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