Comparing the copper binding features of alpha and beta synucleins.

Amyloid aggregation of α-synuclein (AS) is one of the hallmarks of Parkinson's disease (PD). Copper ions specifically bind at the N-terminus of AS, accelerating protein aggregation. Its protein homolog ß-synuclein (BS) is also a copper binding protein, but it inhibits AS aggregation. Here, a comparative spectroscopic study of the Cu2+ binding properties of AS and BS has been performed, using electronic absorption, circular dichroism (CD) and electronic paramagnetic resonance (EPR). Our comparative spectroscopic study reveals striking similarities between the Cu2+ binding features of the two proteins. The Cu2+ binding site at the N-terminal group of BS protein, modeled by the BS (1-15) fragment is identical to that of AS; however, its rate of reduction is three times faster as compared to the AS site, consistent with BS having an additional Met residue in its Met1-Xn-Met5-Xn-Met10 motif. The latter is also evident in the cyclic voltammetry studies of the Cu-BS complex. On the other hand, the Cu2+ binding features of the His site in both proteins, as modeled by AS(45-55) and BS(60-70), are identical, indicating that the shift in the His position does not affect its coordination features. Finally, replacement of Glu46 by Ala does not alter Cu2+ binding to the His site, suggesting that the familial PD E46K mutation would not impact copper-induced aggregation. While further studies of the redox activity of copper bound to His50 in AS are required to understand the role of this site in metal-mediated aggregation, our study contributes to a better understanding of the bioinorganic chemistry of PD.

Excess membrane binding of monomeric alpha-, beta- and gamma-synuclein is invariably associated with inclusion formation and toxicity.

α-Synuclein (αS) has been well-documented to play a role in human synucleinopathies such as Parkinson's disease (PD) and dementia with Lewy bodies (DLB). First, the lesions found in PD/DLB brains-Lewy bodies and Lewy neurites-are rich in aggregated αS. Second, genetic evidence links missense mutations and increased αS expression to familial forms of PD/DLB. Third, toxicity and cellular stress can be caused by αS under certain experimental conditions. In contrast, the homologs ß-synuclein (ßS) and γ-synuclein (γS) are not typically found in Lewy bodies/neurites, have not been clearly linked to brain diseases and have been largely non-toxic in experimental settings. In αS, the so-called non-amyloid-ß component of plaques (NAC) domain, constituting amino acids 61-95, has been identified to be critical for aggregation in vitro. This domain is partially absent in ßS and only incompletely conserved in γS, which could explain why both homologs do not cause disease. However, αS in vitro aggregation and cellular toxicity have not been firmly linked experimentally, and it has been proposed that excess αS membrane binding is sufficient to induce neurotoxicity. Indeed, recent characterizations of Lewy bodies have highlighted the accumulation of lipids and membranous organelles, raising the possibility that ßS and γS could also become neurotoxic if they were more prone to membrane/lipid binding. Here, we increased ßS and γS membrane affinity by strategic point mutations and demonstrate that these proteins behave like membrane-associated monomers, are cytotoxic and form round cytoplasmic inclusions that can be prevented by inhibiting stearoyl-CoA desaturase.

Temperature is a key determinant of alpha- and beta-synuclein membrane interactions in neurons.

