Biometals as conformational modulators of α-synuclein photochemical crosslinking
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Metal dyshomeostasis has long been linked to Parkinson’s disease (PD), and the amyloidogenic protein α-synuclein (αS) is universally recognized as a key player in PD pathology. Structural consequences upon coordination of copper and iron to αS have gained attention due to significant dyshomeostasis of both metals in the PD brain. Protein–metal association can navigate protein folding in distinctive pathways based on the identity of the bio-metal in question. In this work, we employed photo-chemical crosslinking of unmodified proteins (PICUP) to evaluate these potential metal ion-induced structural alterations in the folding dynamics of N-terminally acetylated αS (NAcαS) following metal coordination. Through fluorescence analysis and immunoblotting analyses following photoirradiation, we discovered that coordination of iron obstructs copper-promoted crosslinking. The absence of intra-molecular crosslinking upon iron association further supports its C-terminal coordination site and suggests a potential role for iron in mitigating nearby post-translational modification of tyrosine residues. Decreased fluorescence emission upon synergistic coordination of both copper and iron highlighted that although copper acts as a conformational promotor of NAcαS crosslinking, iron inhibits analogous conformational changes within the protein. The metal coordination preferences of NAcαS suggest that both competitive binding sites as well as dual metal coordination contribute to the changes in folding dynamics, unveiling unique structural orientations for NAcαS that have a direct and measureable influence on photoinitiated dityrosine crosslinks. Moreover, our findings have physiological implications in that iron overload, as is associated with PD-insulted brain tissue, may serve as a conformational block of copper-promoted protein oxidation.
Keywordsα-Synuclein Dityrosine Photochemical crosslinking Iron Copper
Research reported in this publication was supported by NIH R01 GM134015, the National Center for Advancing Translational Sciences Clinical and Translational Science Award (UL1TR002649), and the Center for Clinical and Translational Research Endowment Fund of Virginia Commonwealth University, as well as by the VCU College of Humanities and Sciences. We would also like to express our appreciation to the United States Secret Service for donation of the Hitachi F-4500 fluorescence spectrophotometer and Hitachi U-3310 UV/Vis spectrophotometer to the Lucas lab.
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