Alpha-synuclein is known to bind to small unilamellar vesicles (SUVs) via its N terminus, which forms an amphipathic alpha-helix upon membrane interaction. Here we show that calcium binds to the C terminus of alpha-synuclein, therewith increasing its lipid-binding capacity. Using CEST-NMR, we reveal that alpha-synuclein interacts with isolated synaptic vesicles with two regions, the N terminus, already known from studies on SUVs, and additionally via its C terminus, which is regulated by the binding of calcium. Indeed, dSTORM on synaptosomes shows that calcium mediates the localization of alpha-synuclein at the pre-synaptic terminal, and an imbalance in calcium or alpha-synuclein can cause synaptic vesicle clustering, as seen ex vivo and in vitro. This study provides a new view on the binding of alpha-synuclein to synaptic vesicles, which might also affect our understanding of synucleinopathies.
As an intrinsically disordered protein, monomeric alpha-synuclein (aSyn) occupies a large conformational space. Certain conformations lead to aggregation prone and non-aggregation prone intermediates, but identifying these within the dynamic ensemble of monomeric conformations is difficult. Herein, we used the biologically relevant calcium ion to investigate the conformation of monomeric aSyn in relation to its aggregation propensity. We observe that the more exposed the N-terminus and the beginning of the NAC region of aSyn are, the more aggregation prone monomeric aSyn conformations become. Solvent exposure of the Nterminus of aSyn occurs upon release of C-terminus interactions when calcium binds, but the level of exposure and aSyn's aggregation propensity is sequence and post translational modification dependent. Identifying aggregation prone conformations of monomeric aSyn and the environmental conditions they form under will allow us to design new therapeutics targeted to the monomeric protein.
Understanding the mechanisms behind amyloid protein aggregation in diseases, such as Parkinson’s and Alzheimer’s disease, is often hampered by the reproducibility of in vitro assays. Yet, understanding the basic mechanisms of protein misfolding is essential for the development of novel therapeutic strategies. We show here, that for the amyloid protein α-synuclein (aSyn), a protein involved in Parkinson’s disease (PD), chromatographic buffers and storage conditions can significantly interfere with the overall structure of the protein and thus affect protein aggregation kinetics. We apply several biophysical and biochemical methods, including size exclusion chromatography (SEC), dynamic light scattering (DLS), and atomic force microscopy (AFM), to characterize the high molecular weight conformers formed during protein purification and storage. We further apply hydrogen/deuterium-exchange mass spectrometry (HDX-MS) to characterize the monomeric form of aSyn and reveal a thus far unknown structural component of aSyn at the C-terminus of the protein. Furthermore, lyophilizing the protein greatly affected the overall structure of this monomeric conformer. We conclude from this study that structural polymorphism may occur under different storage conditions, but knowing the structure of the majority of the protein at the start of each experiment, as well as the factors that may influence it, may pave the way to an improved understanding of the mechanism leading to aSyn pathology in PD.
In Parkinson’s disease and other synucleinopathies, α-synuclein misfolds and aggregates. Its intrinsically disordered nature, however, causes it to adopt several meta-stable conformations stabilized by internal hydrogen bonding. Because they interconvert on short timescales, monomeric conformations of disordered proteins are difficult to characterize using common structural techniques. Few techniques can measure the conformations of monomeric α-synuclein, including millisecond hydrogen/deuterium-exchange mass spectrometry (HDX-MS). Here, we demonstrate a new approach correlating millisecond HDX-MS data with aggregation kinetics to determine the localized structural dynamics that underpin the self-assembly process in full-length wild-type monomeric α-synuclein. Our custom instrumentation and software enabled measurement of the amide hydrogen-exchange rates on the millisecond timescale for wild-type α-synuclein monomer up to residue resolution and under physiological conditions, mimicking those in the extracellular, intracellular, and lysosomal cellular compartments. We applied an empirical correction to normalize measured hydrogen-exchange rates and thus allow comparison between drastically different solution conditions. We characterized the aggregation kinetics and morphology of the resulting fibrils and correlate these with structural changes in the monomer. Applying a correlative approach to connect molecular conformation to aggregation in α-synuclein for the first time, we found that the central C-terminal residues of α-synuclein are driving its nucleation and thus its aggregation. We provide a new approach to link the local structural dynamics of intrinsically disordered proteins to functional attributes, which we evidence with new details on our current understanding of the relationship between the local chemical environment and conformational ensemble bias of monomeric α-synuclein.
