Determining the mechanism of SNARE complex assembly through site-directed spin labeling

Information transfer in the central nervous system is facilitated by the release of neurotransmitter from a presynaptic neuron. This neurotransmitter release is the result of a membrane fusion event between a synaptic vesicle and the presynaptic plasma membrane. Neurotransmitter then binds to receptors on the postsynaptic neuron, which relays the signal between neurons. Membrane fusion is mediated by proteins termed Soluble NSF Attachment REceptors or SNAREs, which assemble together in a four-helix bundle. The assembly of the SNAREs releases energy, which drives the two membranes together, leading to fusion. There are three SNARE proteins: syntaxin and SNAP-25, attached to the plasma membrane, and synaptobrevin, attached to the vesicle membrane. SNAP-25 contributes two of the four helices to the SNARE complex, SN1 and SN2. The current mechanism and sequence of SNARE assemble is not understood. Here, site-directed spin labeling paired with Electron Paramagnetic Spectroscopy was used to examine the order in which the four motifs assemble into the complex, and revealed several important features of SNARE assembly. First, the SN1 and SN2 segments of SNAP-25 behave differently. The SN1 segment of SNAP-25 exhibits a pronounced increase in backbone ordering from the N- to the C-terminus that is not seen in the SN2 segment. Both the SN1 and SN2 segments of SNAP-25 assemble with syntaxin; however, while the association of the SN1 segment with syntaxin produces a stable 2:2 (SN1:syntaxin) complex, the complex formed between SN2 and syntaxin is largely disordered. Synaptobrevin does not bind syntaxin alone, but associates with syntaxin in the presence of either SN1 or SN2; however, the synaptobrevin:syntaxin:SN2 complex remains disordered. Taken together, these data suggest that synaptobrevin and syntaxin do not assemble in the absence of SNAP-25, and that the SN2 segment of SNAP-25 is the last to enter the SNARE complex. Second, two proposed complexes involving the SNARE regulatory protein, Munc18, were tested: a SNAP-25:syntaxin:Munc18 complex vs a synaptobrevin:syntaxin:Munc18 complex. Only formation of a SNAP-25:syntaxin:Munc18 template complex was detected. Distances between SN1 and SN2 were obtained in numerous conditions allowing for an assessment of the conformation of SNAP-25 in the template complex. SN2 appears to be mostly disengaged when interacting with syntaxin and Munc18, providing further evidence that SN2 is incorporated into the SNARE complex subsequent to SN1. Altogether, these data distinguish between two proposed pathways for SNARE assembly, and demonstrate that the contributions from SN1 and SN2 of SNAP-25 are not equivalent.