Different regions of synaptic vesicle membrane regulate VAMP2 conformation for the SNARE assembly

Chuchu Wang,Wenbin Liu,Zheng-Jiang Zhu,Bin Cai,Qinglu Zhong,Dan Li,Shengnan Zhang,Xiao Hu,Shouqiao Hou,Jia Tu,Zhijun Liu,Cong Liu,Guohui Li,Lin He,Jiajie Diao,Zhenying Liu

doi:10.1038/s41467-020-15270-4

Abstract

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.

Highlights

Vesicle associated membrane protein 2 (VAMP2/synaptobrevin2), a core sensitive-factor attachment receptor (SNARE) protein residing on synaptic vesicles (SVs), forms helix bundles with syntaxin-1 and SNAP25 for the SNARE assembly
Confocal microscopy further visualized that the delivered VAMP2 distributed along with the cell membrane skeleton in both cell types (Fig. 1e), which is in sharp comparison to α-synuclein, that was identified to be evenly distributed in the cytosol in the previous in-cell NMR study12 as well as in our experiment (Supplementary Fig. 2)
Consistent with a recent in vitro solution NMR study on the pre-fusion state of VAMP227, we observed a transient and dynamic membrane binding of VAMP2 with the affinity increasing from the N-terminal proline-rich domain to the C-terminal juxta-membrane domain

Summary

Introduction

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. 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. The assembly of SNARE complex in neurons is driven by the formation of a stable four-helix bundle between VAMP2 ( known as synaptobrevin2), syntaxin-1, and SNAP25, and modulated by multiple factors including proteins (e.g., synaptotagmin-12, Munc183, complexin4), lipids (e.g., sphingosine, PIP26) and metabolic ions (e.g., calcium). Theillet et al developed an effective approach to deliver isotope-labeled α-synuclein into human cells by electroporation, which is potentially a general technique for the delivery of intrinsically disordered proteins. We deliver the intrinsically disordered N-terminal cytosolic tail of VAMP2 into human cells by electroporation and observe that it associates with membranes. We further dissect that the membrane association and activity of VAMP2 SNARE motif are elegantly tuned by the different regions of SV membrane, which adds a spatial dimension on the complex modulation of SNARE machinery

Methods

Results

Conclusion