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.

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.

Deciphering the mechanisms of HPV E6 mutations in the destabilization of E6/E6AP/p53 complex.

In epithelial tumors, oncoprotein E6 binds with the ubiquitin ligase E6AP to form E6/E6AP heterodimer; then this heterodimer recruits p53 to form E6/E6AP/p53 heterotrimer and induces p53 degradation. Recent experiments demonstrated that three E6 single-site mutants (F47R, R102A, and L50E) can inhibit the E6/E6AP/p53 heterotrimer formation and rescue p53 from the degradation pathway. However, the molecular mechanism underlying mutation-induced heterotrimer inhibition remains largely elusive. Herein, we performed extensive molecular dynamics simulations (totally ∼13 µs) on both heterodimer and heterotrimer to elucidate at an atomic level how each p53-degradation-defective HPV16 E6 mutant reduces the structural stabilities of the two complexes. Our simulations reveal that the three E6 mutations destabilize the structure of E6/E6AP/p53 complex through distinct mechanisms. Although F47RE6 mutation has no effect on the structure of E6/E6AP heterodimer, it results in an electrostatic repulsion between R47E6 and R290p53, which is unfavorable for E6-p53 binding. R102AE6 mutation destabilizes the structure of E6/E6AP heterodimer and significantly disrupts hydrophobic and cation-π interactions between F47E6 and E286p53/L298p53/R290p53. L50EE6 mutation impairs both E6 interdomain interactions (especially F47-K108 cation-π interaction) and E6-E6AP intermolecular interactions important for the stabilization of E6/E6AP heterodimer. This study identifies the intra- and intermolecular interactions crucial for the complex stability, which may provide mechanistic insights into the inhibition of complex formation by the three HPV16 E6 mutations.

A Comprehensive Insight into the Mechanisms of Dopamine in Disrupting Aß Protofibrils and Inhibiting Aß Aggregation.

Fibrillary aggregates of amyloid-ß (Aß) are the pathological hallmark of Alzheimer's disease (AD). Clearing Aß deposition or inhibiting Aß aggregation is a promising approach to treat AD. Experimental studies reported that dopamine (DA), an important neurotransmitter, can inhibit Aß aggregation and disrupt Aß fibrils in a dose-dependent manner. However, the underlying molecular mechanisms still remain mostly elusive. Herein, we investigated the effect of DA on Aß42 protofibrils at three different DA-to-Aß molar ratios (1:1, 2:1, and 10:1) using all-atom molecular dynamics simulations. Our simulations demonstrate that protonated DA at a DA-to-Aß ratio of 2:1 exhibits stronger Aß protofibril disruptive capacity than that at a molar-ratio of 1:1 by mostly disrupting the F4-L34-V36 hydrophobic core. When the ratio of DA-to-Aß increases to 10:1, DA has a high probability to bind to the outer surface of protofibril and has negligible effect on the protofibril structure. Interestingly, at the same DA-to-Aß ratio (10:1), a mixture of protonated (DA+) and deprotonated (DA0) DA molecules significantly disrupts Aß protofibrils by the binding of DA0 to the F4-L34-V36 hydrophobic core. Replica-exchange molecular dynamics simulations of Aß42 dimer show that DA+ inhibits the formation of ß-sheets, K28-A42/K28-D23 salt-bridges, and interpeptide hydrophobic interactions and results in disordered coil-rich Aß dimers, which would inhibit the subsequent fibrillization of Aß. Further analyses reveal that DA disrupts Aß protofibril and prevents Aß dimerization mostly through π-π stacking interactions with residues F4, H6, and H13, hydrogen bonding interactions with negatively charged residues D7, E11, E22 and D23, and cation-π interactions with residues R5. This study provides a complete picture of the molecular mechanisms of DA in disrupting Aß protofibril and inhibiting Aß aggregation, which could be helpful for the design of potent drug candidates for the treatment/intervention of AD.

Natural stereoisomeric flavonoids exhibit different disruptive effects and the mechanism of action on Aß42 protofibril.