Aggregation of α-synuclein (αS) leads to the hallmark neuropathology of Parkinson's disease (PD) and related synucleinopathies. αS has been described to exist in both cytosolic and membrane-associated forms, the relative abundance of which has remained unsettled. To study αS under the most relevant conditions by a quantitative method, we cultured and matured rodent primary cortical neurons for >17 days and determined αS cytosol:membrane distribution via centrifugation-free sequential extractions based on the weak ionic detergent digitonin. We noticed that at lower temperatures (4 °C or room temperature), αS was largely membrane-associated. At 37 °C, however, αS solubility was markedly increased. In contrast, the extraction of control proteins (GAPDH, cytosolic; calnexin, membrane) was not affected by temperature. When we compared the relative distribution of the synuclein homologs αS and ß-synuclein (ßS) under various conditions that differed in temperature and digitonin concentration (200-1200 µg/ml), we consistently found αS to be more membrane-associated than ßS. Both proteins, however, exhibited temperature-dependent membrane binding. Under the most relevant conditions (37 °C and 800 µg/ml digitonin, i.e., the lowest digitonin concentration that extracted cytosolic GAPDH to near completion), cytosolic distribution was 49.8% ± 9.0% for αS and 63.6% ± 6.6% for ßS. PD-linked αS A30P was found to be largely cytosolic, confirming previous studies that had used different methods. Our work highlights the dynamic nature of cellular synuclein behavior and has important implications for protein-biochemical and cell-biological studies of αS proteostasis, such as testing the effects of genetic and pharmacological manipulations.

Widespread occurrence of the droplet state of proteins in the human proteome.

A wide range of proteins have been reported to condensate into a dense liquid phase, forming a reversible droplet state. Failure in the control of the droplet state can lead to the formation of the more stable amyloid state, which is often disease-related. These observations prompt the question of how many proteins can undergo liquid-liquid phase separation. Here, in order to address this problem, we discuss the biophysical principles underlying the droplet state of proteins by analyzing current evidence for droplet-driver and droplet-client proteins. Based on the concept that the droplet state is stabilized by the large conformational entropy associated with nonspecific side-chain interactions, we develop the FuzDrop method to predict droplet-promoting regions and proteins, which can spontaneously phase separate. We use this approach to carry out a proteome-level study to rank proteins according to their propensity to form the droplet state, spontaneously or via partner interactions. Our results lead to the conclusion that the droplet state could be, at least transiently, accessible to most proteins under conditions found in the cellular environment.

HLA DR2b-binding peptides from human endogenous retrovirus envelope, Epstein-Barr virus and brain proteins in the context of molecular mimicry in multiple sclerosis.

The aetiology of multiple sclerosis (MS) is as yet poorly understood. Multiple mechanisms in different disease stages are responsible for immunopathology in MS. HLA Class II DR2b (DRB1*1501 ß, DRA1*0101 α) is the strongest genetic risk factor for MS. Remnants of ancient retroviruses in the human genome, termed human endogenous retroviruses (HERV), and Epstein-Barr virus (EBV) infection are also associated with MS. In silico analyses of human endogenous retroviral envelope (HERV env) proteins and three myelin proteins that are principal targets of an autoimmune response in MS showed sequence similarities between potential TH epitopes within pairs of viral and myelin peptides predicted to bind HLA DR2b. This led to the proposal that such molecular mimicry may potentially trigger MS. HLA DR2b binding characteristics of previously identified peptides from the three myelin proteins and HERV env proteins as well as additional in silico predicted peptides from other encephalitogenic brain proteins and EBV proteins were studied to further investigate molecular mimicry. Peptides containing potential TH epitopes from the myelin oligodendrocyte glycoprotein and HERV env previously predicted to bind HLA DR2b as well as other pertinent potential HLA DR2b-restricted TH epitopes were confirmed to bind HLA DR2b molecules. Molecular modelling of HLA DR2b in complex with high affinity peptides derived from MOG and HERV env proteins showed that their binding could occur in a similar manner to a HLA DR2b-binding peptide containing a known TH epitope. A structurally related pair of peptides predicted to bind HLA DR2b from the EBV protein EBNA1 and ß synuclein, a brain protein implicated in MS, were also shown to similarly bind HLA DR2b. The findings justify investigating CD4+ T cell responses to the identified peptides.

Vibrational Circular Dichroism Sheds New Light on the Competitive Effects of Crowding and ß-Synuclein on the Fibrillation Process of α-Synuclein.

