Membrane Binding Induces Distinct Structural Signatures in the Mouse Complexin-1C-Terminal Domain.

[Display omitted] • The role of the complexin CTD in regulating SV exocytosis is controversial due to conflicting functional studies. • The isolated mCpx1 CTD is largely disordered, but forms two discontinuous helical structures in the presence of micelles. • This helical structure also forms in the presence of SUVs, as confirmed by CD, ESR and fluorescence spectroscopies. • The mCpx1 CTD interacts with LUVs in a lipid dependent fashion. • Differences between the mCpx1 and wCpx1 CTDs suggest a mechanistic basis for functional differences between the proteins. 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. 1 1 Abbreviations 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. [ABSTRACT FROM AUTHOR]