Mapping of Photochemically-Derived Dityrosine across Fe-Bound N-Acetylated α-Synuclein.

Parkinson's disease (PD) is the second most common neurological disease and belongs to a group of neurodegenerative disorders called synucleinopathies in which pathological aggregates of N-terminally acetylated α-synuclein (NAcα-Syn) accumulate in various regions of the brain. In PD, these NAcα-Syn aggregates have been found to contain covalent dityrosine crosslinks, which can occur either intermolecularly or intramolecularly. Cerebral metal imbalance is also a hallmark of PD, warranting investigations into the effects of brain biometals on NAcα-Syn. NAcα-Syn is an intrinsically disordered protein, and metal-mediated conformational modifications of this structurally dynamic protein have been demonstrated to influence its propensity for dityrosine formation. In this study, a library of tyrosine-to-phenylalanine (Y-to-F) NAcα-Syn constructs were designed in order to elucidate the nature and the precise residues involved in dityrosine crosslinking of Fe-bound NAcα-Syn. The structural capacity of each mutant to form dityrosine crosslinks was assessed using Photo-Induced Cross-Linking of Unmodified Proteins (PICUP), demonstrating that coordination of either FeIII or FeII to NAcα-Syn inhibits dityrosine crosslinking among the C-terminal residues. We further demonstrate that Y39 is the main contributor to dityrosine formation of Fe-bound NAcα-Syn, while Y125 is the main residue involved in dityrosine crosslinks in unmetalated NAcα-Syn. Our results confirm that iron coordination has a global effect on NAcα-Syn structure and reactivity.

Navigating the dynamic landscape of alpha-synuclein morphology: a review of the physiologically relevant tetrameric conformation.

N-acetylated α-synuclein (αSyn) has long been established as an intrinsically disordered protein associated with a dysfunctional role in Parkinson's disease. In recent years, a physiologically relevant, higher order conformation has been identified as a helical tetramer that is tailored by buried hydrophobic interactions and is distinctively aggregation resistant. The canonical mechanism by which the tetramer assembles remains elusive. As novel biochemical approaches, computational methods, pioneering purification platforms, and powerful imaging techniques continue to develop, puzzling information that once sparked debate as to the veracity of the tetramer has now shed light upon this new counterpart in αSyn neurobiology. Nuclear magnetic resonance and computational studies on multimeric αSyn structure have revealed that the protein folding propensity is controlled by small energy barriers that enable large scale reconfiguration. Alternatively, familial mutations ablate tetramerization and reconfigure polymorphic fibrillization. In this review, we will discuss the dynamic landscape of αSyn quaternary structure with a focus on the tetrameric conformation.

C-Terminal CuII Coordination to α-Synuclein Enhances Aggregation.

The structurally dynamic amyloidogenic protein α-synuclein (αS) is universally recognized as a key player in Parkinson's disease (PD). Copper, which acts as a neuronal signaling agent, is also an effector of αS structure, aggregation, and localization in vivo. In humans, αS is known to carry an acetyl group on the starting methionine residue, capping the N-terminal free amine which was a known high-affinity CuII binding site. We now report the first detailed characterization data using electron paramagnetic resonance (EPR) spectroscopy to describe the CuII coordination modes of N-terminally acetylated αS (NAcαS). Through use of EPR hyperfine structure analyses and the Peisach-Blumberg correlation, an N3O1 binding mode was established that involves the single histidine residue at position 50 and a lower population of a second CuII-binding mode that may involve a C-terminal contribution. We additionally generated an N-terminally acetylated disease-relevant variant, NAcH50Q, that promotes a shift in the CuII binding site to the C-terminus of the protein. Moreover, fibrillar NAcH50Q-CuII exhibits enhanced parallel ß-sheet character and increased hydrophobic surface area compared to NAcαS-CuII and to both protein variants that lack a coordinated cupric ion. The results presented herein demonstrate the differential impact of distinct CuII binding sites within NAcαS, revealing that C-terminal CuII binding exacerbates the structural consequences of the H50Q missense mutation. Likewise, the global structural modifications that result from N-terminal capping augment the properties of CuII coordination. Hence, consideration of the effect of CuII on NAcαS and NAcH50Q misfolding may shed light on the extrinsic or environmental factors that influence PD pathology.

Copper Induced Radical Dimerization of α-Synuclein Requires Histidine.

