Computational Insights of Unfolding of N-Terminal Domain of TDP-43 Reveal the Conformational Heterogeneity in the Unfolding Pathway

Abstract

TDP-43 proteinopathies is a disease hallmark that characterizes amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). The N-terminal domain of TDP-43 (NTD) is important to both TDP-43 physiology and TDP-43 proteinopathy. However, its folding and dimerization process is still poorly characterized. In the present study, we have investigated the folding/unfolding of NTD employing all-atom molecular dynamics (MD) simulations in 8 M dimethylsulfoxide (DMSO) at high temperatures. The MD results showed that the unfolding of the NTD at high temperature evolves through the formation of a number of conformational states differing in their stability and free energy. The presence of structurally heterogeneous population of intermediate ensembles was further characterized by the different extents of solvent exposure of Trp80 during unfolding. We suggest that these non-natives unfolded intermediate ensembles may facilitate NTD oligomerization and subsequently TDP-43 oligomerization, which might lead to the formation of irreversible pathological aggregates, characteristics of disease pathogenesis.

Keywords: DMSO; NTD; TDP-43; conformational heterogeneity; unfolding intermediates.

Figure 1

Thermal unfolding analysis of NTD. Time evolutions of (A) Cα- RMSD, (B) radius of gyration, Rg, (C) fraction of native contacts, Nc, and (D) SASA of NTD at temperature 300 K (indigo), 350 K (red), 400 K (green), 450 K (blue), and 500 K (purple). NTD, N-terminal domain; SASA, solvent accessible surface area.

Figure 2

Time evolution plot of NTD secondary structure using DSSP in 8 M DMSO at (A) 300 K, (B) 350 K, (C) 400 K, and (D) 450 K. DMSO, dimethylsulfoxide.

Figure 3

The free energy landscape (FEL) of NTD during thermal unfolding. Free energy contour maps constructed at 400 K from (A) Nc vs. Cα-RMSD, and (B) Nc vs. Rg. Similarly, FEL at 450 K from (C) Nc vs. Cα-RMSD, and (D) Nc vs. Rg. The color is scaled according to kcal mol⁻¹. The dark blue region represents the energy minima and energetically favored protein conformations, and the yellow region represents the unfavorable high-energy conformations. RMSD, root mean square deviation.

Figure 4

FEL of the number of intraprotein H- bonds vs. the number of native contacts at (A) 400 K, and (B) 450 K. The results clearly indicate the large decrease in the number of H-bonds at a higher temperature.

Figure 5

Thermal unfolding of NTD in 8 M DMSO. Time evolution of the solvent-exposed surface area, SASA, of the W80 side chain and Ca-RMSD at (A) 400 K, and (B) 450 K. Each point on this plot is colored according to its time of occurrence according to the color scale shown.

Figure 6

Dynamic cross-correlation map (DCCM) of NTD’s residue motion during MD simulation. The residue motions of NTD at (A,B) 400 K, and (C,D) 450 K during the start and the end of the simulation, respectively. The degrees of the correlation motions and anti-correlation motions are shown in blue and red, respectively.