The primed SNARE–complexin–synaptotagmin complex for neuronal exocytosis

Abstract

Summary Synaptotagmin, complexin and neuronal SNARE proteins mediate evoked synchronous neurotransmitter release, but the molecular mechanisms mediating the cooperation between these molecules remain unclear. Here, we determined 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 interacted with the other side of the SNARE complex via the previously identified primary interface. Mutations that disrupt either interface in solution also severely impaired evoked synchronous release in neurons, suggesting that both interfaces are essential for the primed pre-fusion state. Ca2+ 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 commence, explaining the cooperation between complexin and synaptotagmin-1 in synchronizing evoked release on the sub-millisecond timescale.

Molecular Mechanisms of Synaptic Vesicle Priming by Munc13 and Munc18

“…In contrast, the frequency of spontaneous (mini) release that is measured in electrophysiological experiments depends on both the number of functional synapses as well as the number of synaptic vesicles that are capable of undergoing spontaneous fusion. Moreover, spontaneous fusion in neurons likely depends on another Ca 2+ sensor that is presently not included in our fusion assay (Xu et al, 2009;Zhou et al, 2017). Therefore, our observed Ca 2+ -independent vesicle fusion does not necessarily correlate to spontaneous mini release in vivo.…”

“…Upon inclusion of Munc13, the contacts are largely restricted to point contacts only, i.e., no long contacts are present and very few "dead-end" hemifusion diaphragms were observed prior to Ca 2+ -addition. The ternary SNARE complex interacts with both synaptotagmin-1 and with complexin-1, forming a primed and "locked" complex (Zhou et al, 2015(Zhou et al, , 2017 that is required for maximum probability of Ca 2+ -triggered release. These interactions will not occur if there are improper subconfigurations within the ternary SNARE complex.…”

Hypothesis – buttressed rings assemble, clamp, and release SNAREpins for synaptic transmission

“…The recent Syt‐Cpx‐SNARE structure 24, when combined with the Syt ring structure, lends a likely explanation as to how this could occur. We assume that each of the six SNAREpins will be retained locally by the nearest Syt C2B in the inner ring in the same geometry found in the crystal structure by the ‘primary’ 24, 25 binding site involving the two helices of SNAP‐25 but not VAMP or Syntaxin (Fig. 3: A side view, B top view).…”

“…As shown in Fig. A5, the activation energy must be larger than the binding energies of SNAREpin to the Syt molecules, that is, larger than the sum of the energies of the bonds to both the primary (~ 13  k B T ) and tripartite C2Bs (~ 8  k B T ; energies estimated from the binding constants in 24), that is, larger than 21  k B T . This value is reasonably consistent with the rate of spontaneous release but requires either additional stabilization (potentially via binding to the outer MUN ring) or an additional activation energy barrier, or both, combing to 5 ± 3  k B T .…”

“…Interestingly, the C2B domains of Syt1 (and similar) are all able to bind both the primary and tripartite sites on the SNAREpin‐Cpx complex based on conserved sequences 24. By contrast, the C2B domain of Syt7 (and similar; and all C2A domains of Syts) lacks the sequences needed for primary SNAREpin binding to SNAP‐25, but they all contain the helical extension 24 that mediates tripartite binding to Cpx, VAMP, and Syntaxin.…”