Structural diversity in the membrane-bound hIAPP dimer correlated with distinct membrane disruption mechanisms.

Amyloid deposits of the human islet amyloid polypeptide (hIAPP) have been identified in 90% of patients with type II diabetes. Cellular membranes accelerate the hIAPP fibrillation, and the integrity of membranes is also disrupted at the same time, leading to the apoptosis of ß cells in pancreas. The molecular mechanism of hIAPP-induced membrane disruption, especially during the initial membrane disruption stage, has not been well understood yet. Herein, we carried out extensive all-atom molecular dynamics simulations investigating the hIAPP dimerization process in the anionic POPG membrane, to provide the detailed molecular mechanisms during the initial hIAPP aggregation stage in the membrane environment. Compared to the hIAPP monomer on the membrane, we observed not only an increase of α-helical structures, but also a substantial increase of ß-sheet structures upon spontaneous dimerization. Moreover, the random coiled and α-helical dimer structures insert deep into the membrane interior with a few inter-chain contacts at the C-terminal region, while the ß-sheet-rich structures reside on the membrane surface accompanied by strong inter-chain hydrophobic interactions. The coexistence of α and ß structures constitutes a diverse structural ensemble of the membrane-bound hIAPP dimer. From α-helical to ß-sheet structures, the degree of membrane disruption decreases gradually, and thus the membrane damage induced by random coiled and α-helical structures precedes that induced by ß-sheet structures. We speculate that insertion of random coiled and α-helical structures contributes to the initial stage of membrane damage, while ß-sheet structures on the membrane surface are more involved in the later stage of fibril-induced membrane disruption.

Influence of ALS-linked M337V mutation on the conformational ensembles of TDP-43321-340 peptide monomer and dimer.

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/ß-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 ß-sheet formation, albeit with a minor impact on the average probability of both helix structure and ß-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-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.

Structural Perturbation of Monomers Determines the Amyloid Aggregation Propensity of Calcitonin Variants.

Human calcitonin (hCT) is a polypeptide hormone that participates in calcium-phosphorus metabolism. Irreversible aggregation of 32-amino acid hCT into ß-sheet-rich amyloid fibrils impairs physiological activity and increases the risk of medullary carcinoma of the thyroid. Amyloid-resistant hCT derivatives substituting critical amyloidogenic residues are of particular interest for clinical applications as therapeutic drugs against bone-related diseases. Uncovering the aggregation mechanism of hCT at the molecular level, therefore, is important for the design of amyloid-resistant hCT analogues. Here, we investigated the aggregation dynamics of hCT, non-amyloidogenic salmon calcitonin (sCT), and two hCT analogues with reduced aggregation tendency─TL-hCT and phCT─using long timescale discrete molecular dynamics simulations. Our results showed that hCT monomers mainly adopted unstructured conformations with dynamically formed helices around the central region. hCT self-assembled into helix-rich oligomers first, followed by a conformational conversion into ß-sheet-rich oligomers with ß-sheets formed by residues 10-30 and stabilized by aromatic and hydrophobic interactions. Our simulations confirmed that TL-hCT and phCT oligomers featured more helices and fewer ß-sheets than hCT. Substitution of central aromatic residues with leucine in TL-hCT and replacing C-terminal hydrophobic residue with hydrophilic amino acid in phCT only locally suppressed ß-sheet propensities in the central region and C-terminus, respectively. Having mutations in both central and C-terminal regions, sCT monomers and dynamically formed oligomers predominantly adopted helices, confirming that both central aromatic and C-terminal hydrophobic residues played important roles in the fibrillization of hCT. We also observed the formation of ß-barrel intermediates, postulated as the toxic oligomers in amyloidosis, for hCT but not for sCT. Our computational study depicts a complete picture of the aggregation dynamics of hCT and the effects of mutations. The design of next-generation amyloid-resistant hCT analogues should consider the impact on both amyloidogenic regions and also take into account the amplification of transient ß-sheet population in monomers upon aggregation.

Amyloid Oligomers: A Joint Experimental/Computational Perspective on Alzheimer's Disease, Parkinson's Disease, Type II Diabetes, and Amyotrophic Lateral Sclerosis.

Protein misfolding and aggregation is observed in many amyloidogenic diseases affecting either the central nervous system or a variety of peripheral tissues. Structural and dynamic characterization of all species along the pathways from monomers to fibrils is challenging by experimental and computational means because they involve intrinsically disordered proteins in most diseases. Yet understanding how amyloid species become toxic is the challenge in developing a treatment for these diseases. Here we review what computer, in vitro, in vivo, and pharmacological experiments tell us about the accumulation and deposition of the oligomers of the (Aß, tau), α-synuclein, IAPP, and superoxide dismutase 1 proteins, which have been the mainstream concept underlying Alzheimer's disease (AD), Parkinson's disease (PD), type II diabetes (T2D), and amyotrophic lateral sclerosis (ALS) research, respectively, for many years.