21As an intrinsically disordered protein, monomeric alpha synuclein (aSyn) constantly reconfigures and 22 probes the conformational space. Long-range interactions across the protein maintain its solubility 23 and mediate this dynamic flexibility, but also provide residual structure. Certain conformations lead 24 to aggregation prone and non-aggregation prone intermediates, but identifying these within the 25 dynamic ensemble of monomeric conformations is difficult. Herein, we used the biologically relevant 26 calcium ion to investigate the conformation of monomeric aSyn in relation to its aggregation 27 propensity. By using calcium to perturb the conformational ensemble, we observe differences in 28 structure and intra-molecular dynamics between two aSyn C-terminal variants, D121A and pS129, and 29 the aSyn familial disease mutants, A30P, E46K, H50Q, G51D, A53T and A53E, compared to wild-type 30 (WT) aSyn. We observe that the more exposed the N-terminus and the beginning of the NAC region 31 are, the more aggregation prone monomeric aSyn conformations become. N-terminus exposure 32 occurs upon release of C-terminus interactions when calcium binds, but the level of exposure is 33 specific to the aSyn mutation present. There was no correlation between single charge alterations, 34 2 calcium affinity, or the number of ions bound on aSyn's aggregation propensity, indicating that 35 sequence or post-translation modification (PTM)-specific conformational differences between the N-36 and C-termini and the specific local environment mediate aggregation propensity instead. 37Understanding aggregation prone conformations of monomeric aSyn and the environmental 38 conditions they form under will allow us to design new therapeutics targeted to the monomeric 39 protein, to stabilise aSyn in non-aggregation prone conformations, by either preserving long-range 40 interactions between the N-and C-termini or by protecting the N-terminus from exposure. 41 42 or the environment that can destabilise monomeric conformations to favour aggregation will aid in 58 the design of anti-aggregation therapeutics to stabilise the native aSyn conformation. 59 aSyn is a characteristic IDP with high opposing charge at its termini and low overall hydrophobicity 5 . 60Monomeric aSyn has three characteristic main regions; the N-terminus, aar 1-60, which is overall 61 positively charged, the non-amyloid-β component (NAC) region, aar 61-89, is hydrophobic and forms 62 the core of fibrils during aggregation 7 , and the C-terminus, aar 90-140, is a highly negatively charged 63 region which binds metal ions 8 (Figure 1a). To date, six disease-related mutations have been identified 64 in the SNCA gene, encoding the aSyn protein, A30P, E46K, H50Q, G51D, A53T, A53E, which are a 65 hallmark for hereditary autosomal dominant PD and are primarily linked to early age but also late age 66 of onset (H50Q) 9-15 . However, genetic mutations and multiplications of the SNCA gene only account 67for 5-10% of PD cases and the remaining cases are sporadic (idiopathic) an...
In Parkinson's disease and other synucleinopathies, the intrinsically disordered, presynaptic protein alpha-synuclein misfolds and aggregates. We hypothesise that the exposure of alpha-synuclein to different cellular environments, with different chemical compositions, pH and binding partners, alters its biological and pathological function by inducing changes in molecular conformation. Our custom instrumentation and software enable measurement of the amide hydrogen exchange rates of wild-type alpha-synuclein at amino acid resolution under physiological conditions, mimicking those in the extracellular, intracellular, and lysosomal compartments of cells. We characterised the aggregation kinetics and morphology of the resulting fibrils and correlate these with structural changes in the monomer. Our findings reveal that the C-terminal residues of alpha-synuclein are driving its nucleation and thus its aggregation. Furthermore, the entire NAC region and specific other residues strongly promoted elongation of fibrils. This provides new detail on our current understanding of the relationship between the local chemical environment and monomeric conformations of alpha-synuclein.
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