C-Terminal Cu^II 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 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 (^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 Cu^II-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 Cu^II binding site to the C-terminus of the protein. Moreover, fibrillar ^NAcH50Q-Cu^II exhibits enhanced parallel β-sheet character and increased hydrophobic surface area compared to ^NAcαS-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 ^NAcαS, 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 ^NAcαS and ^NAcH50Q misfolding may shed light on the extrinsic or environmental factors that influence PD pathology.