Interplay between VA M P2 and lipids in regulation of SNARE complex assembly studied by in-cell NMR Chuchu Wang1, Jia Tu1, Shengnan Zhang1, Zhijun Liu2, Jiajie Diao3, Zheng-Jiang Zhu1, Cong Liu1 1 Interdisciplinary Research Center on Biology and Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai 200032, China. 2 National Center for Protein Science, Shanghai 201210, China. 3Department of Cancer Biology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267, USA 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 membrane1. The SNARE motif of VAMP2, which is essential in mediating SNARE complex formation, was found to bind certain types of membrane mimic (e.g. DPC micelles rather than nanodisc) in vitro2,3 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 membrane binding 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 reveal that VAMP2 recognizes different lipid molecules by distinct binding pattern, which results in different influences on VAMP2-mediated SNARE complex assembly. Thus the distinct local lipid environment on the SV membrane fine-tunes the conformation of VAMP2 for SNARE complex assembly and regulates SV trafficking. 1. T. Südhof, J. Rizo J. Synaptic vesicle exocytosis. Cold Spring Harb Perspect Biol. 3(12). 2011 2. K. D. Brewer, W. Li, B. E. Horne, J. Rizo, Reluctance to membrane binding enables accessibility of the synaptobrevin SNARE motif for SNARE complex formation. Proceedings of the National Academy of Sciences 108, 12723, 2011. 3. J. F. Ellena et al., Dynamic structure of lipid-bound synaptobrevin suggests a nucleation-propagation mechanism for trans- SNARE complex formation. Proceedings of the National Academy of Sciences 106, 20306,2009 .