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.

介导细胞内膜融合事件的四螺旋束可溶性N-乙基马来酰亚胺敏感融合蛋白（NSF）附着蛋白受体（SNARE）复合物，在其原本为疏水核心的区域中央，存在一个高度保守的离子层。该离子层的功能尚未明确，其组成为：突触融合蛋白（syntaxin）与两种突触体相关蛋白25 kDa（SNAP-25）家族螺旋上的谷氨酰胺（Q）残基，以及囊泡相关膜蛋白上的精氨酸（R）残基，比例为3Q:1R。本研究表明，突触融合蛋白上的离子层谷氨酰胺残基，对于α-可溶性NSF附着蛋白（α-SNAP）与NSF介导的复合物有效解离至关重要。当该残基发生突变后，SNARE复合物仍可与α-SNAP及NSF结合，并通过NSF的ATP水解作用被释放，但复合物无法再解离为SNARE单体。由此可见，离子层的功能之一——尤其是突触融合蛋白上的谷氨酰胺残基——是将NSF的ATP水解过程与融合复合物的解离过程相偶联。本研究提出，α-SNAP与NSF可通过与突触融合蛋白上的谷氨酰胺残基发生特异性相互作用，驱动离子层发生构象变化，最终促使SNARE复合物解离。