On the role of sidechain size and charge in the aggregation of A β 42 with familial mutations

Abstract

The aggregation of the amyloid-β (Aβ) peptide is linked to the pathogenesis of Alzheimer's disease (AD). In particular, some point mutations within Aβ are associated with early-onset familial Alzheimer's disease. Here we set out to explore how the physical properties of the altered side chains, including their sizes and charges, affect the molecular mechanisms of aggregation. We focus on Aβ42 with familial mutations-A21G (Flemish), E22K (Italian), E22G (Arctic), E22Q (Dutch), and D23N (Iowa)-which lead to similar or identical pathology with sporadic AD or severe cerebral amyloid angiopathy. Through global kinetic analysis, we find that for the E22K, E22G, E22Q, and D23N mutations, the acceleration of the overall aggregation originates primarily from the modulation of the nucleation processes, in particular secondary nucleation on the surface of existing fibrils, whereas the elongation process is not significantly affected. Remarkably, the D23 position appears to be responsible for most of the charge effects during nucleation, while the size of the side chain at the E22 position plays a more significant role than its charge. Thus, we have developed a kinetic approach to determine the nature and the magnitude of the contribution of specific residues to the rate of individual steps of the aggregation reaction, through targeted mutations and variations in ionic strength. This strategy can help rationalize the effect of some disease-related mutations as well as yield insights into the mechanism of aggregation and the transition states of the wild-type protein.

Keywords: aggregation mechanism; amyloid; driving forces; kinetic analysis; self-assembly.

Fig. 1.

Location of residues K16, V18, A21, E22, and D23 in the A$\beta$42 WT fibril model (5KK3.pdb) resolved by Colvin et al. (15). The structure within the fibril of two monomers within the same plane are shown in A, where the second monomer is displayed in paler color. The hydrophobic patch of one monomer is shown in a zoom-in top view (B) and side view (C). The image was prepared using MOLMOL (16) and shows 70% of the van der Waals radius.

Fig. 2.

Cryo-transmission electron microscopy (TEM) images of fibrils formed by A$\beta$42 WT or familial mutants. The samples initially contained 10 $\mathrm{\mu }$M monomeric A$\beta$ peptide, 6 $\mathrm{\mu }$M of ThT, 20 mM sodium phosphate, 200 $\mathrm{\mu }$M EDTA, and 0.02% NaN₃ at pH 8.0 and were imaged after reaching the plateau of ThT fluorescence. (Scale bar, 100 nm.)

Fig. 3.

Concentration-dependent aggregation kinetics data of A$\beta$42 WT and five familial mutants—A21G, E22K, E22Q, E22G, and D23N—at 37 °C. ThT fluorescence was monitored as a function of time for initially monomeric samples with peptide concentrations in the range of 0.4 to 10 $\mathrm{\mu }$M. Each color represents the average fluorescence signal intensity of four replicates at the same peptide concentration. All samples contain 6 $\mathrm{\mu }$M ThT, 20 mM sodium phosphate, 200 $\mathrm{\mu }$M EDTA, and 0.02% NaN₃, at pH 8.0. The mutation sites of the five familial mutants are shown below the WT sequence (residues 17 to 27) in the yellow panel. Fits of these data are shown in Fig. 5, their half times are shown in SI Appendix, Fig. S4.

Fig. 4.

Half time of fibril formation as a function of monomer concentration on double logarithmic axes. (A) Previously published data of A$\beta$42 WT at different ionic strengths from 32 mM (lightest blue) to 312 mM (darkest blue) (28). Monomer dependence decreases with increasing ionic strength. (B) A$\beta$42 WT and each of the five familial mutants; representative aggregation curves are shown in Fig. 3. Each data point is an average over several repeats of the aggregation experiments, with 3 to 4 replicates at each concentration (resulting error bars are shown in SI Appendix, Fig. S5). The scaling exponent was estimated by fitting a power function to the data for each mutant (Eq. 1). The half time is concentration dependent, and all familial mutants except for A21G show a decreased monomer dependence (higher scaling exponent) compared with A$\beta$42 WT.

Fig. 5.

Global fitting of the data shown in Fig. 3 for A$\beta$42 WT and five familial mutants—A21G, D23N, E22Q, E22K, and E22G. All data are well reproduced by global fits of models in which the majority of new aggregates is produced by fibril-surface catalyzed formation of nuclei from monomer. Furthermore, this process was found to display saturation, similar to that observed previously for A$\beta$40 (29), for E22G, E22Q, E22K, and D23N. A schematic of the processes considered in this model and how they fit together in the aggregation reaction network is shown at the bottom. Lower concentrations of A$\beta$42 WT and A21G are excluded from the fitting due to issues with reproducibility for reactions with such long half times.

Fig. 6.

Results of the global fitting of each mutant. Error bars are standard deviations over replicates and repeats of the experiment. (A) The combined rate constant of primary nucleation and elongation increases significantly, by up to over two orders of magnitude. (B) The combined rate constant of secondary nucleation and elongation also increases significantly, approximately paralleling the combined rate constant of primary nucleation and elongation. This corresponds to the low concentration limit where saturation effects are negligible; see E for the high concentration limit. (C) Estimate of the elongation rate constant from the TEM measurements (errors in that case are standard errors of the mean over the dimensions of the measured fibrils) and fitted rate constants. The values obtained all closely resemble those determined for the WT protein. (D) $\sqrt{K_M}$, the monomer concentration at which secondary nucleation is half saturated. The region of monomer concentrations sampled in this study is marked in green. A$\beta$42 WT and A21G show little saturation effects, while D23N is fully saturated at all monomer concentrations sampled. Thus, for D23N, we can only obtain an upper bound for $\sqrt{K_M}$, which is displayed as an empty circle in the plot. (E) Rate of conversion of secondary nuclei. This corresponds to the high concentration limit where the system is fully saturated. (F) Illustration of the degree of saturation, using the value of $\sqrt{K_M}$ from D. The region of monomer concentrations sampled in this study is marked in green.

Fig. 7.

Seeding experiments. (A) Normalized ThT fluorescence as a function of time of mutant A21G, E22Q, E22G, E22K, and D23N. Seeds of the same peptide were added at time 0 at concentrations of 0.04%, 0.2%, 1%, 5%, 10%, and 30% monomer equivalents, and the monomer concentration is indicated in each panel. All samples contain 6 $\mathrm{\mu }$M of ThT, 20 mM sodium phosphate, and 200 $\mathrm{\mu }$M of EDTA at pH 8.0. Data at additional monomer concentrations are shown in SI Appendix, Fig. S6. (B) Aggregation half time as a function of the logarithm of the seed concentration for A21G, E22Q, E22G, E22K, and D23N, at one or three different monomer concentrations as indicated in each panel.

Fig. 8.

Effect of varying ionic strength on the half times of aggregation for the different mutants. Half times are plotted against initial monomer concentration on a double logarithmic plot. Bottom Right compares the combined rate constant of secondary nucleation and elongation for the different mutants and ionic strengths. Lower, standard, and higher ionic strengths are 24 mM, 60 mM, and 162 mM, respectively. While D23N is not significantly affected by changes in ionic strength, both E22K and E22Q are sped up significantly at higher ionic strengths, suggesting that charge still plays a role during the aggregation of these mutants.