Cu(II) and dopamine bind to α-synuclein and cause large conformational changes

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Abstract

α-Synuclein (AS) is an intrinsically disordered protein that can misfold and aggregate to form Lewy bodies in dopaminergic neurons, a classic hallmark of Parkinson's disease. The binding of Cu(II) and dopamine to AS was evaluated by nanopore analysis with α-hemolysin. In the absence of Cu(II), wild-type AS (1 μm) readily translocated through the pore with a blockade current of − 85 pA, but mostly bumping events were observed in the presence of 25 μm Cu(II). A binding site in the N-terminus was confirmed, because Cu(II) had no effect on the event profile of a peptide consisting of the C-terminal 96–140 residues. In the presence of dopamine (25 μm), the translocation events at − 85 pA shifted to − 80 pA, which also represents translocation events, because the event time decreases with increasing voltage. Events at − 80 pA were also observed for the mutant A30P AS in the presence of dopamine. Event profiles for an N-terminal 1–60-residue peptide and a C-terminal 96–140-residue peptide were both altered in the presence of 25 μm dopamine. In contrast, dopamine had little effect on the CD spectrum of AS, and a single binding site with a Ka of 3.5 × 103 m−1 was estimated by isothermal titration calorimetry. Thus, dopamine can interact with both the N-terminus and the C-terminus. Two-dimensional NMR spectroscopy of AS in the presence of dopamine showed that there were significant changes in the spectra in all regions of the protein. According to these findings, a model is presented in which dopamine induces folding between the N-terminus and C-terminus of AS. Partially folding conformations such as this may represent important intermediates in the misfolding of AS that leads to fibrillization.

The misfolding of α-synuclein is implicated in the pathogenesis of Parkinson's disease. Both Cu(II) and dopamine bind to AS and cause large conformational changes which are readily detected by nanopore analysis. Dopamine can interact with both the N- and C-termini as demonstrated by two-dimensional NMR suggesting that dopamine induces folding between the N- and C-termini of AS.

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