Naphthoquinone-dopamine hybrids disrupt α-synuclein fibrils by their intramolecular synergistic interactions with fibrils and display a better effect on fibril disruption.

α-Synuclein (αSyn) is an intrinsically disordered protein and its abnormal aggregation into amyloid fibrils is the main hallmark of Parkinson's disease (PD). The disruption of preformed αSyn fibrils using small molecules is considered as a potential strategy for PD treatment. Recent experiments have reported that naphthoquinone-dopamine hybrids (NQDA), synthesized by naphthoquinone (NQ) and dopamine (DA) molecules, can significantly disrupt αSyn fibrils and cross the blood-brain barrier. To unravel the fibril-disruptive mechanisms at the atomic level, we performed microsecond molecular dynamics simulations of αSyn fibrils in the absence and presence of NQDA, NQ, DA, or NQ+DA molecules. Our simulations showed that NQDA reduces the ß-sheet content, disrupts K45-E57 and E46-K80 salt-bridges, weakens the inter-protofibril interaction, and thus destabilizes the αSyn fibril structure. NQDA has the ability to form cation-π and H-bonding interactions with K45/K80, and form π-π stacking interactions with Y39/F94. Those interactions between NQDA and αSyn fibrils play a crucial role in disaggregating αSyn fibrils. Moreover, we found that NQDA has a better fibril destabilization effect than that of NQ, DA, and NQ+DA molecules. This is attributed to the synergistic fibril-binding effect between NQ and DA groups in NQDA molecules. The DA group can form strong π-π stacking interactions with aromatic residues Y39/F94 of the αSyn fibril, while the DA molecule cannot. In addition, NQDA can form stronger cation-π interactions with residues K45/K80 than those of both NQ and DA molecules. Our results provide the molecular mechanism underlying the disaggregation of the αSyn fibril by NQDA and its better performance in fibril disruption than NQ, DA, and NQ+DA molecules, which offers new clues for the screening and development of promising drug candidates to treat PD.

Destabilization mechanism of R3-R4 tau protofilament by purpurin: a molecular dynamics study.

The accumulation of tau protein aggregates is a common feature observed in many neurodegenerative diseases. However, the structural characteristics of tau aggregates can vary among different tauopathies. It has been established that the structure of the tau protofilament in Chronic traumatic encephalopathy (CTE) is similar to that of Alzheimer's disease (AD). In addition, a previous study found that purpurin, an anthraquinone, could inhibit and disassemble the pre-formed 306VQIVYK311 isoform of AD-tau protofilament. Herein, we used all-atom molecular dynamic (MD) simulation to investigate the distinctive features between CTE-tau and AD-tau protofilament and the influence of purpurin on CTE-tau protofilament. Our findings revealed notable differences at the atomic level between CTE-tau and AD-tau protofilaments, particularly in the ß6-ß7 angle and the solvent-accessible surface area (SASA) of the ß4-ß6 region. These structural disparities contributed to the distinct characteristics observed in the two types of tau protofilaments. Our simulations substantiated that purpurin could destabilize the CTE-tau protofilament and decrease ß-sheet content. Purpurin molecules could insert the ß4-ß6 region and weaken the hydrophobic packing between ß1 and ß8 through π-π stacking. Interestingly, each of the three rings in purpurin exhibited unique binding preferences with the CTE-tau protofilament. Overall, our study sheds light on the structural distinctions between CTE-tau and AD-tau protofilaments, as well as the destabilizing mechanism of purpurin on CTE-tau protofilament, which may be helpful to the development of drugs to prevent CTE.

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.

Molecular Insights into the Self-Assembly of Block Copolymer Suckerin Polypeptides into Nanoconfined ß-Sheets.

Suckerin in squid sucker ring teeth is a block-copolymer peptide comprised of two repeating modules-the alanine and histidine-rich M1 and the glycine-rich M2. Suckerin self-assemblies display excellent thermo-plasticity and pH-responsive properties, along with the high biocompatibility, biodegradability, and sustainability. However, the self-assembly mechanism and the detailed role of each module are still elusive, limiting the capability of applying and manipulating such biomaterials. Here, the self-assembly dynamics of the two modules and two minimalist suckerin-mimetic block-copolymers, M1-M2-M1 and M2-M1-M2, in silico is investigated. The simulation results demonstrate that M2 has a stronger self-association but weaker ß-sheet propensities than M1. The high self-assembly propensity of M2 allows the minimalist block-copolymer peptides to coalesce with microphase separation, enabling the formation of nanoconfined ß-sheets in the matrix formed by M1-M2 contacts. Since these glycine-rich fragments with scatted hydrophobic and aromatic residues are building blocks of many other block-copolymer peptides, the study suggests that these modules function as the "molecular glue" in addition to the flexible linker or spacer to drive the self-assembly and microphase separation. The uncovered molecular insights may help understand the structure and function of suckerin and also aid in the design of functional block-copolymer peptides for nanotechnology and biomedicine applications.

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.

Exploiting Minimalistic Backbone Engineered γ-Phenylalanine for the Formation of Supramolecular Co-Polymer.

