Molecular mechanism of misfolding and aggregation of Aβ(13-23)

Abstract

The misfolding and self-assembly of the amyloid-beta (Aβ) peptide into aggregates is a molecular signature of the development of Alzheimer's disease, but molecular mechanisms of the peptide aggregation remain unknown. Here, we combined Atomic Force Microscopy (AFM) and Molecular Dynamics (MD) simulations to characterize the misfolding process of an Aβ peptide. Dynamic force spectroscopy AFM analysis showed that the peptide forms stable dimers with a lifetime of ∼1 s. During MD simulations, isolated monomers gradually adopt essentially similar nonstructured conformations independent from the initial structure. However, when two monomers approach their structure changes dramatically, and the conformational space for the two monomers become restricted. The arrangement of monomers in antiparallel orientation leads to the cooperative formation of β-sheet conformation. Interactions, including hydrogen bonds, salt bridges, and weakly polar interactions of side chains stabilize the structure of the dimer. Under the applied force, the dimer, as during the AFM experiments, dissociates in a cooperative manner. Thus, misfolding of the Aβ peptide proceeds via the loss of conformational flexibility and formation of stable dimers suggesting their key role in the subsequent Aβ aggregation process.

Figure 1

Characterization of Aβ (13-23) fibril by AFM. 100 μM Aβ (13-23) was incubated for 24 hours at 37 °C and the fibrils were imaged by AFM. The mean height of the fibril was 0.6520 ± 0.0484 nm. The white scale bar is 200 nm.

Figure 2

Single molecular force spectroscopy. (a) Schematics of the experimental setup. The peptide was immobilized on AFM tips and mica surfaces through N-terminal cysteine. Bifunctional PEG (about 77 PEG repeats, long linker) was used to attach the peptides to mica surface. MAS (5 repeats of PEG, short linker) was utilized to connect peptides to AFM tips. (b) A typical force curve illustrating the rupture event force curves (grey line) recorded at pH 6 with 500 nm/s pulling rate, black line is from the worm-like chain model fitting; the insert figure shows distribution of rupture force (bar) and fitting results with probability function (line). The mean value of force was 48.62 ± 8.38 pN. (c) The DFS analysis for Aβ (13-23) acquired at pH 2. Forces obtained from different pulling rates are plotted against logarithmic apparent loading rates (ALR). Seven ALR values were used to generate the plot. Each data point is an average of three independent experiments. The data set was approximated by the Bell-Evans model as described in reference . The intercept on the x-axis was used for the calculation of the off-rate constant producing the lifetime value of 1.06 ± 0.95 s. The large variance of this value is due to a logarithmic dependence of the off-rate constant value on the experimentally determined intercept value.

Figure 3

Initial and central structures of MD simulations of monomer structures I-III. (a) Tube representations of the backbone of the initial structures I-III for the three independent MD simulations. (b) The central structure of the largest cluster of the simulations. Random meander is cyan; α-helix is dark blue; 3₁₀-helix is yellow; turn is yellow and H-bonds are indicated by yellow dotted lines. N and C indicate N- and C-termini, respectively.

Figure 4

Evolution of the distance between the center of mass of Cys13 of chain A and the center of mass of Cys13 of chain B in during the 400 ns MD simulation of the dimer structure. Snapshots of the dimer backbone structures from the trajectory are placed inside the plot. (a) 1, 0 ns; 2, 20 ns; 3, 97.9 ns; 4, 221 ns; 5, 300 ns; 6, 359 ns. Backbone conformation of the peptide chain is as follows: cyan is random meander; yellow is 3₁₀-helix; green is β-turn/bend, red arrow is β-sheet and H-bonds are yellow dotted lines. N and C indicate the N-and C-termini, respectively. (b) Inter-molecular interactions (E_int) during the 400 ns MD simulation of the dimer structure. The grey line shows E_int at every 10 ps, the black line is the running average at 5 ns intervals. (c) Antiparallel backbone structure of the central structure of the largest cluster of the last 50 ns of the MD simulation. In chain A the backbone carbon atoms are in green. H-bonds are yellow dotted lines. N and C indicate the N-and C-termini, respectively.

Figure 5

Force curves acquired at 5 nm/ns pulling rate from SMD simulation. (a) Pulling the center of mass of Cys13 of monomer A (COM13A) along the z-axis. The central structure of the largest cluster of the last 50 ns of the MD simulation of the dimer is in a rectangular box. For clarity the water molecules are not shown. The dimension of the box is 6.555 nm × 4.376 nm × 18 nm. The pulling direction is indicated by a dashed arrow. Backbone conformation of the peptide chain is as follows: cyan is random meander; green is β-turn/bend, red arrow is β-sheet. Numbers inside the force curve panels indicate the time (b) and distance (c) locations of the characteristic peaks. Arrows and numbers on panel b indicate the snapshots in figure 6.

Figure 6

Force-induced dissociation pathway of the dimer structure during SMD simulation (5 nm/ns pulling rate). The snapshots of dimer structure are from 1, 0.2 ns; 2, 0.48 ns; 3, 0.49 ns; 4, 0.5 ns; 5, 0.54 ns; 6, 0.6 ns; 7, 0.7 ns; 8, 0.9 ns and 9, 1.2 ns of the SMD trajectory. The numbers correspond to the position numbers shown in Figure 5b. (a) H-bond breaking. Red arrow indicates β–sheet structure and H-bonds are yellow dotted lines. N and C indicate the N- and C-terminal ends, respectively. (b) Changes in the number of inter-chain H-bonds. (c) Force induced dissociation of the inter-chain salt bridge between Lys16 of chain A and Asp23 of chain B. (d) Distance between the center-of-masses of εNH₃⁺ of Lys16 of chain A and βCOO- of Asp23 of chain B groups. (e) Weakly polar interaction between side chain of Phe19 of chain B and side chain of His14 of chain A. (f) Distance between the C^βH group of His14 of chain A and the center of the phenyl ring of Phe19 of chain B.

Figure 7

WHAM analysis of the umbrella sampling simulation. (a) Converged umbrella histograms of 31 configurations, each derived from 20 ns simulation. (b) Potential of mean force (PMF). ΔG¹_bindA,B corresponds to H-bonds and salt bridge breaking and ΔG²_bindA,B is associated with the breaking of weakly polar interactions and a salt bridge.