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N-Terminal Deletions Modify the Cu 2+ Binding Site in Amyloid-β

2005; American Chemical Society; Volume: 44; Issue: 14 Linguagem: Inglês

10.1021/bi047611e

1943-295X

Jesse W. Karr, Henrietta Akintoye, Lauren J. Kaupp, Veronika A. Szalai,

Computational Drug Discovery Methods

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.