An Extended Helical Conformation in Domain 3a of Munc18-1 Provides a Template for SNARE ( Soluble N- Ethylmaleimidesensitive Factor Attachment Protein Receptor) Complex Assembly*

Background: Munc18-1 is required for membrane fusion, but the underlying mechanism is unknown. Results: Distinct point mutations in domain 3a of Munc18-1 differentially affect the conformation of helix 12, VAMP2 binding, and membrane fusion. Conclusion: A conformational switch in helix 12 promotes SNAREpin assembly via the VAMP2 interaction. Significance: The Munc18-1-VAMP2 interaction may represent a general molecular mechanism of how SM proteins accelerate membrane fusion. 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 -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.