Site-Specific Interactions of Cu(II) with α and β-Synuclein: Bridging the Molecular Gap between Metal Binding and Aggregation

Abstract

The aggregation of alpha-synuclein (AS) is a critical step in the etiology of Parkinson's disease (PD) and other neurodegenerative synucleinopathies. Protein-metal interactions play a critical role in AS aggregation and might represent the link between the pathological processes of protein aggregation and oxidative damage. Our previous studies established a hierarchy in AS-metal ion interactions, where Cu(II) binds specifically to the protein and triggers its aggregation under conditions that might be relevant for the development of PD. In this work, we have addressed unresolved structural details related to the binding specificity of Cu(II) through the design of site-directed and domain-truncated mutants of AS and by the characterization of the metal-binding features of its natural homologue beta-synuclein (BS). The structural properties of the Cu(II) complexes were determined by the combined application of nuclear magnetic resonance, electron paramagnetic resonance, UV-vis, circular dichroism spectroscopy, and matrix-assisted laser desorption ionization mass spectrometry (MALDI MS). Two independent, noninteracting copper-binding sites with significantly different affinities for the metal ion were detected in the N-terminal regions of AS and BS. MALDI MS provided unique evidence for the direct involvement of Met1 as the primary anchoring residue for Cu(II) in both proteins. Comparative spectroscopic analysis of the two proteins allowed us to deconvolute the Cu(II) binding modes and unequivocally assign the higher-affinity site to the N-terminal amino group of Met1 and the lower-affinity site to the imidazol ring of the sole His residue. Through the use of competitive chelators, the affinity of the first equivalent of bound Cu(II) was accurately determined to be in the submicromolar range for both AS and BS. Our results prove that Cu(II) binding in the C-terminal region of synucleins represents a nonspecific, very low affinity process. These new insights into the bioinorganic chemistry of PD are central to an understanding of the role of Cu(II) in the fibrillization process of AS and have implications for the molecular mechanism by which BS might inhibit AS amyloid assembly.

Interaction Between α-Synuclein and Metal Ions, Still Looking for a Role in the Pathogenesis of Parkinson’s Disease

“…A similar result was also obtained in an F4W/H50S mutant, suggesting that the high-affinity N-terminal Cu(II)-binding site revealed by W4 fluorescence quenching is not perturbed by H50 that is not likely to be involved (Lee et al 2008). Two independent non-interacting copperbinding sites were also detected in the N-terminal region in another study based on a thorough spectroscopic characterization of the a-synuclein-Cu(II) complexes (Binolfi et al 2008). Using a mass spectrometry-based approach, the investigators unequivocally demonstrated the direct role of Met1 as the primary anchoring residue for Cu(II).…”

“…Then, they reached the conclusion that the high-affinity Cu(II)-binding site in a-synuclein (K d \ 1 lM) is the one in which the N-terminal amino nitrogen of Met1 acts as the anchoring group together with Asp2 and maybe a water molecule. They also defined a second, lower-affinity binding motif for Cu(II) (K d * 50 lM) centered around His50 as evaluated in a CD titration of the a-synucleinCu(II) complex in which the Cu(II)-His50 contributions to the spectra were singled out (Binolfi et al 2008). A further contribution to this subject was provided by potentiometric and spectroscopic studies carried out on truncated N-terminal and H50 region models of a-synuclein fragments.…”

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

“…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. [20][21][22][27][28][29][30][31][32][33][34][35][36] To characterize the Cu II -binding properties, 65 CuCl 2 was first titrated into a solution of aSyn56 at pH 7.4 and analyzed using X-band EPR spectroscopy. Between 0-1 equiv Cu II , two sets of hyperfine features could be distinguished (Figure 1 a), indicating the presence of more than one coordination mode.…”

Oligomeric alpha‐synuclein and its role in neuronal death

“…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.…”