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

Abstract

The aggregation of alpha-synuclein (AS) is a critical step in the etiology of Parkinson's disease (PD) and other neurodegenerative synucleinopathies. This process is selectively enhanced by copper in vitro and the interaction is proposed to play a potential role in vivo. Presently, the identity of the Cu(I) binding sites in AS and their relative affinities are under debate. In this work we have addressed unresolved details related to the structural binding specificity and affinity of Cu(I) to full-length AS. We demonstrated conclusively that: (i) the binding preferences of Cu(I) for the Met-binding sites at the N-(K d = 20 μM) and C-terminus (K d = 270 μM) of AS are widely different: (ii) the imidazole ring of His-50 acts as an effective anchoring residue (K d = 50 μM) for Cu(I) binding to AS; and (iii) no major structural rearrangements occur in the protein upon Cu(I) binding. Overall, our work shows that Cu(I) binding to the N-and C-terminal regions of AS are two independent events, with substantial differences in their affinities, and suggest that protein oxidative damage derived from a misbalance in cellular copper homeostasis would target preferentially the N-terminal region of AS. This knowledge is key to understanding the structural-aggregation basis of the copper catalyzed oxidation of AS.

Interaction of Cu(i) with the Met-X3-Met motif of alpha-synuclein: binding ligands, affinity and structural features

“…3B), consistent with previous studies. 42,50 Interestingly, the value reported for the Cu(I)-complex in the non-acetylated protein was c K d1 = 7.8 AE 1.0 nM, 48 in line with the affinity differences found for Cu(I) binding to the N-acetylated and free amine P1AS peptides. The value of c K d1 for complexation of Cu(I) to the Met-X 3 -Met site in M5I/H50A AcaS and the D2A aS were 63 AE 5 nM and 14 AE 2 nM, respectively.…”

“…NMR-based studies revealed that the main anchoring groups for Cu(I) binding -Met1/Met5 (site 1), His50 (site 2) and Met116/Met127 (site 3)-were preserved in the acetylated form of the protein 42,[47][48][49] (Fig. 1).…”

Copper(I/II), α/β‐Synuclein and Amyloid‐β: Menage à Trois?

“…These apparent affinities of Cu I for truncated or full-length aS were in the lower-mm range. [21,23,38] Thus, affinitieslower than the estimated Cu I affinities here wereobtained.…”

“…In the case of Cu I ,t he affinity of Ab remains am atter of debate, [36,37] and for aS only apparent binding constants (not taking the presence of buffer and reducing agent into account) are reported. [38] In addition, the impact of the additional Met residue in bS on the Cu I affinity is not known.…”

Effects of alpha‐synuclein post‐translational modifications on metal binding

“…Indeed, the catalytic oxidase activity of the AS‐Cu(II) complexes was shown to yield formation of hydroxyl radicals and AS oxidation (Wang et al ; Meloni and Vašák ). An NMR study demonstrated that the redox cycling of copper bound to AS can generate reactive oxygen species, leading to site‐specific metal‐catalyzed oxidation on methionine residues under physiologically relevant conditions (Miotto et al, , ). Met1 and Met5 residues can be oxidized rapidly (Met1 faster than Met5) to a sulfoxide species after air exposure of the AS‐Cu(I) complexes, whereas Met116 and Met127 remain unaffected (Miotto et al, , ).…”