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

Abstract

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.

Interaction of β-amyloid(1-40) peptide with pairs of metal ions: An electrospray ion trap mass spectrometric model study

“…These results are concordant with those obtained previously on working with individual metal ions [29][30][31][32]. Although blood and internal biochemical environment is characterized by a…”

“…Several spectroscopic studies have indicated that the copper-binding site of Aβ consists of the three histidine residues His6, His13 and His14 and an as yet undefined fourth ligand; proposed ligands include Tyr10 [19,[38][39][40], the Nterminal nitrogen [30] or an as yet unidentified carboxylate side chain [31,32].…”

Truncated Amyloid-β(11–40/42) from Alzheimer Disease Binds Cu2+ with a Femtomolar Affinity and Influences Fiber Assembly

“…In particular, the loss of main-chain amide coordination to be replaced by side-chain coordination from a histidine imidazole of a second A␤ molecule is likely to cause a profound change in the appearance of the CD bands, which is not observed. The ability of amyloid fibers to accommodate Cu binding has also been observed for A␤ and A␤ (35,(73)(74)(75)(76). Interestingly, Cu 2ϩ can diffuse into and load on to all A␤ molecules within the fibers and facilitate the Cu 2ϩ coordination with close to 1:1 stoichiometry.…”

Concentration Dependent Cu2+Induced Aggregation and Dityrosine Formation of the Alzheimer's Disease Amyloid-β Peptide

“…The identity of the fourth ligand includes tyrosine at position 10 (Tyr10) (16,(19)(20)(21), the N-terminal nitrogen (22), or an undefined carboxylate side chain (23). Our EPR studies indicate that increasing the Cu 2+ above ∼0.3 mol/mol peptide-induced line broadening in the Cu 2+ EPR spectra suggests the presence of dipolar or exchange effects (17,19).…”