The primed SNARE–complexin–synaptotagmin complex for neuronal exocytosis

Synaptotagmin, complexin, and neuronal SNARE (soluble N -ethylmaleimide sensitive factor attachment protein receptor) proteins mediate evoked synchronous neurotransmitter release, but the molecular mechanisms mediating the cooperation between these molecules remain unclear. Here we determine crystal structures of the primed pre-fusion SNARE–complexin–synaptotagmin-1 complex. These structures reveal an unexpected tripartite interface between synaptotagmin-1 and both the SNARE complex and complexin. Simultaneously, a second synaptotagmin-1 molecule interacts with the other side of the SNARE complex via the previously identified primary interface. Mutations that disrupt either interface in solution also severely impair evoked synchronous release in neurons, suggesting that both interfaces are essential for the primed pre-fusion state. Ca 2+ binding to the synaptotagmin-1 molecules unlocks the complex, allows full zippering of the SNARE complex, and triggers membrane fusion. The tripartite SNARE–complexin–synaptotagmin-1 complex at a synaptic vesicle docking site has to be unlocked for triggered fusion to start, explaining the cooperation between complexin and synaptotagmin-1 in synchronizing evoked release on the sub-millisecond timescale. An atomic model of the primed pre-fusion SNARE–complexin–synaptotagmin-1 complex in neuronal exocytosis accounting for vesicle priming and cooperation in synchronizing and activating evoked release on the sub-millisecond timescale. Three-way complex primes synapses for membrane fusion For rapid neurotransmitter release upon the arrival of an action potential, synaptic vesicles are 'primed' to undergo synchronous fusion with the pre-synaptic membrane, but the molecular basis of such priming is unknown. Now, with two large co-crystal structures, Axel Brunger and colleagues reveal a new and unexpected three-way interface between the proteins synaptotagmin (Syt1), complexin (Cpx) and the SNARE complex, beside a previously identified primary interface involving another molecule of Syt1 with the same SNARE complex. Through a combination of mutagenesis, biochemistry and electrophysiology, the authors show how this tripartite interface locks the primed complex into a state of low fusion probability and how action-potential-driven Ca 2+ binds to the Syt1 molecules to unlock the complex, allowing full zippering of the SNARE complex and triggering membrane fusion in a highly synchronized fashion on the sub-millisecond timescale.