Site-Specific interactions of Cu(II) with Alpha and Beta-Synuclein: Bridging the molecular gap between metal binding and aggregation.

ANDRÉS BINOLFI; GONZALO R. LAMBERTO; ROSARIO DURÁN; LILIANA QUINTANAR; CARLOS W. BERTONCINI; JOSE M SOUZA; CARLOS CERVEÑANSKY; MARKUS ZWECKSTETTER; CHRISTIAN GRIESINGER; CLAUDIO O. FERNÁNDEZ

Angra dos Reis, Brazil

Congreso; 12th Nuclear Magnetic Resonance Users Meeting and 3rd Iberoamerican NMR Meeting; 2009

The aggregation of a-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 neurodegeneration 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.1,2 In this work we have addressed structural unresolved 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 b-synuclein (BS). The structural properties of the Cu(II) complexes were determined by the combined application of Nuclear Magnetic Resonance (NMR), Electron Paramagnetic Resonance (EPR), Mass Spectrometry (MALDI-MS), UV-visible spectroscopy and Circular Dichrosim (CD).3Two independent, non-interacting copper-binding sites could be deflected at the N-terminal region of AS and BS, with significant difference in their affinities for the metal ion. (Figure 1). A third, unspecific, Cu(II) binding site was detected at the C-terminal region of both proteins where other divalent metal ions bind with very low affinity (Figure 1).2Further characterization of the different Cu(II) complexes of AS and BS was performed by EPR spectroscopy to obtain the ligand donor set at each independent site. MALDI-MS provided unique evidences for the direct involvement of Met 1 as the primary anchoring site for Cu(II) (table 1).The comparative spectroscopic analysis between both proteins allowed us to deconvolute the Cu(II) binding modes at the N-terminal region and to assign unequivocally the high affinity site to the α-amino group of Met 1 and the low affinity site to the imidazol ring of the sole His residue. Using competitive chelators the affinity of the first equivalent of bound Cu(II) was accurately determined to be in the sub-micromolar range for both AS and BS.3 Interestingly, the affinity of AS for Cu(II) is similar to that of other target proteins involved in the etiology of neurodegenerative disorders, suggesting that perturbation in Cu(II) metabolism may constitute a more widespread element in neurodegeneration than recognized previously. Our results prove that Cu(II) binding at the C-terminal region of synucleins represents a non-specific, very low affinity process. These new insights into the bioinorganic chemistry of PD are central to understand the role of Cu(II) in the fibrillization process of AS and have implications on the molecular mechanism by which BS might inhibit AS amyloid assembly.