The effects of crowding, using the crowding agent Ficoll 70, and the presence of ß-synuclein on the fibrillation process of α-synuclein were studied by spectroscopic techniques, transmission electron microscopy, and thioflavin T assays. This combined approach, in which all techniques were applied to the same original sample, generated an unprecedented understanding of the effects of these modifying agents on the morphological properties of the fibrils. Separately, crowding gives rise to shorter mutually aligned fibrils, while ß-synuclein leads to branched, short fibrils. The combination of both effects leads to short, branched, mutually aligned fibrils. Moreover, it is shown that the nondestructive technique of vibrational circular dichroism is extremely sensitive to the length and the higher-order morphology of the fibrils.

Comparative Analysis of the Conformation, Aggregation, Interaction, and Fibril Morphologies of Human α-, ß-, and γ-Synuclein Proteins.

The human synuclein (syn) family is comprised of α-, ß-, and γ-syn proteins. α-syn has the highest propensity for aggregation, and its aggregated forms accumulate in Lewy bodies (LB) and Lewy neurites, which are involved in Parkinson's disease (PD). ß- and γ-syn are absent in LB, and their exact role is still enigmatic. ß-syn does not form aggregates under physiological conditions (pH 7.4), while γ-syn is associated with neural and non-neural diseases like breast cancer. Because of their similar regional distribution in the brain, natively unfolded structure, and high degree of sequence homology, studying the effect of the environment on their conformation, interactions, fibrillation, and fibril morphologies has become important. Our studies show that high temperatures, low pH values, and high concentrations increase the rate of fibrillation of α- and γ-syn, while ß-syn forms fibrils only at low pH. Fibril morphologies are strongly dependent on the immediate environment of the proteins. The high molar ratio of ß-syn inhibits the fibrillation in α- and γ-syn. However, preformed seed fibrils of ß- and γ-syn do not affect fibrillation of α-syn. Surface plasmon resonance data show that interactions between α- and ß-syn, ß- and γ-syn, and α- and γ-syn are weak to moderate in nature and can be physiologically significant in counteracting several adverse conditions in the cells that trigger their aggregation. These studies could be helpful in understanding collective human synuclein behavior in various protein environments and in the modulation of the homeostasis between ß-syn and healthy versus corrupt α- and γ-syn that can potentially affect PD pathology.

Effects of phosphatidylcholine membrane fluidity on the conformation and aggregation of N-terminally acetylated α-synuclein.

Membrane association of α-synuclein (α-syn), a neuronal protein associated with Parkinson's disease (PD), is involved in α-syn function and pathology. Most previous studies on α-syn-membrane interactions have not used the physiologically relevant N-terminally acetylated (N-acetyl) α-syn form nor the most naturally abundant cellular lipid, i.e. phosphatidylcholine (PC). Here, we report on how PC membrane fluidity affects the conformation and aggregation propensity of N-acetyl α-syn. It is well established that upon membrane binding, α-syn adopts an α-helical structure. Using CD spectroscopy, we show that N-acetyl α-syn transitions from α-helical to disordered at the lipid melting temperature (Tm ). We found that this fluidity sensing is a robust characteristic, unaffected by acyl chain length (Tm = 34-55 °C) and preserved in its homologs ß- and γ-syn. Interestingly, both N-acetyl α-syn membrane binding and amyloid formation trended with lipid order (1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) > 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/sphingomyelin/cholesterol (2:2:1) ≥ DOPC), with gel-phase vesicles shortening aggregation kinetics and promoting fibril formation compared to fluid membranes. Furthermore, we found that acetylation enhances binding to PC micelles and small unilamellar vesicles with high curvature (r ∼16-20 nm) and that DPPC binding is reduced in the presence of cholesterol. These results confirmed that the exposure of hydrocarbon chains (i.e. packing defects) is essential for binding to zwitterionic gel membranes. Collectively, our in vitro results suggest that N-acetyl α-syn localizes to highly curved, ordered membranes inside a cell. We propose that age-related changes in membrane fluidity can promote the formation of amyloid fibrils, insoluble materials associated with PD.

Multi-Pronged Interactions Underlie Inhibition of α-Synuclein Aggregation by ß-Synuclein.

