User:Jbryan13/SNARE (protein)

Assembly of the SNARE complex
SNARE proteins must assemble into trans-SNARE complexes so that they can provide the force that is necessary for vesicle fusion. The four α-helix domains (1 each from synaptobrevin and syntaxin, and 2 from SNAP-25) come together to form a coiled-coil motif. The rate-limiting step in the assembly process is the association of the syntaxin SNARE domain, since it is usually found in a “closed” state where it is incapable of interacting with other SNARE proteins. When syntaxin is in an open state, trans-SNARE complex formation begins with the association of the four SNARE domains at their N-termini. The SNARE domains proceed in forming a coiled-coil motif in the direction of the C-termini of their respective domains. The SM protein Munc18 is thought to play a role in assembly of the SNARE complex, although the exact mechanism by which it acts is still under debate. It is known that the clasp of Munc18 locks syntaxin in a closed conformation by binding to its α-helical SNARE domains, which inhibits syntaxin from entering SNARE complexes (thereby inhibiting fusion). The clasp is also capable, however, of binding the entire four-helix bundle of the trans-SNARE complex. One hypothesis suggests that, during SNARE-complex assembly, the Munc18 clasp releases closed syntaxin, remains associated with the N-terminal peptide of syntaxin (allowing association of the syntaxin SNARE domain with other SNARE proteins), and then reattaches to the newly formed four-helix SNARE complex. This possible mechanism of dissociation and subsequent re-association with the SNARE domains could be calcium-dependent. This supports the idea that Munc18 plays a key regulatory role in vesicle fusion; under normal conditions the SNARE complex will be prevented from forming by Munc18, but when triggered the Munc18 will actually assist in SNARE-complex assembly and thereby act as a fusion catalyst.

Zippering of the SNARE complex and fusion pore opening
The force that brings two membranes together during fusion is believed to come from the conformational change in trans-SNARE complexes to form cis-SNARE complexes. The current hypothesis that describes this process is referred to as SNARE “zippering.”

When the trans-SNARE complex is formed, the SNARE proteins are still found on opposing membranes. As the SNARE domains continue coiling in a spontaneous process, they form a much tighter, more stable four-helix bundle. The energy that is released as the complex goes to a more stable conformation is used to overcome the repulsive forces between the vesicle and the cell membrane and allow a fusion pore to open. Once fusion has taken place, the fully coiled SNARE proteins are found on the same membrane. This is referred to as the cis-SNARE complex.

The interaction of the SNARE transmembrane (TM) domains with the vesicle and target membranes leads to the formation of a fusion pore. According to one theory, as the SNARE complex zippers, the tightening helix bundle puts torsional force on the TM domains of synaptobrevin and syntaxin. This causes the TM domains to tilt within the separate membranes as the proteins coil more tightly. The TM domains eventually come together between the membranes, linking the membranes in the process. This is a relatively unstable state for the TM domains, and the lipid bilayers consequently rearrange to a more stable configuration around them. As a result of the lipid rearrangement, a fusion pore opens and allows the chemical contents of the vesicle to leak into the outside environment.

Disassembly of the SNARE complex
The energy input that is required for SNARE-mediated fusion to take place comes from SNARE-complex disassembly. The suspected energy source is N-ethylmaleimide-sensitive factor (NSF), an ATPase that is involved with membrane fusion. NSF homohexamers, along with the NSF cofactor α-SNAP, bind and dissociate the SNARE complex by coupling the process with ATP hydrolysis. This process allows for reuptake of synaptobrevin for further use in vesicles, whereas the other SNARE proteins remain associated with the cell membrane.

The dissociated SNARE proteins have a higher energy state than the more stable cis-SNARE complex. It is believed that the energy that drives fusion is derived from the transition to a lower energy cis-SNARE complex. The ATP hydrolysis-coupled dissociation of SNARE complexes is an energy investment that can be compared to “cocking the gun” so that, once vesicle fusion is triggered, the process takes place spontaneously and at optimum velocity. A comparable process takes place in muscles, in which the myosin heads must first hydrolyze ATP in order to adapt the necessary conformation for interaction with actin and the subsequent power stroke to occur.