Influence of ALS‐linked M337V mutation on the conformational ensembles of TDP‐43_321–340 peptide monomer and dimer

Abstract

The transactive response (TAR) DNA/RNA‐binding protein 43 (TDP‐43) can self‐assemble into both functional stress granules via liquid–liquid phase separation (LLPS) and pathogenic amyloid fibrillary aggregates that are closely linked to amyotrophic lateral sclerosis. Previous experimental studies reported that the low complexity domain (LCD) of TDP‐43 plays an essential role in the LLPS and aggregation of the full‐length protein, and it alone can also undergo LLPS to form liquid droplets mainly via intermolecular interactions in the 321–340 region. And the ALS‐associated M337V mutation impairs LCD's LLPS and facilitates liquid–solid phase transition. However, the underlying atomistic mechanism is not well understood. Herein, as a first step to understand the M337V‐caused LLPS disruption of TDP‐43 LCD mediated by the 321–340 region and the fibrillization enhancement, we investigated the conformational properties of monomer/dimer of TDP‐43321–340 peptide and its M337V mutant by performing extensive all‐atom explicit‐solvent replica exchange molecular dynamic simulations. Our simulations demonstrate that M337V mutation alters the residue regions with high helix/β‐structure propensities and thus affects the conformational ensembles of both monomer and dimer. M337V mutation inhibits helix formation in the N‐terminal Ala‐rich region and the C‐terminal mutation site region, while facilitating their long β‐sheet formation, albeit with a minor impact on the average probability of both helix structure and β‐structure. Further analysis of dimer system shows that M337V mutation disrupts inter‐molecular helix–helix interactions and W334‐W334 π‐π stacking interactions which were reported to be important for the LLPS of TDP‐43 LCD, whereas enhances the overall peptide residue‐residue interactions and weakens peptide‐water interactions, which is conducive to peptide fibrillization. This study provides mechanistic insights into the M337V‐mutation‐induced impairment of phase separation and facilitation of fibril formation of TDP‐43 LCD.

Dissecting the Effect of ALS Mutation G335D on the Early Aggregation of the TDP-43 Amyloidogenic Core Peptide: Helix-to-β-Sheet Transition and Conformational Shift

“…In order to identify regions with high helix/β structure probability in WT and G335D dimers, we then calculated helix and β probabilities of each residue. As shown for the WT dimer in Figure a, residues in 321 AMMAAAQAAL 330 region have the highest helix probability, followed by 335 GMMGMLASQ 343 region, consistent with previous studies reporting that residues 321–330 in TDP-43 LCD have the highest propensity to transiently populate a helical structure. , Strikingly, helix probabilities of residues in the 321–330 region in the G335D dimer are rather lower than those in the WT dimer, in agreement with a recent well-tempered metadynamics simulation study of the TDP-43 311–350 monomer . In addition, the average length of α-helix in the G335D dimer (8.4) is shorter than that in the WT dimer (9.0), and the probability of longer α-helix (with a length ≥15) almost disappears in the G335D dimer (Figure b).…”

“…41 Amber99SB-ILDN force field 42 was used for proteins, and the TIP3P model 43 was used for water molecules, in accordance with recent studies demonstrating that this force field can well simulate the aggregation dynamics and conformational ensembles of fragments of the TDP-43 LCD. 19,28,30,33,37,44,45 For MD simulations, WT or the G335D TDP-43 311−360 dimer was placed in the center of a truncated octahedral box with a minimum distance between the protein to the box wall being 1.2 nm. The shortest distance between two hexagons of the truncated octahedral box is 8.09 nm.…”

“…All simulations were performed using the GROMACS-2018.3 package with the PLUMED2 plugin . Amber99SB-ILDN force field was used for proteins, and the TIP3P model was used for water molecules, in accordance with recent studies demonstrating that this force field can well simulate the aggregation dynamics and conformational ensembles of fragments of the TDP-43 LCD. ,,,,,, …”

ALS-Linked A315T and A315E Mutations Enhance β-Barrel Formation of the TDP-43307–319 Hexamer: A REST2 Simulation Study

“…REST2 can accelerate simulation convergence and enhance the sampling with tremendously reduced computational cost compared with temperature replica exchange molecular dynamics, and it has been widely used in the investigation of the conformational ensembles of intrinsically disordered proteins. − All REST2 simulations in this work were performed using the GROMACS 2018.3 software package patched with the open-source, community-developed PLUMED library, version 2.6.0 . The AMBER99SB-ILDN force field was used in our study in accordance with previous studies showing that this force field can well simulate the aggregation and protofilament stability of fragments of the TDP-43 LCD. ,,,− Each system was placed in a cubic box (8.88 × 8.88 × 8.88 nm 3 ) filled with TIP3P water molecules . Na + and Cl – ions were added in order to neutralize the systems and mimic the physiological salt concentration of 0.15 M. Prior to production REST2 runs, we performed 0.1 ns simulations in the NVT (310 K) ensemble, followed by 0.1 ns simulations in the NPT (310 K, 1 bar) ensemble, to equilibrate the solvent and ions around the protein.…”

An ALS-associated mutation dysregulates microglia-derived extracellular microRNAs in a sex-specific manner

“…These alterations can lead to progressive motor dysfunction ( Gordon et al, 2019 ). In terms of its influence on cellular processes, the M337V mutation affects the conformational properties of the TDP-43 protein, resulting in an impairment in the phase separation ability of the protein and an increased tendency towards fibril formation, which is significant in ALS pathology ( Zeng et al, 2023 ). Furthermore, studies using mouse models have shown that overexpression of human TARDBP M337V leads to worse disease features compared to those in mice expressing wildtype human TARDBP .…”