In Vitro Configuration of Munc13-1 Bridging of Phospholipid Bilayers at Resting Conditions

Abstract

Neurotransmitter release is an intricate process involving multiple proteins that govern synaptic vesicle exocytosis. While SNAREs drive membrane fusion, chaperone proteins regulate their function, ensuring fusion happens in a precise and controllable manner. Munc13-1 is an elongated, multi-domain protein participating in docking through plasma membrane interactions via the N-terminal C1, C2B domains and vesicle tethering via the C-terminal C2C domain and SNARE priming by opening Syntaxin-1A, thus promoting SNARE-pin formation. In addition, it was recently proposed that Munc13-1 is a core component of molecular architecture at the synaptic vesicle release site due to its ability to form a supramolecular assembly (clusters). Stability of these clusters could imply ordered spatial arrangement of Munc13-1 molecules but to date, no structural evidence has been reported. Here we report the spatial arrangement of the truncated version of Munc13-1 consisting of C1, C2B, MUN (Δ1408-1442, EF) and C2C domains (Munc13L) on the surface of negatively charged phospholipid membranes. Using cryo-tomography, we found that Munc13L forms hexagonal 2D crystal in Ca2+ free conditions, bridging two membrane bilayers. Subtomograms extracted from the observed crystal were aligned and averaged to yield a 3D map at 14 Å resolution. Fitting of existing crystal structures of C1, C2B and MUN domains revealed that Munc13L exists in two conformational states in the crystal. One form is extended, connecting two bilayers to form a triskelion-like structure by the interaction of C-terminal ends of MUN domains, while the other form is supporting the extended form using the interaction of its C2C domain with MUN region of the supported molecule. These interactions are novel and require further investigation to determine the physiological role and its transformation when activated by calcium.