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

Abstract

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.

Figure 1

Features of the α-synuclein primary structure identifying (A) the three consensus segments and (B) the amino acid sequence associated with each segment. Residues 9 – 97, encompassing the amphipathic repeat region and the NAC, forms an extended helix when associated with lipid membranes.

Figure 2

X-band EPR spectra of α-syn at pH=7.4 with 1, 2, and 4 equivalents of Cu²⁺. Specra were collected at 111 K, ν = 9.44GHz, with a sweep width of 1200 G. The inset shows EPR detected Cu²⁺ as a function of added Cu²⁺ and demonstrates saturation at approximately 2.0 eq. However, competition studies find that the second equivalent is weakly coordinated.

Figure 3

X-Band EPR spectra of α-syn, mutants and truncated species. Vertical lines correspond to the parallel hyperfine features of wild type α-syn. α-Syn(1–97) gives a spectrum that superimposes on wild type, but all other species show significant variation. α-Syn(1–10) and α-syn(H50A) gives equivalent spectra, but distinct from wild type, demonstrating involvement of His50 in the coordination sphere.

Figure 4

Three pulse ESEEM spectra of α-syn with 1.0 Eq. Cu²⁺ and 2.0 Eq. Cu²⁺, α-syn(1–97) with 2.0 Eq. Cu²⁺ and prion protein sequence HGGGW with 1.0 Eq. Cu²⁺. These spectra reveal the expected quadrupolar transitions associated with an imidazole remote nitrogen and demonstrate coordination by His50. α-syn(H50A) fails to give an ESEEM spectrum. α-Syn(1–97) with 2.0 Eq. Cu²⁺ gives additional weak features at 2.0 and 2.8 MHz (arrows), similar to peaks observed in HGGGW and assigned to an amide nitrogen coordinated through the backbone carbonyl.

Figure 5

Coordination features of the primary Cu²⁺ site in α-syn identified here in bond line (top) and stick (bottom) models. Competition studies show that this complex exhibits a dissociation constant of approximately 0.1 nM.