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Comparison of the prefusion and postfusion structures of complexin associated with SNAREs reveals a conformational switch in complexin, which is triggered by the zippering of three key Asp residues in the “switch” region of v-SNAREs into t-SNAREs. This causes complexin to move its accessory helix, thereby releasing the clamped state. This switch is triggered when the calcium sensor synaptotagmin binds calcium ions. 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.

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. The nervous system sends information around the body in the form of electrical signals that travel through cells called neurons. These signals cannot pass across the small gaps—called synapses—that separate neighboring neurons. Instead, when electrical signals reach the synapse, chemicals called neurotransmitters are released across the gap and trigger an electrical signal in the next neuron. Neurotransmitters are stored within neurons in small envelopes of membrane known as synaptic vesicles. They are released when the vesicles fuse with the membrane that surrounds the neuron. This fusion process must be tightly controlled to ensure that information is passed between the neurons at the right time. Complexin is a small protein that controls vesicle fusion by binding to a group of proteins called the SNARE complex. It contains two structured sections called the central helix and the accessory helix, which are both important for vesicle fusion. The central helix is able to bind to the SNARE proteins, and it has the same sequence of amino acids—the building blocks of proteins—in all animals. However, the sequence of amino acids in the accessory helix varies widely across different animals and it is not clear whether it performs the same role in all of them. Radoff et al. studied complexin in the nematode worm C. elegans, and found that when its accessory helix is replaced with the amino acid sequence from the mouse one, it can still properly control vesicle fusion. Indeed, complexin can still work properly when its accessory helix is replaced with an artificial protein helix that has a similar shape. These experiments suggest that the overall structure of the accessory helix is more important than its exact sequence of amino acids. Radoff et al. propose that its role in vesicle fusion is to stabilize the structure of the central helix to allow it to bind to the SNARE proteins. The next challenge is to understand how vesicle fusion is prevented when complexin binds to the SNARE proteins.

Structural studies of complexin bound to a mimetic of a prefusion SNARE complex provide insight into how complexin 'clamps' SNARE complex assembly, thereby inhibiting membrane fusion. While the central helix of complexin is anchored to a SNARE complex, its accessory helix extends away and bridges to a second SNARE complex, generating a zigzag array incompatible with fusion. 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.

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 pre-fusion SNAREpin lacking the portion of the v-SNARE which 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. That the accessory helix competes with the v-SNARE for t-SNARE binding was expected, but surprisingly, the interaction occurs inter-molecularly. Thus complexin organizes the SNAREs into a zig-zag topology which, when interposed between the vesicle and plasma membranes, is incompatible with fusion.

SNARE proteins are the engines that drive membrane fusion throughout the cell. They provide this energy by zippering up into a parallel four helix bundle in a thermodynamically favored process. Because the zippering of SNAREs is spontaneous, fusion events occur immediately upon a vesicle interacting with its target membrane. But, in certain circumstances, such as in synaptic vesicles, spontaneous fusion is not desired, so a clamp protein is necessary to prevent this fusion until signaled to do otherwise. In synapses, this protein is called Complexin and a second protein, called Synaptotagmin, releases the clamp upon a rapid influx of calcium, the hallmark of an action potential. How Complexin clamps is a subject of great interest in the field, and an area of active research. What is known is that a so-called Accessory helix (residues 28-47) is responsible for clamping, while another, Central Helix (reisudes 48-70) is responsible for physically binding to the helix. A recently solved crystal structure revealed how CPX might behave before the SNAREs fully zipper, namely that the accessory helix extends away from the SNAREs at a 45° angle. But, because of the packing of the crystal, it is entirely possible that the crystal is an artifact of packing, and/or truncation. In this thesis, my work first validates the crystal structure, using a FRET pair I developed for this purpose. I establish that the angled-out positioning of the accessory helix does, in fact, occur in solution, and is not due to crystal packing or the truncation of the VAMP2 (the neuronal vesicle-associated SNARE), but rather is due to the fact that its C-terminus is not present. I describe a mechanism by which Complexin can clamp. Further, I demonstrate that the residues in VAMP2 which are responsible for the switch from the \"open\" to the \"closed\" conformation are a patch of asparatates in VAMP2 (residues 64, 65, an 68). I also establish that these three aspartates are responsible for the release of the clamp and that without them, Complexin cannot be brought into the angled-in configuration. I propose a model for how the clamp might be released by Synaptotagmin.