N-Terminal deletions modify the Cu2+ binding site in amyloid-beta.
Copper is implicated in the in vitro formation and toxicity of Alzheimer's disease amyloid plaques containing the beta-amyloid (Abeta) 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 Cu(2+) binding site in native Abeta has been examined. Peptides that contain the proposed binding site for Cu(2+)-three histidines (H6, H13, and H14) and a tyrosine (Y10)-but lack one to three N-terminal amino acids, do not bind Cu(2+) in the same coordination environment as the native peptide. EPR spectra of soluble Abeta with stoichiometric amounts of Cu(2+) show type 2 Cu(2+) EPR spectra for all peptides. The ligand donor atoms to Cu(2+) are 3N1O when Cu(2+) is bound to any of the Abetapeptides (Abeta16, Abeta28, Abeta40, and Abeta42) that contain the first 16 amino acids of full-length Abeta. When a Y10F mutant of Abeta is used, the coordination environment for Cu(2+) remains 3N1O and Cu(2+) 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 Cu(2+) in Abeta fibrils or in the Y10F mutant. Further, we find that Cu(2+) cannot be removed from Cu(2+)-containing fibrils by washing with buffer, but that Cu(2+) binds to fibrils initially assembled without Cu(2+) in the same coordination environment as in fibrils assembled with Cu(2+). Together, these results indicate (1) that the O-atom donor ligand to Cu(2+) in Abeta is not tyrosine, (2) that the native Cu(2+) binding site in Abeta is sensitive to small changes at the N-terminus, and (3) that Cu(2+) binds to Abetafibrils in a manner that permits exchange of Cu(2+) into and out of the fibrillar architecture.
Karr
Jesse W
JW
Department of Chemistry & Biochemistry, University of Maryland, Baltimore County, 1000 Hilltop Circle, Baltimore, Maryland 21250, USA.
Akintoye
Henrietta
H
Kaupp
Lauren J
LJ
Szalai
Veronika A
VA
D000595:Amino Acid Sequence; D016229:Amyloid beta-Peptides / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D001665:Binding Sites; D003300:Copper / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D046529:Microscopy, Electron, Transmission; D008969:Molecular Sequence Data; D013050:Spectrometry, Fluorescence
D016229:Amyloid beta-Peptides; D003300:Copper