The Interaction of Munc18-1 Helix 11 and 12 with the Central Region of the VAMP2 SNARE Motif Is Essential for SNARE Templating and Synaptic Transmission

• Published in: eNeuro 2020-11, Vol.7 (6), p.ENEURO.0278-20.2020
• Main Authors: André, Timon, Classen, Jessica, Brenner, Philipp, Betts, Matthew J, Dörr, Bernhard, Kreye, Susanne, Zuidinga, Birte, Meijer, Marieke, Russell, Robert B, Verhage, Matthijs, Söllner, Thomas H
• Format: Article
• Language: English
• Subjects: Animals; Membrane Fusion; Munc18 Proteins - genetics; Munc18 Proteins - metabolism; New Research; Protein Binding; Rats; SNARE Proteins - genetics; SNARE Proteins - metabolism; Synaptic Transmission; Vesicle-Associated Membrane Protein 2 - genetics; Vesicle-Associated Membrane Protein 2 - metabolism
• ISSN: 2373-2822; 2373-2822
• DOI: 10.1523/ENEURO.0278-20.2020

Sec1/Munc18 proteins play a key role in initiating the assembly of N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes, the molecular fusion machinery. Employing comparative structure modeling, site specific crosslinking by single amino acid substitutions with the photoactivatable unnatural amino acid p-Benzoyl-phenylalanine (Bpa) and reconstituted vesicle docking/fusion assays, we mapped the binding interface between Munc18-1 and the neuronal v-SNARE VAMP2 with single amino acid resolution. Our results show that helices 11 and 12 of domain 3a in Munc18-1 interact with the VAMP2 SNARE motif covering the region from layers -4 to +5. Residue Q301 in helix 11 plays a pivotal role in VAMP2 binding and template complex formation. A VAMP2 binding deficient mutant, Munc18-1 Q301D, does not stimulate lipid mixing in a reconstituted fusion assay. The neuronal SNARE-organizer Munc13-1, which also binds VAMP2, does not bypass the requirement for the Munc18-1·VAMP2 interaction. Importantly, Munc18-1 Q301D expression in deficient neurons severely reduces synaptic transmission, demonstrating the physiological significance of the Munc18-1·VAMP2 interaction.