Nucleation and Growth of Amyloid Fibrils

Abstract

The formation of amyloid fibrils is a complex phenomenon that remains poorly understood at the atomic scale. Herein, we perform extended unbiased all-atom simulations in explicit solvent of a short amphipathic peptide to shed light on the three mechanisms accounting for fibril formation, namely, nucleation via primary and secondary mechanisms, and fibril growth. We find that primary nucleation takes place via the formation of an intermediate state made of two laminated β-sheets oriented perpendicular to each other. The amyloid fibril spine subsequently emerges from the rotation of these β-sheets to account for peptides that are parallel to each other and perpendicular to the main axis of the fibril. Growth of this spine, in turn, takes place via a dock-and-lock mechanism. We find that peptides dock onto the fibril tip either from bulk solution or after diffusing on the fibril surface. The latter docking pathway contributes significantly to populate the fibril tip with peptides. We also find that side chain interactions drive the motion of peptides in the lock phase during growth, enabling them to adopt the structure imposed by the fibril tip with atomic fidelity. Conversely, the docked peptide becomes trapped in a local free energy minimum when docked-conformations are sampled randomly. Our simulations also highlight the role played by nonpolar fibril surface patches in catalyzing and orienting the formation of small cross-β structures. More broadly, our simulations provide important new insights into the pathways and interactions accounting for primary and secondary nucleation as well as the growth of amyloid fibrils.

Figure 1:

Amino acid sequence, β-sheet, and amyloid fibril from an amphipathic peptide. (a) Non-polar phenylalanine (F), positively charged lysine (K), and negatively charged glutamic acid (E) are the three amino acids used to account for the amphipathic Ac-(FKFE)₂-NH₂ peptide. (b) Non-polar and charged residues are segregated to different faces of anti-parallel β-sheet. (c) packing of non-polar faces of two β-sheets against each other accounts for the cross-β structure of amyloid fibrils. Non-polar edges and tips are highlighted in the figure.

Figure 2:

Observation of primary nucleation in two independent simulated trajectories. The time dependence of various quantities are computed to characterize the spontaneous formation of cross-β structures. These quantities are (a and l) the largest cluster size in the simulation box, (b and m) the number of residues per peptide in a β-structure, the number of peptides comprising (c and n) largest and (d and o) second largest β-sheet in the simulation box, (e and p) solvent accessible surface area (SASA) of non-polar residues, and (f and q) dihedral angle between peptides of largest and second largest β-sheets. The formation of a stable oligomer and the existence of a perpendicular cross-β structure are highlighted by gray and orange rectangles. Conformations of the largest cluster at select times are shown in panels g-k, and r-u for the two independent trajectories. A different color is used for each peptide for visualization purposes.

Figure 3:

Unbiased pathways accounting for fibril elongation. (a) The trajectory of the peptide as it interacts with the fibril before docking to its tip is shown. The conformations of the peptide (in red) are depicted at every 20 ns. Numbered arrows show the progression of the peptide in the simulation. The fibril is depicted using a cartoon representation in blue and a van der Waals representation for phenylalanine side chains in cyan. The time dependence of five quantities are computed to characterize the peptide-fibril complex: (b) minimum distance between atoms of the fibril and the peptide, (c) inter- and (d) intra-backbone hydrogen bonds, (e) radius of gyration of the peptide, and (f) number of water molecules around peptide and fibril. Sample configurations of the peptide-fibril complex when the peptide is (g) fully solvated, (h) bound to the fibril surface, (i) docked and (j) locked onto the fibril tip. Arrows highlight possible transitions between the different states.

Figure 4:

Locking of the peptide to the fibril tip. The time dependence of the number of inter-backbone hydrogen bonds between peptide and fibril at (a) 350 K and (f) 325 K. The structure of the peptide-fibril system in trajectory number 4 at 350 K is represented at time (b) 0.16 μs, c) 0.37 μs, d) 0.57 μs, and e) 0.90 μs. The structure of the peptide-fibril system of trajectory number 5 at 325 K is shown at time (g) 0.09 μs, (h) 0.11 μs, (i) 0.2 μs, and (j) 1.01 μs. A van der Waals representation is used for non-polar residues of the fibril (white) and peptide (blue). Negatively charged glutamic acid and positively charged lysine are represented in red and green, respectively.

Figure 5:

Final conformations of 2 μs long simulations where six peptides initially randomly located in the simulation box are allowed to interact with a pre-formed fibril. A van der Waals representation is used for non-polar residues of the fibril (white) and peptide (blue). A cartoon representation is used for the backbone of the fibril (orange) and peptide (blue).

Figure 6:

Sequence of events leading to the formation of a nucleus on the surface of a preformed fibril (in orange and white) from six peptides deposited randomly in the simulation box. a) Events leading to the formation of the structures in Fig. 5e. b) Events leading to the structure in Fig. 5f. Each peptide is shown using a different color.

Figure 7:

Growth of a new nucleus (blue) on the surface of a preformed fibril (white and yellow) through the consecutive addition of six peptides to the simulation box. Peptides are colored in pink, green, red, orange, and cyan according to the order in which they are added to the simulation box. After each addition of six peptides, a 0.5 μs or a 1 μs simulation (as indicated in the figure) is performed to allow peptides to bind to the fibril. Final configurations are shown with the backbone of the preformed fibril illustrate in orange using a cartoon representation. A van der Waals representation is used for non-polar residues.

Figure 8:

Growth of parent fibril and seed. Orange and blue lines correspond to the number of peptides incorporated onto the fibril and seed, respectively. Dashed horizontal lines indicate the instant when six new peptides are introduced to the solution. Labels a-e refer to configurations in Fig. 7.

Figure 9:

Schematic representation of the intermediate states and pathways accounting for fibril formation of Ac-(FKFE)₂-NH₂ peptides in our simulations. BLUE: primary nucleation proceeds via the formation of perpendicular cross-β structures. Rotation of β-sheets in the latter leads to the emergence of the amyloid spine. RED (Upper): docking proceeds with peptides diffusing in solution or on the fibril edge to land on the tip. RED (Bottom): step-wise-locking proceeds with the alignment of side chains at different extremities of the peptide with the fibril. Positively charged residues (K) and negatively charged residues (E) are shown in blue and red, respectively. GREEN: peptides (light blue) aggregate at the non-polar edge of the fibril where new fibrils are nucleated.