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

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.

Impact of membrane curvature on amyloid aggregation.

The misfolding, amyloid aggregation, and fibril formation of intrinsically disordered proteins/peptides (or amyloid proteins) have been shown to cause a number of disorders. The underlying mechanisms of amyloid fibrillation and structural properties of amyloidogenic precursors, intermediates, and amyloid fibrils have been elucidated in detail; however, in-depth examinations on physiologically relevant contributing factors that induce amyloidogenesis and lead to cell death remain challenging. A large number of studies have attempted to characterize the roles of biomembranes on protein aggregation and membrane-mediated cell death by designing various membrane components, such as gangliosides, cholesterol, and other lipid compositions, and by using various membrane mimetics, including liposomes, bicelles, and different types of lipid-nanodiscs. We herein review the dynamic effects of membrane curvature on amyloid generation and the inhibition of amyloidogenic proteins and peptides, and also discuss how amyloid formation affects membrane curvature and integrity, which are key for understanding relationships with cell death. Small unilamellar vesicles with high curvature and large unilamellar vesicles with low curvature have been demonstrated to exhibit different capabilities to induce the nucleation, amyloid formation, and inhibition of amyloid-ß peptides and α-synuclein. Polymorphic amyloidogenesis in small unilamellar vesicles was revealed and may be viewed as one of the generic properties of interprotein interaction-dominated amyloid formation. Several mechanical models and phase diagrams are comprehensively shown to better explain experimental findings. The negative membrane curvature-mediated mechanisms responsible for the toxicity of pancreatic ß cells by the amyloid aggregation of human islet amyloid polypeptide (IAPP) and binding of the precursors of the semen-derived enhancer of viral infection (SEVI) are also described. The curvature-dependent binding modes of several types of islet amyloid polypeptides with high-resolution NMR structures are also discussed.

Membrane-induced initial structure of α-synuclein control its amyloidogenesis on model membranes.

Amyloid fibrillation causes serious neurodegenerative diseases and amyloidosis; however, the detailed mechanisms by which the structural states of precursor proteins in a lipid membrane-associated environment contribute to amyloidogenesis still remains to be elucidated. We examined the relationship between structural states of intrinsically-disordered wild-type and mutant α-synuclein (αSN) and amyloidogenesis on two-types of model membranes. Highly-unstructured wild-type αSN (αSNWT) and a C-terminally-truncated mutant lacking negative charges (αSN103) formed amyloid fibrils on both types of membranes, the model membrane mimicking presynaptic vesicles (Mimic membrane) and the model membrane of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC membrane). Unstructured αSNWT and αSN103 both bound to Mimic membranes in a helical conformation with similar binding affinity. Promotion and then inhibition of amyloidogenesis of αSNWT were observed as the concentration of Mimic lipids increased. We explain this by the two-state binding model: at lower lipid concentrations, binding of αSNWT to membranes enhances amyloidogenicity by increasing the local concentration of membrane-bound αSN and so promoting amyloid nucleation; at higher lipid concentrations, membrane-bound αSNWT is actually in a sense diluted by increasing the number of model membranes, which blocks amyloid fibrillation due to an insufficient bound population for productive nucleation. Meanwhile, αSN103 formed amyloid fibrils over the whole concentration of Mimic lipids used here without inhibition, revealing the importance of helical structures for binding affinity and negatively charged unstructured C-terminal region for modulating amyloidogenesis. We propose that membrane binding-induced initial conformations of αSN, its overall charge states, and the population of membrane-bound αSN are key determinants of amyloidogenesis on membranes.

Model membrane size-dependent amyloidogenesis of Alzheimer's amyloid-ß peptides.

