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 (αS), the physiologically relevant form in humans. We demonstrate that under aerobic conditions, Fe(II) commits αS to a PD-relevant oligomeric assembly, verified by the oligomer-selective A11 antibody, that does not have any parallel β-sheet character but contains a substantial right-twisted antiparallel β-sheet component based on CD analyses and descriptive deconvolution of the secondary structure. ThisαS-Fe oligomer does not develop into the β-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 β-sheet fibril formation, also lacks coordination to this antiparallel conformer. We further show that this oligomeric state is not observed when O is excluded, indicating a role for iron(II)-mediated O chemistry in locking this dynamic protein into a conformation that may have physiological or pathological implications.

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 Cu I -bound N-terminally acetylated αS ( NAc αS) activates O 2 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 Cu I /O 2 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 Cu I coordination site is necessary to stabilize the transition of Cu I to Cu II as is a required mechanistic outcome of Cu I /O 2 reactivity. The aggregation propensity of NAc H50Q−Cu I is also enhanced in comparison to NAc αS−Cu I , suggesting a potential functional role for both copper and intermolecular cross-linking in attenuating NAc αS fibrillization.

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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.

The structurally dynamic amyloidogenic protein αsynuclein (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 Cu II binding site. We now report the first detailed characterization data using electron paramagnetic resonance (EPR) spectroscopy to describe the Cu II coordination modes of N-terminally acetylated αS ( NAc αS). 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 Cu II -binding mode that may involve a C-terminal contribution. We additionally generated an N-terminally acetylated disease-relevant variant, NAc H50Q, that promotes a shift in the Cu II binding site to the Cterminus of the protein. Moreover, fibrillar NAc H50Q-Cu II exhibits enhanced parallel β-sheet character and increased hydrophobic surface area compared to NAc αS-Cu II and to both protein variants that lack a coordinated cupric ion. The results presented herein demonstrate the differential impact of distinct Cu II binding sites within NAc αS, revealing that C-terminal Cu II 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 Cu II coordination. Hence, consideration of the effect of Cu II on NAc αS and NAc H50Q misfolding may shed light on the extrinsic or environmental factors that influence PD pathology.