Local structure and global patterning of Cu2+ binding in fibrillar amyloid-β [Aβ(1-40)] protein

Abstract

The amyloid-β (Aβ) protein forms fibrils and higher-order plaque aggegrates in Alzheimer's disease (AD) brain. The copper ion, Cu(2+), is found at high concentrations in plaques, but its role in AD etiology is unclear. We use high-resolution pulsed electron paramagnetic resonance spectroscopy to characterize the coordination structure of Cu(2+) in the fibrillar form of full-length Aβ(1-40). The results reveal a bis-cis-histidine (His) equatorial Cu(2+) coordination geometry and participation of all three N-terminal His residues in Cu(2+) binding. A model is proposed in which Cu(2+)-His6/His13 and Cu(2+)-His6/His14 sites alternate along the fibril axis on opposite sides of the β-sheet fibril structure. The local intra-β-strand coordination structure is not conducive to Cu(2+)/Cu(+) redox-linked coordination changes, and the global arrangement of Cu sites precludes facile multielectron and bridged-metal site reactivity. This indicates that the fibrillar form of Aβ suppresses Cu redox cycling and reactive oxygen species production. The configuration suggests application of Cu(2+)-Aβ fibrils as an amyloid architecture for switchable electron charge/spin coupling and redox reactivity.

Figure 1

CW-EPR spectra of Cu²⁺-Aβ complexes. (A) Soluble Cu²⁺-Aβ(1-16), experiment and simulation (dashed line), (B) Fibrillar Cu²⁺-Aβ(1-40), experiment and simulation (dashed line), (C) ¹³C,¹⁵N-His13-Cu²⁺-Aβ(1-40), (D) ¹³C,¹⁵N-His14-Cu²⁺-Aβ(1-40). Acquisition parameters: microwave frequency = 9.45 GHz; microwave power = 2 mW; modulation amplitude = 1 mT; modulation frequency = 100 kHz; time constant = 10.24 ms; conversion time = 81.92 ms; T = 120 K. Spectra A and B are an average of 10 scans, and spectra C and D are an average of 25 scans. Simulation parameters for spectra A and B are presented in Table 1.

Figure 2

Three-pulse ESEEM waveforms of Cu²⁺-Aβ complexes. (A) Soluble Cu²⁺-Aβ(1-16), experiment and simulation (dashed line), (B) Fibrillar Cu²⁺-Aβ(1-40), experiment and simulation (dashed line), (C) ¹³C,¹⁵N-His13-Cu²⁺-Aβ(1-40), (D) ¹³C,¹⁵N-His14-Cu²⁺-Aβ(1-40). The vertical scale bar corresponds to 25% of the echo amplitude at τ + T = 8 μs. Acquisition Parameters: microwave frequency, 8.750 GHz, B₀ = 303.0 mT, T = 6 K, τ = 310 ns. Spectrum A is an average of 10 scans, spectrum B is an average of 16 scans, and spectra C and D are an average of 25 scans. Simulation parameters for waveforms A and B are presented in Table 2.

Figure 3

FT spectra of the three-pulse ESEEM waveforms of Cu²⁺-Aβ complexes. (A) Soluble Cu²⁺-Aβ(1-16), experiment and simulation (dashed line), (B) Fibrillar Cu²⁺-Aβ(1-40), experiment and simulation (dashed line), (C) ¹³C,¹⁵N-His13-Cu²⁺-Aβ(1-40), (D) ¹³C,¹⁵N-His14-Cu²⁺-Aβ(1-40). Inset: Expanded view of double quantum harmonic region, for (A) soluble Cu²⁺-Aβ(1-16) and (B) fibrillar Cu²⁺-Aβ(1-40). Acquisition Parameters: microwave frequency, 8.750 GHz, B₀ = 303 mT, T = 6 K, τ = 310 ns. Spectrum A is an average of 10 scans, spectrum B is an average of 16 scans, and spectra C and D are an average of 25 scans. Simulation parameters for spectra A and B are presented in Table 2.

Figure 4

Model for the mutual orientation of the histidine imidazole remote ¹⁴N dipolar superhyperfine principal axis systems in (A) soluble Cu²⁺-Aβ(1-16) and (B) fibrillar Cu²⁺-Aβ(1-40). Figure axes represent the direction cosines, relative to the reference x, y, z-axis system (brown). The dotted surfaces show the 99% confidence interval, that corresponds to the Euler angles, [α_A, β_A, γ_A].

Figure 5

Three-pulse ESEEM waveforms of (A) Fibrillar Cu²⁺-Aβ(1-40), (B) ¹³C,¹⁵N-His13-Cu²⁺-Aβ(1-40), (C) ¹³C,¹⁵N-His14-Cu²⁺-Aβ(1-40), (D) envelope division; B divided by A, (E) envelope division; C divided by A. The vertical scale bar corresponds to 25% of the echo amplitude at τ + T = 8 μs. Aquisition Parameters: microwave frequency, 8.750 GHz, B₀ = 303 mT, T = 6 K, τ = 310 ns.

Figure 6

Model for the coordination of Cu²⁺ in the fibrillar Aβ(1-40) peptide. The model is based on the quaternary structure for the ordered residues 9-40 of Aβ(1-40), which was determined by SS-NMR (Protein Data Bank ID, 2LMN). (A) Protein secondary structure cartoon, showing global view down the fibril axis. The Cu²⁺ ions are represented as orange spheres. (B) Side-on view of the N-terminal region, showing the patterning of Cu²⁺ sites along the fibril axis. Purple and green loops represent His6/His13 and His6/His14 sites, respectively. (C) Local Cu²⁺ coordination site geometry.

Scheme 1