Zinc as chaperone-mimicking agent for retardation of amyloid β peptide fibril formation

Abstract

Metal ions have emerged to play a key role in the aggregation process of amyloid β (Aβ) peptide that is closely related to the pathogenesis of Alzheimer's disease. A detailed understanding of the underlying mechanistic process of peptide-metal interactions, however, has been challenging to obtain. By applying a combination of NMR relaxation dispersion and fluorescence kinetics methods we have investigated quantitatively the thermodynamic Aβ-Zn(2+) binding features as well as how Zn(2+) modulates the nucleation mechanism of the aggregation process. Our results show that, under near-physiological conditions, substoichiometric amounts of Zn(2+) effectively retard the generation of amyloid fibrils. A global kinetic profile analysis reveals that in the absence of zinc Aβ40 aggregation is driven by a monomer-dependent secondary nucleation process in addition to fibril-end elongation. In the presence of Zn(2+), the elongation rate is reduced, resulting in reduction of the aggregation rate, but not a complete inhibition of amyloid formation. We show that Zn(2+) transiently binds to residues in the N terminus of the monomeric peptide. A thermodynamic analysis supports a model where the N terminus is folded around the Zn(2+) ion, forming a marginally stable, short-lived folded Aβ40 species. This conformation is highly dynamic and only a few percent of the peptide molecules adopt this structure at any given time point. Our findings suggest that the folded Aβ40-Zn(2+) complex modulates the fibril ends, where elongation takes place, which efficiently retards fibril formation. In this conceptual framework we propose that zinc adopts the role of a minimal antiaggregation chaperone for Aβ40.

Keywords: Alzheimer’s disease; NMR relaxation; aggregation kinetics; amyloid beta peptide; zinc ion interactions.

Fig. 1.

(A) Aggregation kinetics of Aβ₄₀ alone (Left) and Aβ₄₀:Zn²⁺ 10:1 (Right) at different peptide concentrations with global fits of normalized aggregation traces of 10 (red), 12 (orange), 14 (green), 16 (cyan), 18 (blue), and 20 (violet) μM Aβ₄₀. (B) Variance weighted mean ${\tau }_{1/2}$ values, with and without Zn²⁺, exhibit the same half-time exponent γ (Left). The correlation plot of the fitting parameters $\log \left({\tau }_{1/2}\right)$ vs. $\log \left(r_{max}\right)$ features a slope of −1.0 $\pm$ 0.1 with $R^2$ = 0.90 (Right).

Fig. 2.

(A) Aggregation traces of 20 μM Aβ₄₀ with different Zn²⁺ concentrations were globally fitted with $\sqrt{k_{+}{k}_2}$ as a free parameter whereas $\sqrt{k_n/k_2}$ was constrained for all [Zn²⁺]. (B) Mean ${\tau }_{1/2}$ values show an exponential [Zn²⁺] dependence: ${\tau }_{1/2}={\tau }_0\cdot \exp \left(\left[{\mathrm{Zn}}^{2+}\right]/c_e\right)$. (C) ThT end-point fluorescence at different [Zn²⁺]. (D) The relative elongation rate from the global fit analysis displayed in A (colored points), with $k_{+}$ as the sole free fitting parameter, and elongation rates determined as the initial slopes (black squares) from preseeded aggregation kinetics.

Fig. 3.

(A) Translational diffusion coefficient, $D_t$, of 75 μM Aβ₄₀ alone and in the presence of 20 and 40 μM Zn²⁺ at 281 K (error bars: SD of five or more measurements). (B) Relaxation dispersion profiles of selected residues for 75 μM Aβ in the presence of 20 μM Zn²⁺ (circles) at 278 (red), 281 (orange), 284 (green), 287 (blue), and 290 K (violet) and Aβ alone at 281 K (black squares). (C and D) The fitting parameters absolute chemical shift differences $\left|\mathrm{\Delta }\delta \right|$ and exchange rate $k_{ex}$ from the global fit. Chemical shift differences were constrained to the same values for all temperatures and fitted for all (orange) and only the eight residues with significant relaxation dispersion amplitudes (gray). The stars mark signals with overlap in the spectrum. (E) The Gibbs free energy, obtained from the zinc-bound folded population, $p_B$, fitted to Eq. 1 (red). For higher temperature the Gibbs free energy is also fitted to $\mathrm{\Delta }G\left(T\right)=\mathrm{\Delta }{H}^0-T\mathrm{\Delta }{S}^0$ (blue). Error bars in C–E were estimated from fitting errors.

Fig. 4.

Aβ₄₀ self-assembly follows a monomer-dependent secondary nucleation pathway and fibril formation is efficiently retarded in the presence of zinc ions by primarily inhibiting fibril-end elongation in a simplistic model. Aβ₄₀ in solution transiently forms a low-populated dynamic complex with Zn²⁺ where the peptide’s N terminus is wrapped around the zinc ion. A simplistic mechanistic model is that Zn²⁺ inhibits elongation by modulation of the encounter complex between Aβ₄₀ and the fibril end.