Structural Properties of Syntaxin-1A and its Changes Following Binding of Munc-18

Abstract

Synaptic vesicle fusion is the process responsible for the signal transduction across neurons in the nervous system. The SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) proteins, syntaxin, synaptobrevin, and SNAP-25 are considered to form a minimal machinery necessary to fuse the neurotransmitter-containing vesicle to the presynaptic membrane. Syntaxin possess a single helix (SNARE motif or H3 domain) and with synaptobrevin and SNAP25 forms the tetra-helical complex required for fusion. It also has an independently folded tri-helical Habc domain. Syntaxin likely exists in a conformational equilibrium between states where the H3 and Habc either are or are not in direct interaction (closed and open conformation respectively). When syntaxin adopts a closed conformation, it is not able to assemble into the SNARE complex. This protein is also regulated by Munc-18, which likely binds to one of its conformations exclusively, thus altering its ability to bind other SNAREs. We are examining the structural properties and conformational equilibrium of syntaxin and how they are modulated with Munc-18 binding. Continuous Wave EPR data is used to probe for ordered secondary structure along the SNARE motif. The results indicate that there is an equilibrium between conformational substates in the H3 domain and evidence for tertiary contact with the Habc domain. Upon Munc-18 binding, conformational substates are shifted towards a more ordered structure and stronger tertiary contact with the Habc. Similar effects were also induced with addition of protective osmolites (glycerol, sucrose). Double Electron-Electron Resonance (DEER) EPR has been used to measure the intramolecular distances between both syntaxin domains. The distances obtained were short, and relatively unchanged when Munc-18 was added. This indicates, that syntaxin adopts a closed conformation regardless of the presence of Munc-18.