Residue-specific binding of Ni(II) ions influences the structure and aggregation of amyloid beta (Aβ) peptides

Alzheimer’s disease (AD) is the most common cause of dementia worldwide. AD brains display deposits of insoluble amyloid plaques consisting mainly of aggregated amyloid-β (Aβ) peptides, and Aβ oligomers are likely a toxic species in AD pathology. AD patients display altered metal homeostasis, and AD plaques show elevated concentrations of metals such as Cu, Fe, and Zn. Yet, the metal chemistry in AD pathology remains unclear. Ni(II) ions are known to interact with Aβ peptides, but the nature and effects of such interactions are unknown. Here, we use numerous biophysical methods—mainly spectroscopy and imaging techniques—to characterize Aβ/Ni(II) interactions in vitro, for different Aβ variants: Aβ(1–40), Aβ(1–40)(H6A, H13A, H14A), Aβ(4–40), and Aβ(1–42). We show for the first time that Ni(II) ions display specific binding to the N-terminal segment of full-length Aβ monomers. Equimolar amounts of Ni(II) ions retard Aβ aggregation and direct it towards non-structured aggregates. The His6, His13, and His14 residues are implicated as binding ligands, and the Ni(II)·Aβ binding affinity is in the low µM range. The redox-active Ni(II) ions induce formation of dityrosine cross-links via redox chemistry, thereby creating covalent Aβ dimers. In aqueous buffer Ni(II) ions promote formation of beta sheet structure in Aβ monomers, while in a membrane-mimicking environment (SDS micelles) coil–coil helix interactions appear to be induced. For SDS-stabilized Aβ oligomers, Ni(II) ions direct the oligomers towards larger sizes and more diverse (heterogeneous) populations. All of these structural rearrangements may be relevant for the Aβ aggregation processes that are involved in AD brain pathology.


Fig. S2
. Second derivatives of IR absorbance of 100 µM Aβ40 wt peptide at pD 12.0, 0°C, with different Ni(II) concentrations. Band positions are 1638.9 cm −1 (zero Ni(II)), 1639.6 cm −1 (0.56 mM Ni(II)), 1639.0 cm −1 (1.56 mM Ni(II)), and 1638.2 cm −1 (2.56 mM Ni(II)). The number of smoothing points for the second derivate was 13. The gray spectrum shows the difference between the second derivatives of the spectra with zero Ni(II) and with 2.56 mM Ni(II). The colors of the other spectra match those of the CD spectra in Fig. 3 in the main manuscript to facilitate the comparison.

S3
The effect of Ni(II) ions on the secondary structure of Aβ peptides From Fig. 3 in the main manuscript, it can be seen that addition of Ni(II) ions induces structural transitions under some conditions. The CD measurements of titrations with Ni(II) acetate show clear transitions with isodichroic points for 10 µM Aβ40 wt (Fig. 3D), and for 10 µM Aβ(4-40) peptide (Fig. 4F), both in 20 mM sodium phosphate buffer, pH 7.3 at 25 °C. Fig. S1 shows the difference spectra for these two titrations, created by subtracting the spectra with no added Ni(II) acetate from the spectra with 256 µM Ni(II) acetate. For both peptides the difference spectra correspond to beta sheet structures (Greenfield & Fasman, 1969), indicating that the structural changes are from random coil (the initial structures) to β-sheet.
The structure-altering effects of Ni(II) ions were investigated also by IR spectroscopy. Fig.   S2 shows the second derivatives of the IR absorbance in the amide I range for synthetic Aβ40 wt peptides at pD 12 (10 mM NaOD) and different concentrations of Ni(II) acetate.   concentrations in the SDS-PAGE sample buffer, and may therefore produce larger amounts of different SDS-induced aggregation "by-products" after exposure to 2% SDS in the sample buffer (Bitan et al, 2005). Another explanation is that the effects of the high SDS concentration from the sample buffer on the oligomer distribution are modulated by the Ni(II) ions. The second derivatives of the IR absorbance of these oligomers are presented in Fig. 8 of the main text. In addition, Fig. S4 shows the absorbance spectra after solvent and baseline subtraction.

Materials and methods: SDS polyacrylamide gel electrophoresis of Aβ42 oligomers
The Aβ42 oligomer samples prepared with 0-500 µM Ni(II) acetate, as described in the materials section, were studied by Sodium Dodecyl Sulfate PolyAcrylamide Gel Electrophoresis (SDS-PAGE). The samples were incubated with the sample buffer containing 2% (w/v) of SDS at room temperature for 5 minutes without heating, loaded on Mini-PROTEAX TGX precast gels (Bio-Rad, USA), and used together with SDS-PAGE running buffer and an electrophoresis system (Bio-Rad, USA). The Precision Plus Protein Dual Color Standard (Bio-Rad, USA) was used as the protein molecular weight marker.
The samples were run at 4 °C for 90 minutes and stained with the Pierce Silver Staining Kit (ThermoFisher Scientific, USA).   S4. Infrared absorbance spectra of SDS-stabilized oligomers after subtraction of a spectrum of the respective SDS-containing solvent, subtraction of a polynomial baseline generated at 4 points (1760, 1720, 1600, 1590 cm−1) and subtraction of residual water vapor bands, when necessary. The color code matches that of Fig. 8 of the main text. The vertical line in both panels indicates the spectral position of the main β-sheet band of the oligomers prepared in 0.2% SDS without Ni(II) ions.