N-Terminal Deletions Modify the Cu2+Binding Site in Amyloid-β

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Abstract
Copper is implicated in the in vitro formation and toxicity of Alzheimer's disease amyloid plaques containing the β-amyloid (Aβ) peptide (Bush, A. I., et al. (2003) Proc. Natl. Acad. Sci. U.S.A.100, 11934). By low temperature electron paramagnetic resonance (EPR) spectroscopy, the importance of the N-terminus in creating the Cu2+ binding site in native Aβ has been examined. Peptides that contain the proposed binding site for Cu2+three histidines (H6, H13, and H14) and a tyrosine (Y10)but lack one to three N-terminal amino acids, do not bind Cu2+ in the same coordination environment as the native peptide. EPR spectra of soluble Aβ with stoichiometric amounts of Cu2+ show type 2 Cu2+ EPR spectra for all peptides. The ligand donor atoms to Cu2+ are 3N1O when Cu2+ is bound to any of the Aβ peptides (Aβ16, Aβ28, Aβ40, and Aβ42) that contain the first 16 amino acids of full-length Aβ. When a Y10F mutant of Aβ is used, the coordination environment for Cu2+ remains 3N1O and Cu2+ EPR spectra of this mutant are identical to the wild-type spectra. Isotopic labeling experiments show that water is not the O-atom donor to Cu2+ in Aβ fibrils or in the Y10F mutant. Further, we find that Cu2+ cannot be removed from Cu2+-containing fibrils by washing with buffer, but that Cu2+ binds to fibrils initially assembled without Cu2+ in the same coordination environment as in fibrils assembled with Cu2+. Together, these results indicate (1) that the O-atom donor ligand to Cu2+ in Aβ is not tyrosine, (2) that the native Cu2+ binding site in Aβ is sensitive to small changes at the N-terminus, and (3) that Cu2+ binds to Aβ fibrils in a manner that permits exchange of Cu2+ into and out of the fibrillar architecture.