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

The aggregation of TAR DNA-binding protein of 43 kDa (TDP-43) into fibrillary deposits is associated with amyotrophic lateral sclerosis (ALS). The 311-360 fragment of TDP-43 (TDP-43311-360), the amyloidogenic core region, can spontaneously aggregate into fibrils, and the ALS-associated mutation G335D has an enhanced effect on TDP-43311-360 fibrillization. However, the molecular mechanism underlying G335D-enhanced aggregation at atomic level remains largely unknown. By utilizing all-atom molecular dynamics (MD) and replica exchange with solute tempering 2 (REST2) simulations, we investigated influences of G335D on the dimerization (the first step of aggregation) and conformational ensemble of the TDP-43311-360 peptide. Our simulations show that G335D mutation increases inter-peptide interactions, especially inter-peptide hydrogen-bonding interactions in which the mutant site has a relatively large contribution, and enhances the dimerization of TDP-43311-360 peptides. The α-helix regions in the NMR-resolved conformation of the TDP-43311-360 monomer (321-330 and 335-343) play an essential role in the formation of the dimer. G335D mutation induces helix unfolding and promotes α-to-ß conversion. G335D mutation alters the conformational distribution of TDP-43311-360 dimers and causes population shift from helix-rich to ß-sheet-rich conformations, which facilitates the fibrillization of the TDP-43311-360 peptide. Our MD and REST2 simulation results suggest that the 321-330 region is of paramount importance to α-to-ß transition and could be the initiation site for TDP-43311-360 fibrillization. Our work reveals the mechanism underlying the enhanced aggregation propensity of the G335D TDP-43311-360 peptide, which provides atomistic insights into the G335D mutation-caused pathogenicity of TDP-43 protein.

Insights into the Atomistic Mechanisms of Phosphorylation in Disrupting Liquid-Liquid Phase Separation and Aggregation of the FUS Low-Complexity Domain.

Fused in sarcoma (FUS), a nuclear RNA binding protein, can not only undergo liquid-liquid phase separation (LLPS) to form dynamic biomolecular condensates but also aggregate into solid amyloid fibrils which are associated with the pathology of amyotrophic lateral sclerosis and frontotemporal lobar degeneration diseases. Phosphorylation in the FUS low-complexity domain (FUS-LC) inhibits FUS LLPS and aggregation. However, it remains largely elusive what are the underlying atomistic mechanisms of this inhibitory effect and whether phosphorylation can disrupt preformed FUS fibrils, reversing the FUS gel/solid phase toward the liquid phase. Herein, we systematically investigate the impacts of phosphorylation on the conformational ensemble of the FUS37-97 monomer and dimer and the structure of the FUS37-97 fibril by performing extensive all-atom molecular dynamics simulations. Our simulations reveal three key findings: (1) phosphorylation shifts the conformations of FUS37-97 from the ß-rich, fibril-competent state toward a helix-rich, fibril-incompetent state; (2) phosphorylation significantly weakens protein-protein interactions and enhances protein-water interactions, which disfavor FUS-LC LLPS as well as aggregation and facilitate the dissolution of the preformed FUS-LC fibril; and (3) the FUS37-97 peptide displays a high ß-strand probability in the region spanning residues 52-67, and phosphorylation at S54 and S61 residues located in this region is crucial for the disruption of LLPS and aggregation of FUS-LC. This study may pave the way for ameliorating phase-separation-related pathologies via site-specific phosphorylation.

ALS-associated A315E and A315pT variants exhibit distinct mechanisms in inducing irreversible aggregation of TDP-43312-317 peptides.

Amyotrophic lateral sclerosis (ALS) is intensively associated with insoluble aggregates formed by transactivation response element DNA-binding protein 43 (TDP-43) in the cytoplasm of neuron cells. A recent experimental study reported that two ALS-linked familial variants, A315E and A315pT (pT, phosphorylated threonine), can induce irreversible aggregation of the TDP-43 312NFGAFS317 segment (TDP-43312-317). However, the underlying molecular mechanism remains largely elusive. Here, we investigated the early aggregation process of the wild type (WT) 312NFGAFS317 segment and its A315E and A315pT variants by performing multiple microsecond all-atom molecular dynamics simulations. Our simulations show that the two variants display lower fluidity than WT, consistent with their decreased labilities observed in previous denaturation assay experiments. Despite each of the two variants carrying one negative charge, unexpectedly, we find that both A315E mutation and A315pT phosphorylation enhance intermolecular interactions and result in the formation of more compact oligomers. Compared to WT, A315E oligomers possess low ß-sheet content but a compact hydrophobic core, while A315pT oligomers have high ß-sheet content and large ß-sheets. Side chain hydrogen-bonding and hydrophobic interactions as well as N312-E315 salt bridges contribute most to the increased aggregation propensity of the A315E mutant. By contrast, main chain and side chain hydrogen-bonding interactions, side chain hydrophobic and aromatic interactions, are crucial to the enhanced aggregation capability of the A315pT variant. These results indicate that glutamate mutation and phosphorylation at position 315 induce the irreversible aggregation of TDP-43312-317 peptides through differential mechanisms, which remind us that we should be careful in the investigation of the phosphorylation effect on protein aggregation by using phosphomimetic substitutions. This study provides mechanistic insights into the A315E/A315pT-induced irreversible aggregation of TDP-43312-317, which may be helpful for the in-depth understanding of ALS-mutation/phosphorylation-associated liquid-to-solid phase transition of TDP-43 protein aggregates.

