The ionic layer is required for efficient dissociation of the SNARE complex by α-SNAP and NSF

The four-helical bundle soluble N-ethylmaleimide-sensitive fusion protein (NSF) attachment protein receptor (SNARE) complex that mediates intracellular membrane fusion events contains a highly conserved ionic layer at the center of an otherwise hydrophobic core. This layer has an undetermined function; it consists of glutamine (Q) residues in syntaxin and the two synaptosomal-associated protein of 25 kDa (SNAP-25) family helices, and an arginine (R) in vesicle-associated membrane protein (a 3Q:1R ratio). Here, we show that the ionic-layer glutamine of syntaxin is required for efficient α-SNAP and NSF-mediated dissociation of the complex. When this residue is mutated, the SNARE complex still binds to α-SNAP and NSF and is released through ATP hydrolysis by NSF, but the complex no longer dissociates into SNARE monomers. Thus, one function of the ionic layer—in particular, the glutamine of syntaxin—is to couple ATP hydrolysis by NSF to the dissociation of the fusion complex. We propose that α-SNAP and NSF drive conformational changes at the ionic layer through specific interactions with the syntaxin glutamine, resulting in the dissociation of the SNARE complex