The intrinsically disordered protein ß-synuclein is known to inhibit the aggregation of its intrinsically disordered homolog, α-synuclein, which is implicated in Parkinson's disease. While ß-synuclein itself does not form fibrils at the cytoplasmic pH 7.4, alteration of pH and other environmental perturbations are known to induce its fibrilization. However, the sequence and structural determinants of ß-synuclein inhibition and self-aggregation are not well understood. We have utilized a series of domain-swapped chimeras of α-synuclein and ß-synuclein to probe the relative contributions of the N-terminal, C-terminal, and the central non-amyloid-ß component domains to the inhibition of α-synuclein aggregation. Changes in the rates of α-synuclein fibril formation in the presence of the chimeras indicate that the non-amyloid-ß component domain is the primary determinant of self-association leading to fibril formation, while the N- and C-terminal domains play critical roles in the fibril inhibition process. Our data provide evidence that all three domains of ß-synuclein together contribute to providing effective inhibition, and support a model of transient, multi-pronged interactions between IDP chains in both processes. Inclusion of such multi-site inhibitory interactions spread over the length of synuclein chains may be critical for the development of therapeutics that are designed to mimic the inhibitory effects of ß-synuclein.

Loss in Toxic Function of Aggregates of α -Synuclein Mutants by a β-Synuclein Derived Peptide.

BACKGROUND: Parkinson's disease (PD) primarily results from a severe and selective damage of dopaminergic neurons. The neuropathological hallmark of the disease is the presence of protein inclusions of α-Synuclein (αS) within the surviving neurons. To date, several researchers have been focused on screening for the inhibitors that are able to block, slow down, or reverse αS aggregation, particularly at its early stages. OBJECTIVES: In this work, we aimed to investigate the effects of a ß-Synuclein derived peptide on oligomerization of pathological mutants of αS (A53T, A30P, E46K). METHOD: The effects of the peptide on aggregation of native and mutant of αS were examined by fluorescence spectroscopy and electron microscopy. The influence of the peptide inhibitor on cell toxicity of aggregation products of αS and its mutants were investigated by MTT assay and flow cytometry. RESULTS: It was shown that the peptide inhibitor effectively blocks the fibrillation of not only the native αS but also the PD related αS mutants in vitro, suggesting a similar mechanism of oligomerization for native and mutants of αS. The peptide inhibitor greatly diminished the cell toxicity of the aggregation products of native and mutants of αS. CONCLUSION: Our findings suggest the therapeutic potential of this peptide for treatment of the rare inherited forms of PD.

Bioinorganic Chemistry of Parkinson's Disease: Affinity and Structural Features of Cu(I) Binding to the Full-Length ß-Synuclein Protein.

Alterations in the levels of copper in brain tissue and formation of α-synuclein (αS)-copper complexes might play a key role in the amyloid aggregation of αS and the onset of Parkinson's disease (PD). Recently, we demonstrated that formation of the high-affinity Cu(I) complex with the N-terminally acetylated form of the protein αS substantially increases and stabilizes local conformations with α-helical secondary structure and restricted motility. In this work, we performed a detailed NMR-based structural characterization of the Cu(I) complexes with the full-length acetylated form of its homologue ß-synuclein (ßS), which is colocalized with αS in vivo and can bind copper ions. Our results show that, similarly to αS, the N-terminal region of ßS constitutes the preferential binding interface for Cu(I) ions, encompassing two independent and noninteractive Cu(I) binding sites. According to these results, ßS binds the metal ion with higher affinity than αS, in a coordination environment that involves the participation of Met-1, Met-5, and Met-10 residues (site 1). Compared to αS, the shift of His from position 50 to 65 in the N-terminal region of ßS does not change the Cu(I) affinity features at that site (site 2). Interestingly, the formation of the high-affinity ßS-Cu(I) complex at site 1 in the N-terminus promotes a short α-helix conformation that is restricted to the 1-5 segment of the AcßS sequence, which differs with the substantial increase in α-helix conformations seen for N-terminally acetylated αS upon Cu(I) complexation. Our NMR data demonstrate conclusively that the differences observed in the conformational transitions triggered by Cu(I) binding to AcαS and AcßS find a correlation at the level of their backbone dynamic properties; added to the potential biological implications of these findings, this fact opens new avenues of investigations into the bioinorganic chemistry of PD.

