Methionine oxidation in α-synuclein inhibits its propensity for ordered secondary structure

Erika Ponzini,Antonella De Palma,Lucilla Cerboni,Antonino Natalello,Rossana Rossi,Rani Moons,Albert Konijnenberg,Joanna Narkiewicz,Giuseppe Legname,Frank Sobott,Pierluigi Mauri,Carlo Santambrogio,Rita Grandori

doi:10.1074/jbc.ra118.001907

Abstract

α-Synuclein (AS) is an intrinsically disordered protein highly expressed in dopaminergic neurons. Its amyloid aggregates are the major component of Lewy bodies, a hallmark of Parkinson's disease (PD). AS is particularly exposed to oxidation of its methionine residues, both in vivo and in vitro Oxidative stress has been implicated in PD and oxidized α-synuclein has been shown to assemble into soluble, toxic oligomers, rather than amyloid fibrils. However, the structural effects of methionine oxidation are still poorly understood. In this work, oxidized AS was obtained by prolonged incubations with dopamine (DA) or epigallocatechin-3-gallate (EGCG), two inhibitors of AS aggregation, indicating that EGCG promotes the same final oxidation product as DA. The conformational transitions of the oxidized and non-oxidized protein were monitored by complementary biophysical techniques, including MS, ion mobility (IM), CD, and FTIR spectroscopy assays. Although the two variants displayed very similar structures under conditions that stabilize highly disordered or highly ordered states, differences emerged in the intermediate points of transitions induced by organic solvents, such as trifluoroethanol (TFE) and methanol (MeOH), indicating a lower propensity of the oxidized protein for forming either α- or β-type secondary structures. Furthermore, oxidized AS displayed restricted secondary-structure transitions in response to dehydration and slightly amplified tertiary-structure transitions induced by ligand binding. This difference in susceptibility to induced folding could explain the loss of fibrillation potential observed for oxidized AS. Finally, site-specific oxidation kinetics point out a minor delay in Met-127 modification, likely due to the effects of AS intrinsic structure.

Highlights

␣-Synuclein (AS) is an intrinsically disordered protein highly expressed in dopaminergic neurons
absence of ligands (AS) susceptibility to covalent modifications in the presence of DA or EGCG has been investigated by incubating the protein with either ligand at 37 °C under mild agitation. 2 mM DA and 200 ␮M EGCG were used as previously characterized conditions enabling effective detection of AS-ligand noncovalent complexes [20]
Several lines of evidence suggest that DA inhibition of AS fibrillation is mediated by methionine oxidation [8]

Summary

Oxidation kinetics

AS susceptibility to covalent modifications in the presence of DA or EGCG has been investigated by incubating the protein with either ligand at 37 °C under mild agitation. 2 mM DA and 200 ␮M EGCG were used as previously characterized conditions enabling effective detection of AS-ligand noncovalent complexes [20]. Three oxidized methionine residues (positions 1, 116, and 127, Table S1) were identified in samples incubated with either DA or EGCG by ESIMS/MS, indicating that the modification corresponds to sulfoxidation (Fig. S1). SDS-PAGE analysis indicates that higher-order species are detectable, upon EGCG treatment Their amount in the samples used here for CD titrations (Fig. 3D) is below 15%, as assessed from densitometry analysis (data not shown). The Amide I absorption and second-derivative spectra of the two proteins in solution at 30 °C were superimposable (Fig. 4, C and D) and were characterized by a main component around 1642 cmϪ1 assigned to random-coil structures [27,28,29, 32]. Both techniques can capture differences under particular conditions, always in the direction of a reduced propensity of oMet to acquire ordered secondary structure

Ligand binding

Discussion

Chemicals and proteins