A novel α-synuclein missense mutation in Parkinson disease.

Alpha-synuclein (SNCA) is central to the pathogenesis of Parkinson disease (PD), with 3 missense mutations reported to date. We report a novel mutation (p.H50Q) in a pathologically proven case.

Coordination of copper to the membrane-bound form of α-synuclein.

Aggregation of the 140-amino acid protein α-synuclein (α-syn) is linked to the development of Parkinson's disease (PD). α-Syn is a copper binding protein with potential function as a regulator of metal-dependent redox activity. Epidemiological studies suggest that human exposure to excess copper increases the incidence of PD. α-Syn exists in both solution and membrane-bound forms. Previous work evaluated the Cu(2+) uptake for α-syn in solution and identified Met1-Asp2 and His50 as primary contributors to the coordination shell, with a dissociation constant of approximately 0.1 nM. When bound to the membrane bilayer, α-syn takes on a predominantly helical conformation, which spatially separates His50 from the N-terminus of the protein and is therefore incompatible with the copper coordination geometry of the solution state. Here we use circular dichroism and electron paramagnetic resonance (continuous wave and pulsed) to evaluate the coordination of copper to the membrane-bound form of α-syn. In this molecular environment, Cu(2+) binds exclusively to the N-terminus of the protein (Met1-Asp2) with no participation from His50. Copper does not alter the membrane-bound α-syn conformation or enhance the release of the protein from the bilayer. The Cu(2+) affinity is similar to that identified for solution α-syn, suggesting that copper coordination is retained in the membrane. Consideration of these results demonstrates that copper exerts its greatest conformational effect on the solution form of α-syn.

The elevated copper binding strength of amyloid-ß aggregates allows the sequestration of copper from albumin: a pathway to accumulation of copper in senile plaques.

Copper coexists with amyloid-ß (Aß) peptides at a high concentration in the senile plaques of Alzheimer's disease (AD) patients and has been linked to oxidative damage associated with AD pathology. However, the origin of copper and the driving force behind its accumulation are unknown. We designed a sensitive fluorescent probe, Aß(1-16)(Y10W), by substituting the tyrosine residue at position 10 in the hydrophilic domain of Aß(1-42) with tryptophan. Upon mixing Cu(II), Aß(1-16)(Y10W), and aliquots of Aß(1-42) taken from samples incubated for different lengths of time, we found that the Cu(II) binding strength of aggregated Aß(1-42) has been elevated by more than 2 orders of magnitude with respect to that of monomeric Aß(1-42). Electron paramagnetic spectroscopic measurements revealed that the Aß(1-42) aggregates, unlike their monomeric form, can seize copper from human serum albumin, an abundant copper-containing protein in brain and cerebrospinal fluid. The significantly elevated binding strength of the Aß(1-42) aggregates can be rationalized by a Cu(II) coordination sphere constituted by three histidines from two adjacent Aß(1-42) molecules. Our work demonstrates that the copper binding affinity of Aß(1-42) is dependent on its aggregation state and provides new insight into how and why senile plaques accumulate copper in vivo.

Coordination features and affinity of the Cu²+ site in the α-synuclein protein of Parkinson's disease.

Parkinson's disease (PD) is the second most prevalent age-related, neurodegenerative disorder, affecting >1% of the population over the age of 60. PD pathology is marked by intracellular inclusions composed primarily of the protein α-synuclein (α-syn). These inclusions also contain copper, and the interaction of Cu(2+) with α-syn may play an important role in PD fibrillogenesis. Here we report the stoichiometry, affinity, and coordination structure of the Cu(2+)-α-syn complex. Electron paramagnetic resonance (EPR) titrations show that monomeric α-syn binds 1.0 equiv of Cu(2+) at the protein N-terminus. Next, an EPR competition technique demonstrates that α-syn binds Cu(2+) with a K(d) of ≈0.10 nM. Finally, EPR and electron spin echo modulation (ESEEM) applied to a suite of mutant and truncated α-syn constructs reveal a coordination sphere arising from the N-terminal amine, the Asp2 amide backbone and side chain carboxyl group, and the His50 imidazole. The high binding affinity identified here, in accord with previous measurements, suggests that copper uptake and sequestration may be a part of α-syn's natural function, perhaps modulating copper's redox properties. The findings further suggest that the long-range interaction between the N-terminus and His50 may have a weakening effect on the interaction of α-syn with lipid membranes, thereby mobilizing monomeric α-syn and hastening fibrillogenesis.

Quantification of the binding properties of Cu2+ to the amyloid beta peptide: coordination spheres for human and rat peptides and implication on Cu2+-induced aggregation.

There is no consensus on the coordinating ligands for Cu(2+) by Abeta. However, the differences in peptide sequence between human and rat have been hypothesized to alter metal ion binding in a manner that alters Cu(2+)-induced aggregation of Abeta. Herein, we employ isothermal titration calorimetry (ITC), circular dichroism (CD), and electron paramagnetic resonance (EPR) spectroscopy to examine the Cu(2+) coordination spheres to human and rat Abeta and an extensive set of Abeta(16) mutants. EPR of the mutant peptides is consistent with a 3N1O binding geometry, like the native human peptide at pH 7.4. The thermodynamic data reveal an equilibrium between three coordination spheres, {NH(2), O, N(Im)(His6), N(-)}, {NH(2), O, N(Im)(His6), N(Im)(His13)}, and {NH(2), O, N(Im)(His6), N(Im)(His14)}, for human Abeta(16) but one dominant coordination for rat Abeta(16), {NH(2), O, N(Im)(His6), N(-)}, at pH 7.4-6.5. ITC and CD data establish that the mutation R5G is sufficient for reproducing this difference in Cu(2+) binding properties at pH 7.4. The substitution of bulky and positively charged Arg by Gly is proposed to stabilize the coordination {NH(2), O-, N(Im)(His6), N(-)} that then results in one dominating coordination sphere for the case of the rat peptide. The differences in the coordination geometries for Cu(2+) by the human and rat Abeta are proposed to contribute to the variation in the ability of Cu(2+) to induce aggregation of Abeta peptides.