Complexins play a critical role in regulating SNARE-mediated exocytosis of synaptic vesicles. Evolutionary divergences in complexin function have complicated our understanding of the role these proteins play in inhibiting the spontaneous fusion of vesicles. Previous structural and functional characterizations of worm and mouse complexins have indicated the membrane curvature-sensing C-terminal domain of these proteins is responsible for differences in inhibitory function. We have characterized the structure and dynamics of the mCpx1 CTD in the absence and presence of membranes and membrane mimetics using NMR, ESR, and optical spectroscopies. In the absence of lipids, the mCpx1 CTD features a short helix near its N-terminus and is otherwise disordered. In the presence of micelles and small unilamellar vesicles, the mCpx1 CTD forms a discontinuous helical structure in its C-terminal 20 amino acids, with no preference for specific lipid compositions. In contrast, the mCpx1 CTD shows distinct compositional preferences in its interactions with large unilamellar vesicles. These studies identify structural divergences in the mCpx1 CTD relative to the wCpx1 CTD in regions that are known to be critical to the wCpx1 CTD’s role in inhibiting spontaneous fusion of synaptic vesicles, suggesting a potential structural basis for evolutionary divergences in complexin function.11Abbreviations used: SVs, synaptic vesicles; NTD, N-terminal domain; AHD, accessory helical domain; CHD, central helix domain; CTD, C-terminal domain; intrinsically disordered (ID); wCpx1, worm (Caenorhabditis elegans) complexin 1; AH, amphipathic helix; CT, C-terminal motif; mCpx1, mouse (Mus musculus) complexin 1; SUV, small unilamellar vesicle; LUV, large unilamellar vesicle; NMR, nuclear magnetic resonance; CD, circular dichroism; ESR, electron spin resonance; cwESR, continuous wave ESR; DEER, double electron–electron resonance; DPC, dodecylphosphocholine; POPC, 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine; POPS, 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine; DOPC, 1,2-dioleoyl-sn-glycero-3-phosphocholine; DOPE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine; DOPS, 1,2-dioleoyl-sn-glycero-3-phospho-L-serine; DO, 1,2-dioleoyl; PO, 1-palmitoyl-2-oleoyl.