title: An extended helical conformation in domain 3a of Munc18-1 provides a template for SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex assembly.
abstract: Munc18-1, a SEC1/Munc18 protein and key regulatory protein in synaptic transmission, can either promote or inhibit SNARE complex assembly. Although the binary inhibitory interaction between Munc18-1 and closed syntaxin 1 is well described, the mechanism of how Munc18-1 stimulates membrane fusion remains elusive. Using a reconstituted assay that resolves vesicle docking, priming, clamping, and fusion during synaptic exocytosis, we show that helix 12 in domain 3a of Munc18-1 stimulates SNAREpin assembly and membrane fusion. A single point mutation (L348R) within helix 12 selectively abolishes VAMP2 binding and the stimulatory function of Munc18-1 in membrane fusion. In contrast, targeting a natural switch site (P335A) at the start of helix 12, which can result in an extended α-helical conformation, further accelerates lipid-mixing. Together with structural modeling, the data suggest that helix 12 provides a folding template for VAMP2, accelerating SNAREpin assembly and membrane fusion. Analogous SEC1/Munc18-SNARE interactions at other transport steps may provide a general mechanism to drive lipid bilayer merger. At the neuronal synapse, Munc18-1 may convert docked synaptic vesicles into a readily releasable pool.
authors.affiliation: From the Heidelberg University Biochemistry Center (BZH), Im Neuenheimer Feld 328, 69120 Heidelberg, Germany and.
authors.forename: Daniel
authors.initials: D
authors.lastname: Parisotto
authors.forename: Maximilian
authors.initials: M
authors.lastname: Pfau
authors.forename: Andrea
authors.initials: A
authors.lastname: Scheutzow
authors.forename: Klemens
authors.initials: K
authors.lastname: Wild
authors.forename: Matthias P
authors.initials: MP
authors.lastname: Mayer
authors.forename: Jörg
authors.initials: J
authors.lastname: Malsam
authors.forename: Irmgard
authors.initials: I
authors.lastname: Sinning
authors.forename: Thomas H
authors.initials: TH
authors.lastname: Söllner
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D051938:Munc18 Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020125:Mutation, Missense; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D051381:Rats; D013329:Structure-Activity Relationship; D050827:Syntaxin 1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D050988:Vesicle-Associated Membrane Protein 2 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D051938:Munc18 Proteins; C497939:Stxbp1 protein, rat; D050827:Syntaxin 1; C494706:Vamp2 protein, rat; D050988:Vesicle-Associated Membrane Protein 2
keywords: Exocytosis; Membrane Fusion; Membrane Reconstitution; Membrane Trafficking; SM Protein; Snare Proteins; Synaptic Vesicle; VAMP2/Synaptobrevin