Structural characterization of copper(II) binding to α-synuclein: Insights into the bioinorganic chemistry of Parkinson's disease

Abstract

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...

Protein-metal interactions of calmodulin and α-synuclein monitored by selective noncovalent adduct protein probing mass spectrometry

“…Since copper is known to facilitate fibril formation, this suggests that copper may serve to stabilize higher order aggregates, which might otherwise dissociate back into monomeric form. This hypothesis is consistent with previous observations demonstrating that the hydrodynamic radius of the protein and the chemical shifts of the backbone amide groups do not change upon Cu 2ϩ binding [25,60]. Additionally, no changes in secondary structure are observed in the far UV-circular dichroism spectrum [64].…”

“…Cu 2ϩ was found in previous work to interact with the N-terminal domain of ␣-synuclein [25]. Specifically, there is evidence that Cu 2ϩ binds to His50 and at least one other nitrogen ligand, provided either by the amide backbone or by the N-terminus.…”

“…Specifically, there is evidence that Cu 2ϩ binds to His50 and at least one other nitrogen ligand, provided either by the amide backbone or by the N-terminus. ␣-Synuclein also contains multiple nonspecific Cu 2ϩ binding sites with low affinity located on the C-terminal domain [25,63].…”

“…Among the most effective metals at accelerating aggregation were Al 3ϩ and Cu 2ϩ . In a separate study by Fernandez et al, the putative Cu 2ϩ binding sites were localized to near residue-specific detail using nuclear magnetic resonance spectroscopy [25]. Recently, it was shown that time-resolved fluorescence energy-transfer experiments with ␣-synuclein can effectively separate and identify unique conformations in the ensemble due to differences in inter-residue distances and contact rates [26].…”

Long-Range Correlated Dynamics in Intrinsically Disordered Proteins

“…Protein samples were prepared by dissolving about 10 mg of the lyophilized proteins in 0.5 mL D 2 O. Wild-type α-synuclein and its mutants as well as β-synuclein were prepared as previously described. 35,36 The Tau protein and its K19 fragment were prepared as reported elsewhere. 37,38 Unfolded hen egg white lysozyme was prepared by dissolving the lyophilized protein from Sigma-Aldrich in D 2 Relaxation Rate Profiles.…”