Stability of Human Serum Amyloid A Fibrils

Abstract

In systemic amyloidosis, serum amyloid A (SAA) fibril deposits cause widespread damages to tissues and organs that eventually may lead to death. A therapeutically intervention therefore has either to dissolve these fibrils or inhibit their formation. However, only recently has the human SAA fibril structure been resolved at a resolution that is sufficient for development of drug candidates. Here, we use molecular dynamic simulations to probe the factors that modulate the stability of this fibril model. Our simulations suggest that fibril formation starts with the stacking of two misfolded monomers into metastable dimers, with the stacking depending on the N-terminal amyloidogenic regions of different chains forming anchors. The resulting dimers pack in a second step into a 2-fold two-layer tetramer that is stable enough to nucleate fibril formation. The stability of the initial dimers is enhanced under acidic conditions by a strong salt bridge and side-chain hydrogen bond network in the C-terminal cavity (residues 23-51) but is not affected by the presence of the disordered C-terminal tail.

Figure 1.

a). Cryo-EM model of the human SAA fibril (PDB ID: 6MST) . The N- and C-termini are marked by blue and red spheres, respectively. The β-strands are colored in yellow, turns in cyan and coil regions in gray. In b) we highlight some key residues involved in the packing of folds or the interaction between layers. In order to avoid crowding of the figure we do not show the sequence of the first eleven residues: R-S-F-F-S-F-L-G-E-A-F (in one letter code).

Figure 2.

a) Initial structure of the fibril fragment F2L2. All simulations of fibril models built from SAA_2-55 chains start from configurations were the individual chains have the same structure as in this model. Representative configurations, taken at the end of one of the three 100 ns long trajectories of fibrils simulated at pH=7, are shown in b) -g) . Specifically, final configurations are shown for F2L1 (b), F2L2 (c), F2L3 (d), F1L1 (e) , F1L2 (f) and F1L3 (g) . N- and C-terminus are marked by blue and red spheres, respectively. The β-strands are colored in yellow, turns in cyan and coil regions in gray.

Figure 3.

a). Average RMSD per chain for various fibril models simulated at pH=7. Only the trajectory leading to the highest final RMSD value is shown. The trajectories are for the F1L1 (red), F1L2 (blue), F2L1 (black), F2L2 (yellow), F1L3 (orange) and F2L3 (pink). Corresponding RMSF values are shown in b). In c) and d) we show the same quantities measured in simulations of the corresponding truncated systems: F1L1− (red), F1L2− (blue), F2L1− (black), F2L2− (yellow), F1L3− (orange)

Figure 4.

Average RMSD per chain values for each of the three trajectories simulated for the SAA_2-55 fibril models a) F1L2, b) F2L2, and for the elongated SAA_2-76 models F1L2+ (c) and F2L2+ (d). In sub figures (c) and (d), only residues 2 - 55 are considered for the calculation of RMSD to allow for comparison between systems of different chain length.

Figure 5.

Average RMSF of residues as measured in simulations of fibril models at either neutral pH (black) or under acid conditions (pH=4, drawn in red). In a) we consider the single-fold two-layer F1L2 (F1L2a), in b) the two-fold two-layer F2L2 (F2L2a), in c) the single-fold triple layer F1L3 (F1L3a), and in d) the two-fold-triple-layer F2L3 (F2L3a).

Figure 6.

Intra-chain side-chain distance map for the one-fold two-layer fibrils a) F1L2 (pH=7), b) F1L2A (pH=4), c). F1L2−(without N-terminus); and the corresponding two-fold two-layers fibrils d) F2L2, e) F2L2A and f) F2L2−. Contact distance is encoded by coloring, and the numbers shown in the color legend are in nm. A schematic representation of the contact network is shown for pH = 7 in g) and for acidic conditions (pH=4) in h). Only side chain interactions with an average occupancy per chain larger than 4 % are shown.

Figure 7.

Comparison of the average RMSF of residues in full-sized and truncated (i.e. with N-terminal residues) fibril models.

Figure 8.

Representative structure of largest cluster seen in simulations under neutral conditions of the truncated models F1L2− (a) and F2L2− (b). Configurations from all three trajectories where clustered with a cut-off of 3 Å of systems at pH 7, and the dominant cluster contains 35% N- and C-terminus are marked by blue and red spheres, respectively. The β-strands are colored in yellow, helices in purple, turns in cyan and coil regions in gray.