The accessory helix of complexin functions by stabilizing central helix secondary structure.

The presynaptic protein complexin (CPX) is a critical regulator of synaptic vesicle fusion, but the mechanisms underlying its regulatory effects are not well understood. Its highly conserved central helix (CH) directly binds the ternary SNARE complex and is required for all known CPX functions. The adjacent accessory helix (AH) is not conserved despite also playing an important role in CPX function, and numerous models for its mechanism have been proposed. We examined the impact of AH mutations and chimeras on CPX function in vivo and in vitro using C. elegans. The mouse AH fully restored function when substituted into worm CPX suggesting its mechanism is evolutionarily conserved. CPX inhibitory function was impaired when helix propagation into the CH was disrupted whereas replacing the AH with a non-native helical sequence restored CPX function. We propose that the AH operates by stabilizing CH secondary structure rather than through protein or lipid interactions.

Conformational dynamics of calcium-triggered activation of fusion by synaptotagmin.

Synaptotagmin triggers rapid exocytosis of neurotransmitters from synaptic vesicles in response to Calcium (Ca(2+)) ions. Here, we use a novel Nanodisc-based system, designed to be a soluble mimetic of the clamped synaptic vesicle-bilayer junction, combined with fluorescence resonance energy transfer (FRET) spectroscopy to monitor the structural relationships among SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptor), Synaptotagmin C2 domains, and the lipid bilayer in real time during the Ca(2+)-activation process. We report that Synaptotagmin remains rigidly fixed on the partially assembled SNARE complex with no detectable internal rearrangement of its C2 domains, even as it rapidly inserts into the bilayer. We hypothesize that this straightforward, one-step physical mechanism could explain how this Ca(2+)- sensor rapidly activates neurotransmitter release from the clamped state.

A conformational switch in complexin is required for synaptotagmin to trigger synaptic fusion.

The crystal structure of complexin bound to a prefusion SNAREpin mimetic shows that the accessory helix extends away from the SNAREpin in an 'open' conformation, binding another SNAREpin and inhibiting its assembly, to clamp fusion. In contrast, the accessory helix in the postfusion complex parallels the SNARE complex in a 'closed' conformation. Here we use targeted mutations, FRET spectroscopy and a functional assay that reconstitutes Ca(2+)-triggered exocytosis to show that the conformational switch from open to closed in complexin is needed for synaptotagmin-Ca(2+) to trigger fusion. Triggering fusion requires the zippering of three crucial aspartate residues in the switch region (residues 64-68) of v-SNARE. Conformational switching in complexin is integral to clamp release and is probably triggered when its accessory helix is released from its trans-binding to the neighboring SNAREpin, allowing the v-SNARE to complete zippering and open a fusion pore.

Complexin cross-links prefusion SNAREs into a zigzag array.

Complexin prevents SNAREs from releasing neurotransmitters until an action potential arrives at the synapse. To understand the mechanism for this inhibition, we determined the structure of complexin bound to a mimetic of a prefusion SNAREpin lacking the portion of the v-SNARE that zippers last to trigger fusion. The 'central helix' of complexin is anchored to one SNARE complex, while its 'accessory helix' extends away at ~45° and bridges to a second complex, occupying the vacant v-SNARE binding site to inhibit fusion. We expected the accessory helix to compete with the v-SNARE for t-SNARE binding but found instead that the interaction occurs intermolecularly. Thus, complexin organizes the SNAREs into a zigzag topology that, when interposed between the vesicle and plasma membranes, is incompatible with fusion.