Inhibition of α-Synuclein Fibrillization by Dopamine Is Mediated by Interactions with Five C-Terminal Residues and with E83 in the NAC Region

Fernando E Herrera,Paolo Carloni,Adriana Munoz,Adrian Schmid,Alessandra Chesi,Katerina E Paleologou,Michele Vendruscolo,Hilal A Lashuel,Stefano Gustincich,Arnold Schwartz

doi:10.1371/journal.pone.0003394

Abstract

The interplay between dopamine and α-synuclein (AS) plays a central role in Parkinson's disease (PD). PD results primarily from a severe and selective devastation of dopaminergic neurons in substantia nigra pars compacta. The neuropathological hallmark of the disease is the presence of intraneuronal proteinaceous inclusions known as Lewy bodies within the surviving neurons, enriched in filamentous AS. In vitro, dopamine inhibits AS fibril formation, but the molecular determinants of this inhibition remain obscure. Here we use molecular dynamic (MD) simulations to investigate the binding of dopamine and several of its derivatives onto conformers representative of an NMR ensemble of AS structures in aqueous solution. Within the limitations inherent to MD simulations of unstructured proteins, our calculations suggest that the ligands bind to the 125YEMPS129 region, consistent with experimental findings. The ligands are further stabilized by long-range electrostatic interactions with glutamate 83 (E83) in the NAC region. These results suggest that by forming these interactions with AS, dopamine may affect AS aggregation and fibrillization properties. To test this hypothesis, we investigated in vitro the effects of dopamine on the aggregation of mutants designed to alter or abolish these interactions. We found that point mutations in the 125YEMPS129 region do not affect AS aggregation, which is consistent with the fact that dopamine interacts non-specifically with this region. In contrast, and consistent with our modeling studies, the replacement of glutamate by alanine at position 83 (E83A) abolishes the ability of dopamine to inhibit AS fibrillization.

Highlights

Parkinson’s disease (PD) is the second most common progressive neurodegenerative disorder, affecting 1–2% of the population over 65 [1,2]
Addition of dopamine did not inhibit fibril formation or change the structure of the fibrils formed by both E83A containing mutants (Figure 4, right panels). We have presented both molecular dynamics and in vitro biophysical investigations of complexes formed by AS and dopamine and several of its derivatives in aqueous solution
These calculations, which were based on structures representing about 75% of the conformations obtained by NMR spectroscopy [22], suggest that the ligands bind non-covalently to the C-terminal region including the residues 125YEMPS129

Summary

Introduction

Parkinson’s disease (PD) is the second most common progressive neurodegenerative disorder, affecting 1–2% of the population over 65 [1,2]. The major proteinaceous building block of LBs are insoluble fibrils made up of the a-synuclein (AS) protein [3], suggesting that the aggregation of this protein may play a central role in the development and/or progression of the disease This idea is supported by evidence from genetics, animal modeling, cell culture and biophysical studies: 1) increased production (gene duplication and triplication [4,5]) and/or missense mutations (A53T A30P, and E46K) [6,7,8]) in the gene encoding for AS are linked to autosomal dominant inherited forms of familial PD; 2) several lines of transgenic mice and flies that overexpress wild-type and disease-associated variants of AS show age-dependent formation of AS-containing inclusions, loss of dopaminergic cells and motor abnormalities [9,10]; 3) overexpression of AS causes cell death in cultured dopaminergic neurons and in differentiated neuroblastoma cells [11]; 4) all PD associated mutations have been shown to accelerate and enhance the oligomerization and fibrillogenesis of AS in vitro [12,13]; 5) AS toxicity and fibrillization is influenced by factors that may be relevant to PD, including post-translational modifications, oxidative stress and interaction with toxins and metals [14,15,16,17,18]. Other neurodegenerative diseases are characterized by the accumulation of fibrillar AS, including a LB variant of Alzheimer’s disease, dementia with LB, multiple system atrophy and related diseases, which collectively are referred as a-synucleinopathies [19]

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Results

Conclusion