Evaluation of Essential Dynamics and Fixed-Length Coarse Graining for Multidomain Proteins.

For multiscale modeling of biomolecules, reliable coarse-grained (CG) models can offer great potential to simulate larger temporal and spatial scales than traditional all-atom (AA) models. In this study, we explore the essential dynamics coarse graining (EDCG) and fixed-length coarse graining (FLCG) approaches for constructing highly coarse-grained models for multidomain proteins (MDPs), with 1 to 10 amino acid residues per CG site. In the studies of 13 MDPs, our data indicate that both EDCG and FLCG can preserve the protein dynamics of MDPs. FLCG, which restricts an equal number of residues in each CG site, represents an excellent approximation to EDCG and a straightforward approach for coarse-graining MDPs. Furthermore, FLCG is tested with a class B G-protein-coupled receptor protein, and the agreement with prior experiments suggests its general application to various MDPs in different environments or conditions. Finally, we demonstrate another application of FLCG through progressive backmapping, showcasing the ability to recover from lower-resolution CG models (6 residues/CG site) to higher-resolution ones (1 residue/CG site). These promising outcomes underscore the broad applicability of FLCG to construct highly or ultra-coarse-grained models of complex biomolecules for multiscale simulations.

Elucidating the reversible and irreversible self-assembly mechanisms of low-complexity aromatic-rich kinked peptides and steric zipper peptides.

Many RNA-binding proteins such as fused-in sarcoma (FUS) can self-assemble into reversible liquid droplets and fibrils through the self-association of their low-complexity (LC) domains. Recent experiments have revealed that SYG-rich segments in the FUS LC domains play critical roles in the reversible self-assembly behaviors of FUS. These FUS LC segments alone can self-assemble into reversible kinked fibrils, which are markedly different from the canonical irreversible steric zipper ß-sheet fibrils. However, the molecular determinants underlying the reversible and irreversible self-assembly are poorly understood. Herein we conducted extensive all-atom and coarse-grained molecular dynamics simulations of four representative hexapeptides: two low-complexity aromatic-rich kinked peptides from the amyotrophic lateral sclerosis-related FUS protein, FUS37-42 (SYSGYS) and FUS54-59 (SYSSYG); and two steric zipper peptides from Alzheimer's-associated Aß and Tau proteins, Aß16-21 (KLVFFA) and Tau306-311 (VQIVYK). We dissected their reversible and irreversible self-assembly dynamics, predicted their phase separation behaviors, and elucidated the underpinning molecular interactions. Our simulations showed that alternating stickers (Tyr) and spacers (Gly and Ser) in FUS37-42 and FUS54-59 facilitate the formation of highly dynamic coil-rich oligomers and lead to reversible self-assembly, while consecutive hydrophobic residues of LVFF in Aß16-21 and IVY in Tau306-311 act as hydrophobic patches, favoring the formation of stable ß-sheet-rich oligomers and driving the irreversible self-assembly. Intriguingly, we found that FUS37-42 and FUS54-59 peptides, possessing the same amino acid composition and the same number of sticker and spacer residues, display differential self-assembly propensities. This finding suggests that the self-assembly behaviors of FUS peptides are fine-tuned by the site-specific patterning of spacer residues (Ser and Gly). This study provides significant mechanistic insights into reversible and irreversible peptide self-assembly, which would be helpful for understanding the molecular mechanisms underlying the formation of biological liquid condensates and pathological solid amyloid fibrils.

Dissecting how ALS-associated D290V mutation enhances pathogenic aggregation of hnRNPA2286-291 peptides: Dynamics and conformational ensembles.