Our simulations reveal that two enantiomeric catechins display a better disruptive effect on Aß42 protofibril than their stereoisomer epicatechin. Unexpectedly, we find that catechins adopt both collapsed and extended states, while epicatechin populates only an extended state. Their different protofibril-disruptive effects are mostly attributed to the steric effect caused by the conformational differences.

Liquid-Liquid Phase Separation of Tau Protein Is Encoded at the Monomeric Level.

Liquid-liquid phase separation (LLPS) is involved in both physiological and pathological processes. The intrinsically disordered protein Tau and its K18 construct can undergo LLPS in a distinct temperature-dependent manner, and the LLPS of Tau protein can initiate Tau aggregation. However, the underlying mechanism driving Tau LLPS remains largely elusive. To understand the temperature-dependent LLPS behavior of Tau at the monomeric level, we explored the conformational ensemble of Tau at different temperatures by performing all-atom replica-exchange molecular dynamic simulation on K18 monomer with an accumulated simulation time of 26.4 µs. Our simulation demonstrates that the compactness, ß-structure propensity, and intramolecular interaction of K18 monomer exhibit nonlinear temperature-dependent behavior. 295DNIKHV300/326GNIHHK331/337VEVKSE342 make significant contributions to the temperature dependence of the ß propensity of K18 monomer, while the two fibril-nucleating cores display relatively high ß propensity at all temperatures. At a specific temperature, K18 monomer adopts the most collapsed state with exposed sites for both persistent and transient interactions. Given that more collapsed polypeptide chains were reported to be more prone to phase separate, our results suggest that K18 monomer inherently possesses conformational characteristics favoring LLPS. Our simulation predicts the importance of 295DNIKHV300/326GNIHHK331/337VEVKSE342 to the temperature-dependent conformational properties of K18, which is corroborated by CD spectra, turbidity assays, and DIC microscopy. Taken together, we offer a computational and experimental approach to comprehend the structural basis for LLPS by amyloidal building blocks.

Mechanistic Insights into the Co-Aggregation of Aß and hIAPP: An All-Atom Molecular Dynamic Study.

Patients with Alzheimer's disease (AD) have a high risk of developing Type II diabetes (T2D). The co-aggregation of the two disease-related proteins, Aß and hIAPP, has been proposed as a potential molecular mechanism. However, the detailed Aß-hIAPP interactions and structural characteristics of co-aggregates are mostly unknown at atomic level. Here, we explore the conformational ensembles of the Aß-hIAPP heterodimer and Aß or hIAPP homodimer by performing all-atom explicit-solvent replica exchange molecular dynamic simulations. Our simulations show that the interaction propensity of Aß-hIAPP in the heterodimer is comparable with that of Aß-Aß/hIAPP-hIAPP in the homodimer. Similar hot spot residues of Aß/hIAPP in the homodimer and heterodimer are identified, indicating that both Aß and hIAPP have similar molecular recognition sites for self-aggregation and co-aggregation. Aß in the heterodimer possesses three high ß-sheet probability regions: the N-terminal region E3-H6, the central hydrophobic core region K16-E22, and the C-terminal hydrophobic region I31-A41, which is highly similar to Aß in the homodimer. More importantly, in the heterodimer, the regions E3-H6, F19-E22, and I31-M35 of Aß and the amyloid core region N20-T30 of hIAPP display higher ß-sheet probability than they do in homodimer, implying their crucial roles in the formation of ß-sheet-rich co-aggregates. Our study sheds light on the co-aggregation of Aß and hIAPP at an atomic level, which will be helpful for an in-depth understanding of the molecular mechanism for epidemiological correlation of AD and T2D.

Common cancer mutations R175H and R273H drive the p53 DNA-binding domain towards aggregation-prone conformations.

