Vesicle associated membrane protein 2 (VAMP2/synaptobrevin2), a core SNARE protein residing on synaptic vesicles (SVs), forms helix bundles with syntaxin-1 and SNAP25 for the SNARE assembly. Prior to the SNARE assembly, the structure of VAMP2 is unclear. Here, by using in-cell NMR spectroscopy, we describe the dynamic membrane association of VAMP2 SNARE motif in mammalian cells, and the structural change of VAMP2 upon the change of intracellular lipid environment. We analyze the lipid compositions of the SV membrane by mass-spectrometry-based lipidomic profiling, and further reveal that VAMP2 forms distinctive conformations in different membrane regions. In contrast to the non-raft region, the membrane region of cholesterol-rich lipid raft markedly weakens the membrane association of VAMP2 SNARE motif, which releases the SNARE motif and facilitates the SNARE assembly. Our work reveals the regulation of different membrane regions on VAMP2 structure and sheds light on the spatial regulation of SNARE assembly.

囊泡相关膜蛋白2（VAMP2 / synaptobrevin2）是驻留在突触囊泡（SVs）上的核心SNARE蛋白，与snarinin-1和SNAP25形成螺旋束，用于SNARE组装。在SNARE组装之前，尚不清楚VAMP2的结构。在这里，通过使用细胞内核磁共振波谱，我们描述了哺乳动物细胞中VAMP2 SNARE基序的动态膜结合，以及随着细胞内脂质环境的变化而变化的VAMP2的结构变化。我们通过基于质谱的脂质分析，分析了SV膜的脂质组成，并进一步揭示了VAMP2在不同膜区域形成独特的构象。与非筏区相比，富含胆固醇的脂筏的膜区显着减弱了VAMP2 SNARE基序的膜结合，释放SNARE主题并方便SNARE组装。我们的工作揭示了VAMP2结构上不同膜区域的调控，并阐明了SNARE装配的空间调控。