Aggregation of the neuronal protein α-synuclein (αS) is a critical factor in the pathogenesis of Parkinson's disease. Analytical methods to detect post-translational modifications of αS are under development, yet the mechanistic underpinnings of biomarkers like dityrosine formation within αS have yet to be established. In our work, we demonstrate that CuI-bound N-terminally acetylated αS (NAcαS) activates O2 resulting in both intermolecular dityrosine cross-linking within the fibrillar core as well as intramolecular cross-linking within the C-terminal region. Substitution of the H50 residue with a disease relevant Q mutation abolishes intermolecular dityrosine cross-linking and limits the CuI/O2 promoted cross-linking to the C-terminal region. Such a dramatic change in reaction behavior establishes a previously unidentified role for H50 in facilitating intermolecular cross-linking. Involvement of H50 in the reaction profile implies that long-range histidine coordination with the upstream CuI coordination site is necessary to stabilize the transition of CuI to CuII as is a required mechanistic outcome of CuI/O2 reactivity. The aggregation propensity of NAcH50Q-CuI is also enhanced in comparison to NAcαS-CuI, suggesting a potential functional role for both copper and intermolecular cross-linking in attenuating NAcαS fibrillization.

Mass spectrometry data confirming tetrameric α-synuclein N-terminal acetylation.

Tetrameric α-synuclein (αS) is an elusive multimer of the dynamic neuronal protein implicated in Parkinson׳s disease. Through the data reported herein, we demonstrate that this high molecular weight multimer is N-acetylated. Coexpression of tetrameric αS in Escherichia coli with the NatB acetylase derived from yeast enables access to N-terminally acetylated αS (NAcαS), the native form in humans. Following purification and characterization as previously described by us in "Isolation of Recombinant Tetrameric N-acetylated α-synuclein" (Fernández and Lucas, 2018), the purified protein was excised from a native gel for confirmation of N-terminal acetylation. Through high-resolution mass spectrometry techniques, the identification of this helical tetramer as NAcαS has been clearly demonstrated.

Isolation of recombinant tetrameric N-acetylated α-synuclein.

Parkinson's disease (PD) is multifactorial, likely resulting from an intricate relationship of genetic and environmental factors affecting fundamental cellular processes. Histopathological hallmarks of PD include the development of granular inclusions known as Lewy bodies that are enriched with aggregates of the protein α-synuclein (αS). Historically, αS has been considered a natively unfolded protein prone to amyloidogenic behavior. However, recent studies have revealed a physiologically relevant folded αS tetramer that is both alpha-helical and aggregation-resistant. The two forms are thought to reside in a dynamic coexistence within cells, and it has been suggested that a shift from metastable tetramers to the monomeric form could serve as a mechanism for disease initiation. The underlying pathology causing this type of shift remains unknown, but the importance of understanding tetramer stability and disassembly has therapeutic potential that cannot be overemphasized. Isolation of tetrameric αS is complicated by its dynamic nature, so thorough and detailed biochemical and biophysical studies on this αS conformer have been hampered by accessibility issues. We now report a robust and reliable recombinant expression platform that enables purification of native tetrameric αS without any detergents or other structure-modifying additives.

Iron Redox Chemistry Promotes Antiparallel Oligomerization of α-Synuclein.

Brain metal dyshomeostasis and altered structural dynamics of the presynaptic protein α-synuclein (αS) are both implicated in the pathology of Parkinson's disease (PD), yet a mechanistic understanding of disease progression in the context of αS structure and metal interactions remains elusive. In this Communication, we detail the influence of iron, a prevalent redox-active brain biometal, on the aggregation propensity and secondary structure of N-terminally acetylated αS (NAcαS), the physiologically relevant form in humans. We demonstrate that under aerobic conditions, Fe(II) commits NAcαS to a PD-relevant oligomeric assembly, verified by the oligomer-selective A11 antibody, that does not have any parallel ß-sheet character but contains a substantial right-twisted antiparallel ß-sheet component based on CD analyses and descriptive deconvolution of the secondary structure. This NAcαS-FeII oligomer does not develop into the ß-sheet fibrils that have become hallmarks of PD, even after extended incubation, as verified by TEM imaging and the fibril-specific OC antibody. Thioflavin T (ThT), a fluorescent probe for ß-sheet fibril formation, also lacks coordination to this antiparallel conformer. We further show that this oligomeric state is not observed when O2 is excluded, indicating a role for iron(II)-mediated O2 chemistry in locking this dynamic protein into a conformation that may have physiological or pathological implications.