Binding of VAMP2 to Membranes of Mammalian Cells Probed by In-Cell NMR

Abstract

The synaptic vesicle associated membrane protein 2 (VAMP2), one of the main components of the SNARE complex, regulates the fusion of synaptic vesicles with the presynaptic membrane. The SNARE motif of VAMP2, which is essential in mediating SNARE complex formation, recognizes and binds to only certain types of membrane mimic (e.g. DPC micelles but not nanodisc) in vitro. Whether and how VAMP2 is associated with membrane in vivo are key questions to understand the regulatory mechanism of VAMP2-mediated SNARE complex assembly. In this study, we systemically characterized the structural changes of VAMP2 in different mammalian cells at residue-resolution by using in-cell NMR. Combining with immunofluorescence microscopy, membrane fractionation and in solution NMR, we found that the SNARE motif and juxtamembrane region of VAMP2 partially bind to membranes, while the rest is highly flexible in cellular environment. Our work demonstrates the dynamical binding mode of VAMP2 to membrane in cells, and highlights the potential importance of this binding mode in regulating assembly of SNARE complex.