The p53 protein is a tumor suppressor and the most often mutated protein in human cancers. Recent studies reported that p53 mutants, including two of the common cancer mutants (R175H and R273H), are more prone to aggregation than wild type (WT) p53 and their pathological aggregation can lead to diverse cancers. However, the underlying molecular mechanism is poorly understood. Herein, we investigated the structural and dynamic properties of R175H and R273H mutants of the p53 core domain (p53C) by performing extensive all-atom molecular dynamics simulations. We found that both R175H and R273H mutants exhibit a well preserved ß-sheet structure, but a larger hydrophobic surface area and higher loop flexibility than WT p53C. These conformational properties are consistent with the structural features of aggregation-prone molten-globule states. Our data also provide the details on how the two mutations lead to an increased flexibility of loop2. Moreover, using dynamic network analysis, we identified the allosteric path through which the R273H mutation induces an increased flexibility of the distant N-terminal region of loop2. These results provide mechanistic insights into the high aggregation propensities of R175H and R273H mutants.

Pristine and Hydroxylated Fullerenes Prevent the Aggregation of Human Islet Amyloid Polypeptide and Display Different Inhibitory Mechanisms.

Protein aggregation, involving the formation of dimers, oligomers, and fibrils, is associated with many human diseases. Type 2 diabetes is one of the common amyloidosis and linked with the aggregation of human islet amyloid polypeptide (hIAPP). A series of nanoparticles are reported to be able to interact with proteins and enhance/inhibit protein aggregation. However, the effects of C60 (a model system of hydrophobic nanoparticle) and C60(OH)8 (a hydroxylated fullerene) on hIAPP aggregation remain unknown. In this study, we investigate the influences of pristine fullerene C60 and hydroxylated C60 on the dimerization of hIAPP using molecular dynamics (MD) simulations. Extensive replica exchange molecular dynamics (REMD) simulations show that isolated hIAPP dimers adopt ß-sheet structure containing the amyloid-precursor (ß-hairpin). Both C60 and C60(OH)8 notably inhibit the ß-sheet formation of hIAPP dimer and induce the formation of collapsed disordered coil-rich conformations. Protein-nanoparticle interaction analyses reveal that the inhibition of hIAPP aggregation by C60 is mainly via hydrophobic and aromatic-stacking interactions, while the prevention of hIAPP aggregation by C60(OH)8 is mostly through collective hydrogen bonding and aromatic-stacking interactions. Conventional MD simulations indicate that both C60 and C60(OH)8 weaken the interactions within hIAPP protofibril and disrupt the ß-sheet structure. These results provide mechanistic insights into the possible inhibitory mechanism of C60 and C60(OH)8 toward hIAPP aggregation, and they are of great reference value for the screening of potent amyloid inhibitors.

Molecular Dynamics Simulations Reveal the Inhibitory Mechanism of Dopamine against Human Islet Amyloid Polypeptide (hIAPP) Aggregation and Its Destabilization Effect on hIAPP Protofibrils.

The aberrant self-assembly of human islet amyloid polypeptide (hIAPP) into toxic oligomers, protofibrils, and mature fibrils is associated with the pathogenesis of type 2 diabetes (T2D). Inhibition of hIAPP aggregation and destabilization of preformed hIAPP fibrils are considered as two major therapeutic strategies for treating T2D. Previous experimental studies reported that dopamine prevented the formation of hIAPP oligomers and fibrils. However, the underlying inhibitory mechanism at the atomic level remains elusive. Herein we investigated the conformational ensembles of hIAPP dimer with and without dopamine using replica-exchange molecular dynamics simulations. The simulations demonstrated that dopamine preferentially bound to R11, L12, F15, H18, F23, I26, L27, and Y37 residues, inhibited the formation of ß-sheets in the amyloidogenic regions spanning residues 11RLANFLVH18, 22NFGAIL27, and 30TNVGSNT36, and resulted in more disordered hIAPP dimers, thus hindering the amyloid formation of hIAPP. Protonated and deprotonated dopamine molecules displayed distinct binding capabilities but bound to similar residue sites on hIAPP. Additional microsecond molecular dynamics simulations showed that dopamine mainly bound to the ß1 and turn regions of hIAPP protofibril and destabilized the protofibril structure. This study not only revealed the molecular mechanism of dopamine toward the inhibition of hIAPP aggregation but also demonstrated the protofibril-destabilizing effects of dopamine, which may be helpful for the design of drug candidates to treat T2D.