca2+-triggered release even in the absence of the Ca2+ sensor Synaptotagmin 1. Our results indicate that distinct functional domains of Complexins differentially regulate synaptic exocytosis, and that via the interplay between these domains Complexins play a crucial role in fine-tuning Ca2+-triggered fast neurotransmitter release.

Sec1/munc18-like proteins (SM proteins) and SNARE complexes are probably universally required for membrane fusion. However, the molecular mechanism by which they interact has only been defined for synaptic vesicle fusion where munc18 binds to syntaxin in a closed conformation that is incompatible with SNARE complex assembly. We now show that Sly1, an SM protein involved in Golgi and ER fusion, binds to a short, evolutionarily conserved N-terminal peptide of Sed5p and Ufe1p in yeast and of syntaxins 5 and 18 in vertebrates. In these syntaxins, the Sly1 binding peptide is upstream of a separate, autonomously folded N-terminal domain. These data suggest a potentially general mechanism by which SM proteins could interact with peptides in target proteins independent of core complex assembly and suggest that munc18 binding to syntaxin is an exception.