Cu²⁺Binding Modes of Recombinant α-Synuclein − Insights from EPR Spectroscopy

Abstract

The interaction of the small (140 amino acid) protein, alpha-synuclein (alphaS), with Cu(2+) has been proposed to play a role in Parkinson's disease (PD). While some insight from truncated model complexes has been gained, the nature of the corresponding Cu(2+) binding modes in the full length protein remains comparatively less well characterized. This work examined the Cu(2+) binding of recombinant human alphaS using Electron Paramagnetic Resonance (EPR) spectroscopy. Wild type (wt) alphaS was shown to bind stoichiometric Cu(2+) via two N-terminal binding modes at physiological pH. An H50N mutation isolated one binding mode, whose g parallel, A parallel, and metal-ligand hyperfine parameters correlated well with a {NH2, N(-), beta-COO(-), H2O} mode previously identified in truncated model fragments. Electron spin-echo envelope modulation (ESEEM) studies of wt alphaS confirmed the second binding mode at pH 7.4 involved coordination of His50 and its g parallel and A parallel parameters correlated with either {NH2, N(-), beta-COO(-), N(Im)} or {N(Im), 2 N(-)} coordination observed in alphaS fragments. At pH 5.0, His50-anchored Cu(2+) binding was greatly diminished, while {NH2, N(-), beta-COO(-), H2O} binding persisted in conjunction with another two binding modes. Metal-ligand hyperfine interactions from one of these indicated a 1N3O coordination sphere, which was ascribed to a {NH2, CO} binding mode. The other was characterized by a spectrum similar to that previously observed for diethylpyrocarbonate-treated alphaS and was attributed to C-terminal binding centered on Asp121. In total, four Cu(2+) binding modes were identified within pH 5.0-7.4, providing a more comprehensive picture of the Cu(2+) binding properties of recombinant alphaS.

Oligomeric alpha‐synuclein and its role in neuronal death

“…Our data suggested that simultaneous copper binding to both the N-and C-termini simultaneous could generate oligomeric aSyn. Co-ordination of copper by a-Syn as a result of residues at distant sites has been suggested previously (46). On the basis of these results, we extended our study to the toxicity of fulllength a-Syn and its relation to copper.…”

“…As mentioned earlier, a-Syn binds to copper and iron (46,48,49), and a-Syn aggregation is stimulated in the presence of these metals (37,50). This has led us to examine whether the toxicity of extracellular synuclein proteins was exacerbated in the presence of metals.…”

“…An excellent recent study has looked at the binding of copper to a-Syn using EPR (46). The study shows that there are multiple coordination modes for the two main copper binding sites on the protein, which are pH dependent.…”

The N Terminus of α‐Synuclein Forms CuII‐Bridged Oligomers

“…The first coordination sphere of aSyn Early evidence from model peptides [24] and full-length aSyn140, [25,26] suggested that the protein adopted two coordination modes in the presence of 1 } (mode II). Nevertheless, numerous studies proposed only a single high affinity coordination mode, with disagreement regarding the role of Met1 and His50 and/or overlooking the effect of pH-dependent equilibrium.…”

Bioinorganic chemistry of synucleinopathies: Deciphering the binding features of Met motifs and His-50 in AS–Cu(I) interactions

“…This mechanism is a highly selective, site-specific process that involves interactions of the protein with both oxidation states of the copper ion. Added to the large body of evidence supporting AS-Cu(II) interactions [14][15][16][17][18][19][20], elucidation of the residue-specific basis determining the AS-Cu(I) structural-affinity features is central to establish a connection of the AS-copper interactions with the mechanistic basis-oxidative damage-behind the metal-enhanced AS amyloid assembly. In that direction, we have demonstrated recently that both Met residues in the motif 1 MDVFM 5 constitute key structural determinants for the binding of Cu(I) to the N-terminal region of AS in a Met 1 (S)-Cu(I)-(S)Met 5 coordination environment [21,22].…”