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

Abstract

a-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 a-hemolysin. In the absence of Cu(II), wild-type AS (1 lM) readily translocated through the pore with a blockade current of À 85 pA, but mostly bumping events were observed in the presence of 25 lM 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 lM), the translocation events at À 85 pA shifted to À 80 pA, which also represents translocation events, because the event time decreases with increasing voltage. Events at À 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 lM dopamine. In contrast, dopamine had little effect on the CD spectrum of AS, and a single binding site with a K a of 3.5 9 10 3 M À1 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.

Behavior of α‐synuclein–drug complexes during nanopore analysis with a superimposed AC field

“…The values of I and T provide information about the conformation of AS when it is interacting with the pore . When AS is in the unfolded conformation the molecule translocates through the pore, generating a large I and a long T . Conversely, if AS is in a compact conformation, or if it aggregates, it is more likely to bump against the pore, generating a smaller value of I and a shorter T .…”

“…When AS is in the unfolded conformation the molecule translocates through the pore, generating a large I and a long T . Conversely, if AS is in a compact conformation, or if it aggregates, it is more likely to bump against the pore, generating a smaller value of I and a shorter T . A variety of self‐assembling bacterial pores and solid‐state pores have been investigated but perhaps the most popular of these is the α‐hemolysin toxin derived from Staphylococcus aureus .…”

Role of neurotoxicants and traumatic brain injury in α-synuclein protein misfolding and aggregation

“…Zhang et al , [150] showed that incubating α-Syn with Cu (II) for 60 h produced protofibrillar, annular and fibrillar species; protofibrils are especially cytotoxic. Interestingly, more α-Syn aggregation was seen in dopaminergic neurons following Cu exposure, suggesting that Cu and dopamine work synergistically on the misfolding and aggregation of α-Syn [56, 127]. …”

Quarterly intrinsic disorder digest (April–May–June, 2014)

“…Scarlata and Golebiewska provided a review of the evidence linking a-synuclein aggregation to oxidation and propose a hypothesis that it is the loss of cellular partners under oxidative conditions that promotes aggregation. 70 Buell et al focused on the influence of solution conditions on the nucleation of a-synuclein aggregation. They found that secondary nucleation increases dramatically at pH values below 6, suggesting that some intracellular locations, such as endosomes and lysosomes, may facilitate rapid aggregation.…”

“…They found that both the binding of dopamine and Cu 2C to a-synuclein caused large conformational changes. 70 Tau is another well-known IDP linked to neurodegeneration. Elbaum-Garfinkle et al used disease-promoting mutants of tau to investigate the link between the microtubule binding of Tau and disease.…”