A pH-dependent switch promotes ß-synuclein fibril formation via glutamate residues.

α-Synuclein (αS) is the primary protein associated with Parkinson's disease, and it undergoes aggregation from its intrinsically disordered monomeric form to a cross-ß fibrillar form. The closely related homolog ß-synuclein (ßS) is essentially fibril-resistant under cytoplasmic physiological conditions. Toxic gain-of-function by ßS has been linked to dysfunction, but the aggregation behavior of ßS under altered pH is not well-understood. In this work, we compare fibril formation of αS and ßS at pH 7.3 and mildly acidic pH 5.8, and we demonstrate that pH serves as an on/off switch for ßS fibrillation. Using αS/ßS domain-swapped chimera constructs and single residue substitutions in ßS, we localized the switch to acidic residues in the N-terminal and non-amyloid component domains of ßS. Computational models of ßS fibril structures indicate that key glutamate residues (Glu-31 and Glu-61) in these domains may be sites of pH-sensitive interactions, and variants E31A and E61A show dramatically altered pH sensitivity for fibril formation supporting the importance of these charged side chains in fibril formation of ßS. Our results demonstrate that relatively small changes in pH, which occur frequently in the cytoplasm and in secretory pathways, may induce the formation of ßS fibrils and suggest a complex role for ßS in synuclein cellular homeostasis and Parkinson's disease.

Potential of mean force and molecular dynamics study on the transient interactions between α and ß synuclein that drive inhibition of α-synuclein aggregation.

Self-association of α-synuclein (αS) into pathogenic oligomeric species and subsequent formation of highly ordered amyloid fibrils is linked to the Parkinson's disease. So most of the recent studies are now focused on the development of potential therapeutic strategies against this debilitating disease. ß-synuclein (ßS), a presynaptic protein that co-localizes with αS has been recently reported to act as an inhibitor of αS self-assembly. But the specificity of molecular interaction, nature and location between αS/ßS is not known despite the potential importance of ßS as an inhibitor of αS. We used molecular dynamics and potential of mean force (PMF) to study association of αS/ßS and αS/αS. The calculated PMF indicates that contact wells are significantly deeper and presence of a minimum at αS/ßS separation of 13.5 Å with a free energy barrier of 40 kcal/mol. We observed the dissociation energy barrier to be two times higher for the hetero-dimer (αS/ßS) than the homo-dimer (αS/αS). We also carried out umbrella samplings involving two degrees of freedom (one being the distance between the monomeric units and the other angle between the long axes of the two monomeric chains) and observed similar PMF profile. We noticed relatively stronger range of transient interactions between the monomeric units in hetero-dimer (αS/ßS) than homo-dimer (αS/αS). So our findings suggest that αS readily combines with ßS to form hetero-dimer than combining with itself in forming homo-dimer. Hence we see predominant transient interactions between αS and ßS can be used to drive inhibition of αS aggregation.

ß-Synuclein suppresses both the initiation and amplification steps of α-synuclein aggregation via competitive binding to surfaces.

α-Synuclein is an intrinsically disordered protein that is associated with the pathogenesis of Parkinson's disease through the processes involved in the formation of amyloid fibrils. α and ß-synuclein are homologous proteins found at comparable levels in presynaptic terminals but ß-synuclein has a greatly reduced propensity to aggregate and indeed has been found to inhibit α-synuclein aggregation. In this paper, we describe how sequence differences between α- and ß-synuclein affect individual microscopic processes in amyloid formation. In particular, we show that ß-synuclein strongly suppresses both lipid-induced aggregation and secondary nucleation of α-synuclein by competing for binding sites at the surfaces of lipid vesicles and fibrils, respectively. These results suggest that ß-synuclein can act as a natural inhibitor of α-synuclein aggregation by reducing both the initiation of its self-assembly and the proliferation of its aggregates.