We herein report the mechanism of amyloid formation of amyloid-ß (Aß) peptides on small (SUV) and large unilamellar vesicles (LUVs), which consist of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipids. Although Aß1-42 formed fibrils on SUVs at all POPC concentrations used, the lag time, elongation rate, maximum thioflavin T intensity, and fibrillar morphology were distinct, indicating polymorphic amyloid formation. LUVs, at low POPC concentrations, did not markedly affect fibrillation kinetics; however, increases in POPC concentrations suppressed amyloid formation. No significant differences in the thermal stabilities of Aß1-42 fibrils formed with and without vesicles were observed, although fibrils formed on SUVs showed some differences with dilution. SUVs markedly promoted Aß1-40 fibrillation by condensing Aß1-40, whereas no effects of LUVs on amyloidogenesis were detected. Salts greatly increased Aß1-40 amyloidogenicity on vesicles. We proposed comprehensive models for vesicle size-dependent Aß amyloidogenesis. Inhomogeneous packing defects in SUVs may induce distinct nucleation in the polymorphisms of amyloids and decreasing local concentrations of Aß with higher amounts of LUVs inhibits amyloid formation. We also pointed out that C-terminal hydrophobicity of Aß is important for amyloidogenesis on membranes.

Evolutionary Divergence of the C-terminal Domain of Complexin Accounts for Functional Disparities between Vertebrate and Invertebrate Complexins.

Complexin is a critical presynaptic protein that regulates both spontaneous and calcium-triggered neurotransmitter release in all synapses. Although the SNARE-binding central helix of complexin is highly conserved and required for all known complexin functions, the remainder of the protein has profoundly diverged across the animal kingdom. Striking disparities in complexin inhibitory activity are observed between vertebrate and invertebrate complexins but little is known about the source of these differences or their relevance to the underlying mechanism of complexin regulation. We found that mouse complexin 1 (mCpx1) failed to inhibit neurotransmitter secretion in Caenorhabditis elegans neuromuscular junctions lacking the worm complexin 1 (CPX-1). This lack of inhibition stemmed from differences in the C-terminal domain (CTD) of mCpx1. Previous studies revealed that the CTD selectively binds to highly curved membranes and directs complexin to synaptic vesicles. Although mouse and worm complexin have similar lipid binding affinity, their last few amino acids differ in both hydrophobicity and in lipid binding conformation, and these differences strongly impacted CPX-1 inhibitory function. Moreover, function was not maintained if a critical amphipathic helix in the worm CPX-1 CTD was replaced with the corresponding mCpx1 amphipathic helix. Invertebrate complexins generally shared more C-terminal similarity with vertebrate complexin 3 and 4 isoforms, and the amphipathic region of mouse complexin 3 significantly restored inhibitory function to worm CPX-1. We hypothesize that the CTD of complexin is essential in conferring an inhibitory function to complexin, and that this inhibitory activity has been attenuated in the vertebrate complexin 1 and 2 isoforms. Thus, evolutionary changes in the complexin CTD differentially shape its synaptic role across phylogeny.

Small liposomes accelerate the fibrillation of amyloid ß (1-40).

The deposition of amyloid ß (Aß) peptides is a pathological hallmark of Alzheimer disease. Aß peptides were previously considered to interact specifically with ganglioside-containing membranes. Several studies have suggested that Aß peptides also bind to phosphatidylcholine membranes, which lead to deformation of membranes and fibrillation of Aß. Moreover, the role of membrane curvature, one type of deformation produced by binding of proteins to a membrane, in the binding and fibrillation of Aß remains unclear. To clearly understand the relationship between the binding, consequent membrane deformation, and fibrillation of Aß, we examined the amyloid fibrillation of Aß-(1-40) in the presence of liposomes of various sizes. Membrane curvature increased with a decrease in the size of the liposomes. We used liposomes made of 1,2-dioleoyl-sn-glycero-3-phosphocholine to eliminate electrostatic effects. The results obtained showed that liposomes of smaller sizes (≤50 nm) significantly accelerated the nucleation step, thereby shortening the lag time of fibrillation. On the other hand, liposomes of larger sizes decreased the amount of fibrils but did not notably affect the lag time. The morphologies of fibrils, which were monitored by total internal reflection fluorescence microscopy, atomic force microscopy, and transmission electron microscopy, revealed that the length of Aß-(1-40) fibrils became shorter and the amount of amorphous aggregates became larger as liposomes increased in size. These results suggest that the curvature of membranes coupled with an increase in water-accessible hydrophobic regions is important for binding and concentrating Aß monomers, leading to amyloid nucleation. Furthermore, amyloid fibrillation on membranes may compete with non-productive binding to produce amorphous aggregates.