The aggregation of RNA binding proteins, including hnRNPA1/2, TDP-43 and FUS, is heavily implicated in causing or increasing disease risk for a series of neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). A recent experimental study demonstrated that an ALS-related D290V mutation in the low complexity domain (LCD) of hnRNPA2 can enhance the aggregation propensity of wild type (WT) hnRNPA2286-291 peptide. However, the underlying molecular mechanisms remain elusive. Herein, we investigated effects of D290V mutation on aggregation dynamics of hnRNPA2286-291 peptide and the conformational ensemble of hnRNPA2286-291 oligomers by performing all-atom molecular dynamic and replica-exchange molecular dynamic simulations. Our simulations demonstrate that D290V mutation greatly reduces the dynamics of hnRNPA2286-291 peptide and that D290V oligomers possess higher compactness and ß-sheet content than WT, indicative of mutation-enhanced aggregation capability. Specifically, D290V mutation strengthens inter-peptide hydrophobic, main-chain hydrogen bonding and side-chain aromatic stacking interactions. Those interactions collectively lead to the enhancement of aggregation capability of hnRNPA2286-291 peptides. Overall, our study provides insights into the dynamics and thermodynamic mechanisms underlying D290V-induced disease-causing aggregation of hnRNPA2286-291, which could contribute to better understanding of the transitions from reversible condensates to irreversible pathogenic aggregates of hnRNPA2 LCD in ALS-related diseases.

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

Pathogenic mutations of transactivation response element DNA-binding protein 43 (TDP-43) are closely linked with amyotrophic lateral sclerosis (ALS). It was recently reported that two ALS-linked familial mutants A315T and A315E of TDP-43307-319 peptides can self-assemble into oligomers including tetramers, hexamers, and octamers, among which hexamers were suggested to form the ß-barrel structure. However, due to the transient nature of oligomers, their conformational properties and the atomic mechanisms underlying the ß-barrel formation remain largely elusive. Herein, we investigated the hexameric conformational distributions of the wild-type (WT) TDP-43307-319 fragment and its A315T and A315E mutants by performing all-atom explicit-solvent replica exchange with solute tempering 2 simulations. Our simulations reveal that each peptide can self-assemble into diverse conformations including ordered ß-barrels, bilayer ß-sheets and/or monolayer ß-sheets, and disordered complexes. A315T and A315E mutants display higher propensity to form ß-barrel structures than the WT, which provides atomic explanation for their enhanced neurotoxicity reported previously. Detailed interaction analysis shows that A315T and A315E mutations increase inter-molecular interactions. Also, the ß-barrel structures formed by the three different peptides are stabilized by distinct inter-peptide side-chain hydrogen bonding, hydrophobic, and aromatic stacking interactions. This study demonstrates the enhanced ß-barrel formation of the TDP-43307-319 hexamer by the pathogenic A315T and A315E mutations and reveals the underlying molecular determinants, which may be helpful for in-depth understanding of the ALS-mutation-induced neurotoxicity of TDP-43 protein.

Atomistic Insights into A315E Mutation-Enhanced Pathogenicity of TDP-43 Core Fibrils.

The aggregation of TAR DNA-binding protein of 43 kDa (TDP-43) into fibrillary deposits is implicated in amyotrophic lateral sclerosis (ALS), and some hereditary mutations localized in the low complexity domain (LCD) facilitate the formation of pathogenic TDP-43 fibrils. A recent cryo-EM study reported the atomic-level structures of the A315E TDP-43 LCD (residues 288-319, TDP-43288-319) core fibril in which the protofilaments have R-shaped structures and hypothesized that A315E U-shaped protofilaments can readily convert to R-shaped protofilaments compared to the wild-type (WT) ones. There are no atomic structures of WT protofilaments available yet. Herein, we performed extensive all-atom explicit-solvent molecular dynamics simulations on A315E and WT protofilaments starting from both the cryo-EM-determined R-shaped and our constructed U-shaped structures. Our simulations show that WT protofilaments also adopt the R-shaped structures but are less stable than their A315E counterparts. Except for R293-E315 salt bridges, N312-F316 hydrophobic interactions and F316-F316 π-π stacking interactions are also crucial for the stabilization of the neck region of the R-shaped A315E protofilaments. The loss of R293-E315 salt bridges and the weakened interactions of N312-F316 and F316-F316 result in the reduced stability of the R-shaped WT protofilaments. Simulations starting from U-shaped folds reveal that A315E protofilaments can spontaneously convert to the cryo-EM-derived R-shaped protofilaments, whereas WT protofilaments convert to R-shape-like structures with remodeled neck regions. The R-shape-like WT protofilaments could act as intermediate states slowing down the U-to-R transition. This study reveals that A315E mutation can not only enhance the structural stability of the R-shaped TDP-43288-319 protofilaments but also promote the U-to-R transition, which provides atomistic insights into the A315E mutation-enhanced TDP-43 pathogenicity in ALS.