Engineering metal ion coordination to regulate amyloid fibril assembly and toxicity

Abstract

Protein and peptide assembly into amyloid has been implicated in functions that range from beneficial epigenetic controls to pathological etiologies. However, the exact structures of the assemblies that regulate biological activity remain poorly defined. We have previously used Zn(2+) to modulate the assembly kinetics and morphology of congeners of the amyloid beta peptide (Abeta) associated with Alzheimer's disease. We now reveal a correlation among Abeta-Cu(2+) coordination, peptide self-assembly, and neuronal viability. By using the central segment of Abeta, HHQKLVFFA or Abeta(13-21), which contains residues H13 and H14 implicated in Abeta-metal ion binding, we show that Cu(2+) forms complexes with Abeta(13-21) and its K16A mutant and that the complexes, which do not self-assemble into fibrils, have structures similar to those found for the human prion protein, PrP. N-terminal acetylation and H14A substitution, Ac-Abeta(13-21)H14A, alters metal coordination, allowing Cu(2+) to accelerate assembly into neurotoxic fibrils. These results establish that the N-terminal region of Abeta can access different metal-ion-coordination environments and that different complexes can lead to profound changes in Abeta self-assembly kinetics, morphology, and toxicity. Related metal-ion coordination may be critical to the etiology of other neurodegenerative diseases.

Fig. 1.

Characterization of Cu²⁺–Aβ(13–21) (HHQKLVFFA) congener complexes. (A and B) UV-Vis absorbance (A) and CD spectra (B) of 0.4 mM Cu²⁺ in the presence of 0.4 mM Aβ peptide. Black, Aβ(13–21)K16A; green, Aβ(Q15A); red, Aβ(H14A); and blue, Aβ(Ac-N). (C) CW-EPR spectra of the Cu²⁺–Aβ(13–21)K16A complex with different Cu²⁺-to-peptide ratios. The concentration of Aβ(13–21)K16A peptide is 0.5 mM. (D) Fourier transform (solid line) and simulation (dashed line) of three-pulse ESEEM for the soluble Cu²⁺–Aβ(13–21)K16A complex prepared from 0.4 mM Cu²⁺ in the presence of 0.5 mM Aβ(13–21)K16A.

Fig. 2.

Isotope edited FT-IR. Absorption of amide I bands of Ac-Aβ(13–21)H14A (Ac-HAQKLVFFA) fibrils formed with free peptide (P) and in the presence of Zn²⁺ or Cu²⁺. UL, unlabeled peptide; F20, ¹³CO was introduced into the peptide at Phe-20; L17, ¹³CO was introduced into the peptide at Leu-17.

Fig. 3.

Copper ion analysis in the Ac-Aβ(13–21)H14A (Ac-HAQKLVFFA) fibrillar complex. (A) Fourier transform (solid line) and simulation (dashed line) of three-pulse ESEEM for Cu²⁺-Ac-Aβ(13–21)H14A fibrils. Simulation values are summarized in SI Table 2. (B) The Fourier transform of extended x-ray absorption fine-structure (EXAFS) (Inset) of Cu²⁺-Ac-Aβ(13–21)H14A fibrils. Solid line, experiment; dashed line, fit 8 (values listed in SI Table 4).

Fig. 4.

MEF2 luciferase reporter gene assay. (Left) Aβ(1–42) dose-dependent response. (Right) Cu²⁺–Ac-Aβ(13–21)H14A fibrils are as toxic as Aβ(1–42) and Aβ(10–35) fibrils and Cu²⁺–Aβ(13–21)K16A complex is nontoxic. Peptide concentration is 10 μM.

Fig. 5.

Structural models for the Cu²⁺–peptide complexes. (A) Proposed model for Cu²⁺ coordination with Aβ(13–21)K16A showing only the first four residues (HHQA). (B) Crystal structure for Cu²⁺–HGGGW complex, the N-terminal repeat sequence of human prion protein (63). Only equatorial ligands are illustrated in both models. Purple, Cu ion; green, carbon; red, oxygen; blue, nitrogen; gray, unidentified ligand. (C) Structural models for Cu²⁺ arrangement in Ac-Aβ(13–21)H14A fibrils. (Left) View of three stacked β-sheets. (Right) View down peptide backbone. H-bonds are parallel to fibril long axis, and fibrils consist of parallel in-register β-sheets in both the absence and presence of metal ions. Sheets stack perpendicular to the H-bond direction. Both Zn²⁺ and Cu²⁺ coordinate two histidines along the H-bonding dimension within the same β-sheet. Red, His-13; blue, remaining Ac-Aβ(13–21)H14A residues; magenta, Zn²⁺, Cu²⁺; and green, H-bonds between backbone carbonyl and amide. Amino acid side chains, except His-13, are removed for clarity.