Conformational Changes of Syntaxin-3B in Regulating SNARE Complex Assembly

Joshua Ferrar*, Brandon Choi, Katie Dunleavy, Giovanni Howells, Bishal Misra, and Claire Gething

Biochemistry Department, West Virginia University, Morgantown, WV 26505

Presentation Category: Health Sciences (Poster Presentation #116)

Student’s Major: Biochemistry

Neurotransmitter release of synaptic vesicles relies on the assembly of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex, consisting of syntaxin and SNAP-25 on the plasma membrane, and synaptobrevin on the synaptic vesicle. The formation of the SNARE complex progressively zippers towards the membranes driving membrane fusion between the plasma membrane and the synaptic vesicle. However, the underlying molecular mechanism of SNARE complex regulation is unclear. Here we study the syntaxin-3B isoform found in the retinal ribbon synapses using single-molecule fluorescence resonance energy transfer (smFRET) to monitor the conformational changes of syntaxin-3B that modulates the ternary SNARE complex formation. In comparison to syntaxin-1A located in the conventional synapses, we found that syntaxin-3B is predominantly in the closed conformation inefficiently forming the ternary SNARE complex. Interestingly, a phosphomimetic mutation (T14E) at the N-terminal region of syntaxin-3B promoted ternary SNARE complex assembly in the presence of SNAP-25 and synaptobrevin. In addition, we investigated the effect of Sec1/Munc18 (SM) protein family member, Munc18-1, which is an essential molecular chaperone regulating membrane trafficking and membrane fusion. Similar to syntaxin-1A/Munc18-1 heterodimer complex, Munc18-1 prevented the ternary SNARE complex formation by locking syntaxin-3B in an auto inhibiting closed conformation, which is released by the priming factor, Munc13.

Funding:

Program/mechanism supporting research/creative efforts: a WVU 497-level course