The loss of inhibitory C-terminal conformations in disease associated P123H ß-synuclein.

ß-synuclein (ßS) is a homologue of α-synuclein (αS), the major protein component of Lewy bodies in patients with Parkinson's disease. In contrast to αS, ßS does not form fibrils, mitigates αS toxicity in vivo and inhibits αS fibril formation in vitro. Previously a missense mutation of ßS, P123H, was identified in patients with Dementia with Lewy Body disease. The single P123H mutation at the C-terminus of ßS is able to convert ßS from a nontoxic to a toxic protein that is also able to accelerate formation of inclusions when it is in the presence of αS in vivo. To elucidate the molecular mechanisms of these processes, we compare the conformational properties of the monomer forms of αS, ßS and P123H-ßS, and the effects on fibril formation of coincubation of αS with ßS, and with P123H-ßS. NMR residual dipolar couplings and secondary structure propensities show that the P123H mutation of ßS renders it more flexible C-terminal to the mutation site and more αS-like. In vitro Thioflavin T fluorescence experiments show that P123H-ßS accelerates αS fibril formation upon coincubation, as opposed to wild type ßS that acts as an inhibitor of αS aggregation. When P123H-ßS becomes more αS-like it is unable to perform the protective function of ßS, which suggests that the extended polyproline II motif of ßS in the C-terminus is critical to its nontoxic nature and to inhibition of αS upon coincubation. These studies may provide a basis for understanding which regions to target for therapeutic intervention in Parkinson's disease.

Unveiling transient protein-protein interactions that modulate inhibition of alpha-synuclein aggregation by beta-synuclein, a pre-synaptic protein that co-localizes with alpha-synuclein.

Pathology in Parkinson's disease is linked to self-association of α-Synuclein (αS) into pathogenic oligomeric species and highly ordered amyloid fibrils. Developing effective therapeutic strategies against this debilitating disease is critical and ßS, a pre-synaptic protein that co-localizes with αS, can act as an inhibitor of αS assembly. Despite the potential importance of ßS as an inhibitor of αS, the nature, location and specificity of the molecular interactions between these two proteins is unknown. Here we use NMR paramagnetic relaxation enhancement experiments, to demonstrate that ßS interacts directly with αS in a transient dimer complex with high specificity and weak affinity. Inhibition of αS by ßS arises from transient αS/ßS heterodimer species that exist primarily in head- to- tail configurations while αS aggregation arises from a more heterogeneous and weaker range of transient interactions that include both head-to-head and head-to-tail configurations. Our results highlight that intrinsically disordered proteins can interact directly with one another at low affinity and that the transient interactions that drive inhibition versus aggregation are distinct by virtue of their plasticity and specificity.

Copper(I/II), α/ß-Synuclein and Amyloid-ß: Menage à Trois?

Copper binding to α-synuclein (aS) and to amyloid-ß (Ab) has been connected to Parkinson's and Alzheimer's disease (AD), respectively, because Cu ions can modulate the peptide aggregation, and these Cu ⋅ peptide complexes can catalyse the production of reactive oxygen species (ROS). In a significant proportion of AD brains, aggregation of aS and Ab has been detected, and it was proposed that Ab and aS interact with each other. Thus, we investigated the potential interactions of Ab and aS through their binding of copper(I) and copper(II). Additionally, ß-synuclein (bS) was investigated, due to its additional methionine residue, a potential Cu(I) ligand. We found that: 1) the peptides containing the Cu-binding domains Ab1-16, aS1-15 and bS1-15 have similar affinities towards Cu(II) and towards Cu(I), with Ab1-16 being slightly stronger, 2) in the case of Cu(I), the additional Met residue in bS1-15 increased the affinity slightly, 3) the exchange of Cu(I/II) between the two peptides is rapid (≤ ms), 4) a/bS1-15 and Ab1-16 form a heterodimeric complex with Cu(II), 5) Cu(I) probably promotes a transient ternary complex, 6) the different Cu(I/II) coordination of Ab1-16, aS1-15 and bS1-15 impacts the capacity to produce ROS and to oxidise catechol, and 7) when Ab1-16, aS1-15 and Cu are present, the ROS production more closely resembles that by Ab1-16. The work gives insights into the coordination chemistry of these related peptides, and the relevance of coordination differences, the ternary complex and ROS production are discussed.

