2011 Theses Doctoral

Molecular Mechanisms Controlling Synaptic Vesicle Fusion

Radoff, Daniel Todd

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 truncationIn 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.