Site-Specific Interaction between α‑Synuclein and Membranes Probed by NMR-Observed Methionine Oxidation Rates

α-Synuclein (αS) is an intrinsically disordered protein that is water-soluble but also can bind negatively charged lipid membranes while adopting an α-helical conformation. Membrane affinity is increased by post-translational N-terminal acetylation, a common modification in all eukaryotic cells. In the presence of lipid vesicles containing a small fraction of peroxidized lipids, the N-terminal Met residues in αS (Met1 and Met5) rapidly oxidize while reducing the toxic lipid hydroperoxide to a nonreactive lipid hydroxide, whereas C-terminal Met residues remain unaffected. Met oxidation can be probed conveniently and quantitatively by NMR spectroscopy. The results show that oxidation of Met1 reduces the rate of oxidation of Met5 and vice versa as a result of decreased membrane affinity of the partially oxidized protein. The effect of Met oxidation on the αS–membrane affinity extends over large distances, as in the V49M mutant, oxidation of Met1 and Met5 strongly impacts the oxidation rate of Met49 and vice versa. When not bound to membrane, oxidized Met1 and Met5 of αS are excellent substrates for methionine sulfoxide reductase (Msr), thereby providing an efficient vehicle for water-soluble Msr enzymes to protect the membrane against oxidative damage

Isoindole Linkages Provide a Pathway for DOPAL-Mediated Cross-Linking of α‑Synuclein

3,4-Dihydroxyphenylacetaldehyde (DOPAL) is a toxic and reactive product of dopamine catabolism. In the catecholaldehyde hypothesis for Parkinson’s disease, it is a critical driver of the selective loss of dopaminergic neurons that characterizes the disease. DOPAL also cross-links α-synuclein, the main component of Lewy bodies, which are a pathological hallmark of the disease. We previously described the initial adduct formed in reactions between DOPAL and α-synuclein, a dicatechol pyrrole lysine (DCPL). Here, we examine the chemical basis for DOPAL-based cross-linking. We find that autoxidation of DCPL’s catechol rings spurs its decomposition, yielding an intermediate dicatechol isoindole lysine (DCIL) product formed by an intramolecular reaction of the two catechol rings to give an unstable tetracyclic structure. DCIL then reacts with a second DCIL to give a dimeric, di-DCIL. This product is formed by an intermolecular carbon-carbon bond between the isoindole rings of the two DCILs that generates two structurally nonequivalent and separable atropisomers. Using α-synuclein, we demonstrate that the DOPAL-catalyzed formation of oligomers can be separated into two steps. The initial adduct formation occurs robustly within an hour, with DCPL as the main product, and the second step cross-links α-synuclein molecules. Exploiting this two-stage reaction, we use an isotopic labeling approach to show the predominant cross-linking mechanism is an interadduct reaction. Finally, we confirm that a mass consistent with a di-DCIL linkage can be observed in dimeric α-synuclein by mass spectrometry. Our work elucidates previously unknown pathways of catechol-based oxidative protein damage and will facilitate efforts to detect DOPAL-based cross-links in disease-state neurons