title: Structural characterization of a high affinity mononuclear site in the copper(II)-α-synuclein complex.
abstract: Human α-Synuclein (aS), a 140 amino acid protein, is the main constituent of Lewy bodies, the cytoplasmatic deposits found in the brains of Parkinson's disease patients, where it is present in an aggregated, fibrillar form. Recent studies have shown that aS is a metal binding protein. Moreover, heavy metal ions, in particular divalent copper, accelerate the aggregation process of the protein. In this work, we investigated the high affinity binding mode of truncated aS (1-99) (aS99) with Cu(II), in a stoichiometric ratio, to elucidate the residues involved in the binding site and the role of copper ions in the protein oligomerization. We used Electron Paramagnetic Resonance spectroscopy on the Cu(II)-aS99 complex at pH 6.5, performing both multifrequency continuous wave experiments and pulsed experiments at X-band. The comparison of 9.5 and 95 GHz data showed that at this pH only one binding mode is present. To identify the nature of the ligands, we performed Electron Spin Echo Envelope Modulation, Hyperfine Sublevel Correlation Spectroscopy, and pulsed Davies Electron-Nuclear Double Resonance (Davies-ENDOR) experiments. We determined that the EPR parameters are typical of a type-II copper complex, in a slightly distorted square planar geometry. Combining the results from the different pulsed techniques, we obtained that the equatorial coordination is {N(Im), N(-), H(2)O, O}, where N(im) is the imino nitrogen of His50, N(-) a deprotonated amido backbone nitrogen that we attribute to His50, H(2)O an exchangeable water molecule, and O an unidentified oxygen ligand. Moreover, we propose that the free amino terminus (Met1) participates in the complex as an axial ligand. The MXAN analysis of the XAS k-edge absorption data allowed us to independently validate the structural features proposed on the basis of the magnetic parameters of the Cu(II)-aS99 complex and then to further refine the quality of the proposed structural model.
authors.affiliation: Dipartimento di Scienze Chimiche, Università di Padova, via Marzolo, 1, 35131 Padova, Italy.
authors.forename: Marco
authors.initials: M
authors.lastname: Bortolus
authors.forename: Marco
authors.initials: M
authors.lastname: Bisaglia
authors.forename: Alfonso
authors.initials: A
authors.lastname: Zoleo
authors.forename: Maria
authors.initials: M
authors.lastname: Fittipaldi
authors.forename: Maurizio
authors.initials: M
authors.lastname: Benfatto
authors.forename: Luigi
authors.initials: L
authors.lastname: Bubacco
authors.forename: Anna Lisa
authors.initials: AL
authors.lastname: Maniero
mesh_terms: D056831:Coordination Complexes / Q000737:chemistry* / Q000378:metabolism; D003300:Copper / Q000737:chemistry* / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D006639:Histidine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D016631:Lewy Bodies / Q000378:metabolism; D008667:Metalloproteins / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D051844:alpha-Synuclein / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D056831:Coordination Complexes; D008667:Metalloproteins; D051844:alpha-Synuclein; D006639:Histidine; D003300:Copper