<p>NatA binds inositol hexakisphosphate and other ligands, and exhibits conformational flexibility dependent on the ligand bound.</p> / Dissertation

The pre-synaptic protein α-Synuclein (αS) is often linked to the pathology of Parkinson’s disease (PD), an age-related neurodegenerative disorder. Lewy bodies, the cytopathological hallmarks of PD, are found to be rich in aggregates of misfolded αS protein. Metal dyshomeostasis has also been linked to PD due to the accumulation of iron in the substantia nigra pars compacta, and diminished copper levels reported in this same region. Metal dyshomeostasis in the brain coupled with oxidative stress can enhance the aggregation of αS. Recently, it was confirmed that mammalian αS is universally acetylated at the N-terminus, a common post-translational modification in humans. The consequences of this modification have been understudied, and it is believed to impart a functional role under physiological conditions with respect to membrane-interactions and protein folding. In an attempt to elucidate the pathological mechanism behind PD with respect to the structural dynamics of the protein, our investigations were focused on physiologically prevalent, N-terminally acetylated αS (NAcαS) and its interaction with the most prevalent redox-active metal ions in the brain (iron and copper) under both aerobic and/or anaerobic conditions. The structural features associated with metal-bound NAcαS differed depending on the iron oxidation states, where under aerobic conditions Feᴵᴵ stabilized an oligomer-locked, anti-parallel right-twisted β-sheet conformation that could potentially impart toxicity to neurons. In contrast, Feᴵᴵᴵ promoted a fibrillar structure rich in parallel β-sheets. N-terminal capping also altered the Cuᴵᴵ coordination sphere and had a dramatic effect on protein aggregation. Parallel studies on NAcαS variants with different site mutations near the putative copper binding sites (ex: H50Q and F4W) indicated that preferential binding shifts upon changes in the side chain residues. In depth analysis of the electron structure of Cuᴵᴵ-bound NAcαS using electron paramagnetic resonance spectroscopy (EPR) revealed a coordination sphere of N3O1 that includes the H50 residue in the wild-type protein that shifts to an O4 coordination sphere at the C-terminus upon Cuᴵᴵ binding to the disease-relevant H50Q variant. Immunoblotting analyses revealed that copper-induced redox chemistry promoted O2-activation and the subsequent formation of dityrosine crosslinks, a post-translational modification identified as a biomarker of PD. EPR-detection of tyrosyl radical formation in the presence of Cuᴵ-bound NAcαS further supported this radical coupling mechanism. Intermolecular crosslinks within the fibrillar core of NAcαS as well as intramolecular crosslinks within the C-terminal region underpin the role of metal-dioxygen chemistry in PD-related pathology. The unique structural features resulting from iron vs copper coordination to NAcαS inspired studies directed at the synergistic effect of each individual metal species as revealed by photo-initiated crosslinking of NAcαS. C-terminal intramolecular tyrosine interactions were mainly impacted by the presence of both metals, which each have binding sites around the same region. These findings emphasize that protein dynamics, metal binding site conformational changes, as well as aggregation pathways can deviate drastically upon N-terminal acetylation of αS and that protein-metal interactions may play a vital role in PD etiology.