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

eNeuro. 2020 Nov 5;7(6):ENEURO.0278-20.2020. doi: 10.1523/ENEURO.0278-20.2020. Print Nov/Dec 2020.

Abstract

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 Munc18-1 deficient neurons severely reduces synaptic transmission, demonstrating the physiological significance of the Munc18-1·VAMP2 interaction.

Keywords: Munc18-1; SNARE; VAMP2; crosslinking; membrane fusion; neurotransmission.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Membrane Fusion
  • Munc18 Proteins* / genetics
  • Munc18 Proteins* / metabolism
  • Protein Binding
  • Rats
  • SNARE Proteins* / genetics
  • SNARE Proteins* / metabolism
  • Synaptic Transmission
  • Vesicle-Associated Membrane Protein 2* / genetics
  • Vesicle-Associated Membrane Protein 2* / metabolism

Substances

  • Munc18 Proteins
  • SNARE Proteins
  • Vamp2 protein, rat
  • Vesicle-Associated Membrane Protein 2