Dimerization propensities of Synucleins are not predictive for Synuclein aggregation.

Aggregation and fibril formation of human alpha-Synuclein (αS) are neuropathological hallmarks of Parkinson's disease and other synucleinopathies. The molecular mechanisms of αS aggregation and fibrillogenesis are largely unknown. Several studies suggested a sequence of events from αS dimerization via oligomerization and pre-fibrillar aggregation to αS fibril formation. In contrast to αS, little evidence suggests that γS can form protein aggregates in the brain, and for ßS its neurotoxic properties and aggregation propensities are controversially discussed. These apparent differences in aggregation behavior prompted us to investigate the first step in Synuclein aggregation, i.e. the formation of dimers or oligomers, by Bimolecular Fluorescence Complementation in cells. This assay showed some Synuclein-specific limitations, questioning its performance on a single cell level. Nevertheless, we unequivocally demonstrate that all Synucleins can interact with each other in a very similar way. Given the divergent aggregation properties of the three Synucleins this suggests that formation of dimers is not predictive for the aggregation of αS, ßS or γS in the aged or diseased brain.

Differences in the binding of copper(I) to α- and ß-synuclein.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the presence of abnormal α-synuclein (αS) deposits in the brain. Alterations in homeostasis and metal-induced oxidative stress may play a crucial role in the progression of αS amyloid assembly and pathogenesis of PD. Contrary to αS, ß-synuclein (ßS) is not involved in the PD etiology. However, it has been suggested that the ßS/αS ratio is altered in PD, indicating that a correct balance of these two proteins is implicated in the inhibition of αS aggregation. αS and ßS share similar abilities to coordinate Cu(II). In this study, we investigated and compared the interaction of Cu(I) with the N-terminal portion of ßS and αS by means of NMR, circular dichroism, and X-ray absorption spectroscopies. Our data show the importance of M10K mutation, which induces different Cu(I) chemical environments. Coordination modes 3S1O and 2S2O were identified for ßS and αS, respectively. These new insights into the bioinorganic chemistry of copper and synuclein proteins are a basis to understand the molecular mechanism by which ßS might inhibit αS aggregation.

A relationship between the transient structure in the monomeric state and the aggregation propensities of α-synuclein and ß-synuclein.

α-Synuclein is an intrinsically disordered protein whose aggregation is implicated in Parkinson's disease. A second member of the synuclein family, ß-synuclein, shares significant sequence similarity with α-synuclein but is much more resistant to aggregation. ß-Synuclein is missing an 11-residue stretch in the central non-ß-amyloid component region that forms the core of α-synuclein amyloid fibrils, yet insertion of these residues into ß-synuclein to produce the ßSHC construct does not markedly increase the aggregation propensity. To investigate the structural basis of these different behaviors, quantitative nuclear magnetic resonance data, in the form of paramagnetic relaxation enhancement-derived interatomic distances, are combined with molecular dynamics simulations to generate ensembles of structures representative of the solution states of α-synuclein, ß-synuclein, and ßSHC. Comparison of these ensembles reveals that the differing aggregation propensities of α-synuclein and ß-synuclein are associated with differences in the degree of residual structure in the C-terminus coupled to the shorter separation between the N- and C-termini in ß-synuclein and ßSHC, making protective intramolecular contacts more likely.