ISBN: 978-83-60043-10-3 - eurobic9

Eurobic9, 2-6 September, 2008, Wrocław, Poland
SL26. Site-specific Interactions of Cu(II) with Alpha-Synuclein: Bridging
the Molecular Gap Between Metal Binding and Aggregation
A. Binolfi, a G. R. Lamberto, a R. Duran, b L. Quintanar, c C. W. Bertoncini, d J. M. Souza, e
C. Cerveñansky, b M. Zweckstetter, f C. Griesinger, f a, f
and C. O. Fernández
a Instituto de Biología Molecular y Celular de Rosario, Argentina
b Institut Pasteur de Montevideo e Instituto Clemente Estable, Uruguay
c Centro de Investigación y Estudios Avanzados, Mexico
d Department of Chemistry, University of Cambridge, United Kingdom
e Facultad de Medicina, Universidad de la República, Uruguay
f Max Planck Institute for Biophysical Chemistry, Germany
e-mail: fernandez@ibr.gov.ar; cfernan@gwdg.de
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. 1, 2 In this work we
have addressed structural unresolved details related to the binding specificity of Cu(II) to AS. 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). Two independent, non-interacting copper-binding sites could be
deflected at the N-terminal region of AS, with significant difference in their affinities for the metal ion. MALDI-
MS provided unique evidences for the direct involvement of Met 1 as the primary anchoring residue for Cu(II).
A comparative spectroscopic analysis between different variants of the protein allowed us to deconvolute the
Cu(II) binding modes and to assign unequivocally the high affinity site to the N-terminal amino group of Met1
and the low affinity site to that involving 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 submicromolar range.
Our results prove that Cu(II) binding at the C-terminal region of the protein 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.
Acknowledgement
C.O. Fernández thanks ANPCyT, Fundacion Antorchas, CONICET, Max Planck Society and the Alexander von
Humboldt Foundation for financial support. C.O. Fernández is the head of a Partner Group of the Max Planck
Institute for Biophysical Chemistry (Göttingen).
References
[1] R.M. Rasia, C.W. Bertoncini, D. Marsh, W. Hoyer, D. Cherny, M. Zweckstetter, C. Griesinger, T.M. Jovin,
C.O. Fernández, Proc Natl Acad Sci USA, 102, 4294 (2005).
[2] A. Binolfi, R.M. Rasia, C.W. Bertoncini, M. Ceolin, M. Zweckstetter, C. Griesinger, T.M. Jovin and C.O.
Fernández, J Am Chem Soc, 128, 9893 (2006).
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