Amyloid aggregation of α-synuclein (AS) has been linked to the pathological effects associated with Parkinson's disease (PD). Cu(II) binds specifically at the N-terminus of AS and triggers its aggregation. Site-specific Cu(I)-catalyzed oxidation of AS has been proposed as a plausible mechanism for metal-enhanced AS amyloid formation. In this study, Cu(I) binding to AS was probed by NMR spectroscopy, in combination with synthetic peptide models, site-directed mutagenesis, and C-terminal-truncated protein variants. Our results demonstrate that both Met residues in the motif (1)MDVFM(5) constitute key structural determinants for the high-affinity binding of Cu(I) to the N-terminal region of AS. The replacement of one Met residue by Ile causes a dramatic decrease in the binding affinity for Cu(I), whereas the removal of both Met residues results in a complete lack of binding. Moreover, these Met residues can be oxidized rapidly after air exposure of the AS-Cu(I) complex, whereas Met-116 and Met-127 in the C-terminal region remain unaffected. Met-1 displays higher susceptibility to oxidative damage compared to Met-5 because it is directly involved in both Cu(II) and Cu(I) coordination, resulting in closer exposure to the reactive oxygen species that may be generated by the redox cycling of copper. Our findings support a mechanism where the interaction of AS with copper ions leads to site-specific metal-catalyzed oxidation in the protein under physiologically relevant conditions. In light of recent biological findings, these results support a role for AS-copper interactions in neurodegeneration in PD.

The fibrillation of amyloidogenic proteins is a critical step in the etiology of neurodegenerative disorders such as Alzheimer and Parkinson diseases. There is major interest in the therapeutic intervention on such aberrant aggregation phenomena, and the utilization of polyaromatic scaffolds has lately received considerable attention. In this regard, the molecular and structural basis of the anti-amyloidogenicity of polyaromatic compounds, required to evolve this molecular scaffold toward therapeutic drugs, is not known in detail. We present here biophysical and biochemical studies that have enabled us to characterize the interaction of metal-substituted, tetrasulfonated phthalocyanines ( The misfolding of proteins into a toxic conformation and their deposition as amyloid-like fibrils are proposed to be at the molecular foundation of a number of neurodegenerative disorders including Creutzfeldt-Jakob, Alzheimer, and Parkinson diseases (1-3). A detailed understanding of the mechanism by which proteins of wide structural diversity are transformed into morphologically similar aggregates is therefore of high clinical importance.␣-Synuclein (AS) 6 is a highly soluble, intrinsically disordered protein, expressed predominantly in the neurons of the central nervous system and localized at presynaptic terminals in close proximity to synaptic vesicles. Evidence that AS amyloidogenesis plays a causative role in the development of Parkinson diseases is furnished by a variety of genetic, neuropathological, and biochemical studies (4 -7). Structurally, AS comprises 140 amino acids distributed in three different regions: the amphipathic N terminus (residues 1-60), showing imperfect KTKEGV repeats and involved in lipid binding (8, 9); the highly hydrophobic self-aggregating sequence known as non-A␤ component (residues 61-95), presumed to initiate fibrillation (10); and the acidic C-terminal region (residues 96 -140), rich in Pro, Asp, and Glu residues and critical for blocking rapid AS filament assembly (11,12). In its native monomeric state AS is best described as an ensemble of structurally heterogeneous conformations, with no persistent secondary structure and with long range interresidue interactions that have been shown to stabilize aggregation-autoinhibited conformations (13-15). However, the mechanism(s) underlying the structural transition from the innocuous, monomeric AS to its neurotoxic form still remain poorly described.The use of aggregation inhibitors as molecular probes of the structural and toxic mechanisms related to amyloid formation * This work was supported by the Agencia Nacional de Promoció n Científica y Tecnoló gica, Argentina, Fundacíon Antorchas, Max Planck Society, Alexander von Humboldt Foundation, Fundació n Josefina Prats, and the Government of Santa Fe. The Bruker Avance II 600 MHz NMR spectrometer used in this work was purchased with funds from ANPCyT (PME2003-2006

α-Synuclein (AS) aggregation is associated to neurodegeneration in Parkinson's disease (PD). At the same time, alterations in metal ion homeostasis may play a pivotal role in the progression of AS amyloid assembly and the onset of PD. Elucidation of the structural basis directing AS-metal interactions and their effect on AS aggregation constitutes a key step towards understanding the role of metal ions in AS amyloid formation and neurodegeneration. Despite of the reported evidences that link Zn(2+) with the pathophysiology of PD and the fact that this metal ion was shown to promote AS fibrillation in vitro, neither the structural characterization of the binding sites nor the identification of the amino acids involved in the interaction of Zn(2+) with the protein AS has been carried out. By using NMR spectroscopy, we have addressed here unknown structural details related to the binding of Zn(2+) to the protein AS through the design of site-directed and domain truncated mutants of AS. The binding of zinc to the Aβ peptide was also studied and discussed comparatively. Although the results of this study contribute to the understanding of the structural and molecular basis behind the acceleration of AS fibrillation mediated by Zn(2+), the low affinity that characterizes the interaction of Zn(2+) with AS contrasts strongly with the high-affinity features reported for the binding of this metal ion to other target proteins linked to human amylodosis such as Aβ peptide and the Islet Amyloid Polypeptide (IAPP), challenging the biological relevance of zinc interactions in the pathogenesis of PD.