Local unfolding of Cu, Zn superoxide dismutase monomer determines the morphology of fibrillar aggregates

Abstract

Aggregation of Cu, Zn superoxide dismutase (SOD1) is often found in amyotrophic lateral sclerosis patients. The fibrillar aggregates formed by wild type and various disease-associated mutants have recently been found to have distinct cores and morphologies. Previous computational and experimental studies of wild-type SOD1 suggest that the apo-monomer, highly aggregation prone, displays substantial local unfolding dynamics. The residual folded structure of locally unfolded apoSOD1 corresponds to peptide segments forming the aggregation core as identified by a combination of proteolysis and mass spectroscopy. Therefore, we hypothesize that the destabilization of apoSOD1 caused by various mutations leads to distinct local unfolding dynamics. The partially unfolded structure, exposing the hydrophobic core and backbone hydrogen bond donors and acceptors, is prone to aggregate. The peptide segments in the residual folded structures form the "building block" for aggregation, which in turn determines the morphology of the aggregates. To test this hypothesis, we apply a multiscale simulation approach to study the aggregation of three typical SOD1 variants: wild type, G37R, and I149T. Each of these SOD1 variants has distinct peptide segments forming the core structure and features different aggregate morphologies. We perform atomistic molecular dynamics simulations to study the conformational dynamics of apoSOD1 monomer and coarse-grained molecular dynamics simulations to study the aggregation of partially unfolded SOD1 monomers. Our computational studies of monomer local unfolding and the aggregation of different SOD1 variants are consistent with experiments, supporting the hypothesis of the formation of aggregation "building blocks" via apo-monomer local unfolding as the mechanism of SOD1 fibrillar aggregation.

Figure 1. The thermodynamics of apoSOD1 monomer

(A) In SOD1 monomer, the two metal ions (spheres) are coordinated by residues (sticks) in strands 4, 5, and 7. The amyloid core of wildtype SOD1 is composed of N-terminal strands 1–3 (blue to cyan), middle strand 6 (yellow), and C-terminal strand 8 (red). Other regions are colored gray. The two mutated residues in this study, G37 and I149, are shown in sphere. (B) The regions forming the amyloid core of three typical SOD1 variants: wildtype (WT), G37R, and I149T. The arrows illustrate the strands along the primary sequence. The thick lines highlight the regions found in the proteolysis-resistant amyloid core for each of the three SOD1 variants. (C) The specific heat of the wildtype apoSOD1 as a function of temperature. The specific heat values and corresponding statistical uncertainties (shown as error bars) are computed using WHAM analysis of the replica exchange simulation trajectories (Methods). At temperatures bellow the low-temperature peak, the protein is native-like with the beta-barrel intact. At temperatures higher than the high-temperature peak, the protein is unfolded, without persistent secondary and tertiary structures. At the intermediate temperature between the two peaks, the protein is partially unfolded. (D) The specific heat curves for all three variants feature similar local unfolding and global unfolding thermodynamics.

Figure 2. Average RMSD as a function of temperature

Average RMSD is computed using WHAM analysis. Based on the self-consistently determined density of states ρ(E) and the conditional probability to observe a structure with RMSD of R at a given energy E, P(R|E), the average RMSD and corresponding error bar can be computed accordingly (Methods).

Figure 3. The local unfolding conformational dynamics of apoSOD1 monomer

The locally unfolded conformational ensemble for each SOD1 variant is reconstructed from the simulation trajectories (Methods). The corresponding conformational dynamic parameters, including RMSF(A,C) and RMSD (B,D) per residue are computed based on the reconstructed structural ensemble. In order to estimate the error bars, we split the simulation trajectory into halves and compute the dynamic parameters independently. To avoid overlapping lines, we compare the RMSF and RMSD between WT and G37R (A,C), and between WT and I149T (B,D), separately. The average RMSD per residue is computed with respect to the native states. Due to their large conformational flexibility, we do not include the loops in the RMSD calculation.

Figure 4. Thermodynamics of coarse-grained SOD1 monomer

(A) WT, (B) G37R, and (C) I149T. In the left column, the probability of each residue to form the amyloid core (P^core) is computed from the experimentally identified, proteolysis-resistant peptides in the amyloid core (Methods). In the right column, the specific heat of the coarse-grained SOD1 monomer is computed from replica exchange simulations of monomer folding. Due to the experimentally-based bias potential, the protein features a stable partially folded intermediate state.

Figure 5. The aggregation process of wildtype SOD1 monomers

(A) The initial configuration of eight SOD1 monomers in the simulation box. The monomer concentration is as high as 1mM. The cartoon representation is assigned and illustrated by using MolScript and PyMol, respectively.(B) One monomer (indicated by the arrow) associates with the end of the nascent amyloid-like aggregate and forms the cross-beta core. (C) The associated monomer is incorporated into the aggregate after structural rearrangement. (D) The resulting stable aggregate structure with well-formed cross-beta core.

Figure 6. The fibrillar aggregates of SOD1

(A)WT, (B)G37R and (C) I149T. The first and second columns correspond to the aggregates formed in simulations. Two views are shown by 90° rotation along the axis of the amyloid fibril. The aggregate of G37R contains two cores. The computed fibril diffraction patterns of the aggregates feature the typical “cross-beta” characteristics (third column). The peak along the fibril axis corresponds to the hydrogen bonds between strands, and the peak perpendicular to the axis corresponds to the separation between the adjacent beta-sheet. The fourth column corresponds to the electron microscopic images of aggregates formed in vitro. The SOD1 fibrils were prepared by shaking disulfide-reduced apo-SOD1 at 37 °C, 1,200 rpm for 50 hours (Methods). A bar in each panel represents 0.1 μm.

Figure 7. Screened Debye-Hückel potential function between two opposite single charges

The continuous potential has a Debye length of ~10 Å, assuming the relative permittivity of water 80 and a monovalent salt concentration of 0.1 mM. The step-function in red is the discretized step function utilized in DMD simulations. For two atoms of the same charge, we change the sign of the potential to indicate a repulsive.