The aggregation of ␣-synuclein (AS) is characteristic of Parkinson's disease and other neurodegenerative synucleinopathies. We demonstrate here that Cu(II) ions are effective in accelerating AS aggregation at physiologically relevant concentrations without altering the resultant fibrillar structures. By using numerous spectroscopic techniques (absorption, CD, EPR, and NMR), we have located the primary binding for Cu(II) to a specific site in the N terminus, involving His-50 as the anchoring residue and other nitrogen͞oxygen donor atoms in a square planar or distorted tetragonal geometry. The carboxylate-rich C terminus, originally thought to drive copper binding, is able to coordinate a second Cu(II) equivalent, albeit with a 300-fold reduced affinity. The NMR analysis of AS-Cu ( amyloid ͉ fibrillation ͉ metallobiology T he protein ␣-synuclein (AS) is the main component of neuronal and glial cytoplasmic inclusions, pathologically described as Lewy bodies, that constitute the hallmark lesions of a group of neurodegenerative diseases collectively referred to as synucleinopathies (1, 2). The identification of point mutations and locus triplication in the AS gene as sole causes of familial inherited Parkinson's disease (PD) (3, 4) has stimulated research on the mechanism of AS neurotoxicity.AS comprises 140 amino acids distributed in three different regions: (i) the amphipathic N terminus (residues 1-60); (ii) the highly hydrophobic self-aggregating sequence known as NAC (non-A␤ component, residues 61-95), which is presumed to initiate fibrillation (5); and (iii) the acidic C-terminal region (residues 96-140). In its native monomeric state, AS adopts an ensemble of conformations with no significant secondary structure (6, 7), although long-range interactions have been shown to stabilize an aggregation-autoinhibited global protein architecture (8, 9). The protein undergoes dramatic conformational transitions from its natively unstructured state to an ␣-helical conformation upon interaction with lipid membranes (10, 11) or to the characteristic crossed ␤-conformation in highly organized amyloid-like fibrils under conditions that trigger aggregation (12, 13). Whereas the mechanism for the ␣-helical transition is well understood, the detailed mechanism of amyloid formation remains to be elucidated.The kinetics of fibrillation of AS are consistent with a nucleation-dependent mechanism (14), being modulated by factors and effectors of different types. Among them, low pH and high temperature (15-17), organic solvents (18), heparin (19), polyamines (14,20), and metal cations (21-23) accelerate AS aggregation. Not only do metal cations exert a physiological influence on protein structure, but transition metals have been also frequently recognized as risk factors in neurodegenerative disorders (24, 25). Brain lesions associated with Alzheimer's disease (AD) are rich in Fe(III), Zn(II), and Cu(II) (26). Recent biophysical and structural studies of the amyloid precursor protein and the amyloid-␤ peptide (A␤) have provided stron...

The aggregation of alpha-synuclein (AS) is characteristic of Parkinson's disease and other neurodegenerative synucleinopathies. Interactions with metal ions affect dramatically the kinetics of fibrillation of AS in vitro and are proposed to play a potential role in vivo. We recently showed that Cu(II) binds at the N-terminus of AS with high affinity (K(d) approximately 0.1 microM) and accelerates its fibrillation. In this work we investigated the binding features of the divalent metal ions Fe(II), Mn(II), Co(II), and Ni(II), and their effects on AS aggregation. By exploiting the different paramagnetic properties of these metal ions, NMR spectroscopy provides detailed information about the protein-metal interactions at the atomic level. The divalent metal ions bind preferentially and with low affinity (millimolar) to the C-terminus of AS, the primary binding site being the (119)DPDNEA(124) motif, in which Asp121 acts as the main anchoring residue. Combined with backbone residual dipolar coupling measurements, these results suggest that metal binding is not driven exclusively by electrostatic interactions but is mostly determined by the residual structure of the C-terminus of AS. A comparative analysis with Cu(II) revealed a hierarchal effect of AS-metal(II) interactions on AS aggregation kinetics, dictated by structural factors corresponding to different protein domains. These findings reveal a strong link between the specificity of AS-metal(II) interactions and the enhancement of aggregation of AS in vitro. The elucidation of the structural basis of AS metal binding specificity is then required to elucidate the mechanism and clarify the role of metal-protein interactions in the etiology of Parkinson's disease.