Ordered supramolecular hydrogels assembled by modified aromatic amino acids often exhibit low mechanical rigidity. Aiming to stabilize the hydrogel and understand the impact of conformational freedom and hydrophobicity on the self-assembly process, two building blocks based on 9-fluorenyl-methoxycarbonyl-phenylalanine (Fmoc-Phe) gelator which contain two extra methylene units in the backbone, generating Fmoc-γPhe and Fmoc-(3-hydroxy)-γPhe are designed. Fmoc-γPhe spontaneously assembled in aqueous media forming a hydrogel with exceptional mechanical and thermal stability. Moreover, Fmoc-(3-hydroxy)-γPhe, with an extra backbone hydroxyl group decreasing its hydrophobicity while maintaining some molecular flexibility, self-assembled into a transient fibrillar hydrogel, that later formed microcrystalline aggregates through a phase transition. Molecular dynamics simulations and single crystal X-ray analyses reveal the mechanism underlying the two residues' distinct self-assembly behaviors. Finally, Fmoc-γPhe and Fmoc-(3-OH)-γPhe co-assembly to form a supramolecular hydrogel with notable mechanical properties are demonstrated. It has been believed that the understanding of the structure-assembly relationship will enable the design of new functional amino acid-based hydrogels.

Distinct binding interactions trigger opposite conformational modulations on pathogenic and wildtype Huntingtin exon 1 proteins.

Abnormal elongation of the polyglutamine tract transforms exon 1 of the Huntingtin protein (Htt-exon-1) from wildtype to pathogenic form, and causes Huntington's disease. As an intrinsically disordered protein, the structural ensemble of Htt-exon-1 is highly heterogeneous and the detailed conformation of toxic species is still under debate. Ispinesib, a potential small-molecule drug, has been identified to selectively link the pathogenic Htt-exon-1 into the autophagosome to degrade, thus opening an innovative therapeutic direction. However, the molecular mechanisms behind this selectivity remain largely elusive. Herein, we carry out extensive molecular dynamics simulations with an enhanced sampling method to investigate the ispinesib inducing conformational changes of pathogenic and wildtype Htt-exon-1 and the corresponding binding mechanisms. Our simulations reveal that the ispinesib binding induces opposite conformational changes in pathogenic and wildtype Htt-exon-1, i.e., the 'entropy collapse' with significant reduction of ß-sheets versus the 'entropy expansion' with a slight increase of α-helices. Network analyses further reveal that there are two stable binding sites in the pathogenic Htt-exon-1, while the binding on the wildtype Htt-exon-1 is highly dynamic and non-specific. These dramatic differences originate from the underlying distinct binding interactions. More specifically, stronger hydrogen bonds serve as the specific binding anchors in pathogenic Htt-exon-1, while stronger hydrophobic interactions dominate in the dynamic binding with wildtype Htt-exon-1. Our simulations provide an atomistic mechanism for the ispinesib selective binding on the pathogenic Htt-exon-1, and further shed light on the general mechanisms of small molecule modulation on intrinsically disordered proteins.

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.

Mechanistic insight into the destabilization of p53TD tetramer by cancer-related R337H mutation: a molecular dynamics study.

The p53 protein is a tumor suppressor crucial for cell cycle and genome integrity. In a very large proportion of human cancers, p53 is frequently inactivated by mutations located in its DNA-binding domain (DBD). Some experimental studies reported that the inherited R337H mutation located in the p53 tetramerization domain (p53TD) can also result in destabilization of the p53 protein, and consequently lead to an organism prone to cancer setup. However, the underlying R337H mutation-induced structural destabilization mechanism is not well understood. Herein, we investigate the structural stability and dynamic property of the wild type p53TD tetramer and its cancer-related R337H mutant by performing multiple microsecond molecular dynamics simulations. It is found that R337H mutation destroys the R337-D352 hydrogen bonds, weakens the F341-F341 π-π stacking interaction and the hydrophobic interaction between aliphatic hydrocarbons of R337 and M340, leading to more solvent exposure of all the hydrophobic cores, and thus disrupting the structural integrity of the tetramer. Importantly, our simulations show for the first time that R337H mutation results in unfolding of the α-helix starting from the N-terminal region (residues 335RER(H)FEM340). Consistently, community network analyses reveal that R337H mutation reduces dynamical correlation and global connectivity of p53TD tetramer, which destabilizes the structure of the p53TD tetramer. This study provides the atomistic mechanism of R337H mutation-induced destabilization of p53TD tetramer, which might be helpful for in-depth understanding of the p53 loss-of-function mechanism.

Molecular insights into the oligomerization dynamics and conformations of amyloidogenic and non-amyloidogenic amylin from discrete molecular dynamics simulations.

The amyloid aggregation of human islet amyloid polypeptide (hIAPP) is associated with pancreatic ß-cell death in type 2 diabetes. The S20G substitution of hIAPP (hIAPP(S20G)), found in Japanese and Chinese people, is more amyloidogenic and cytotoxic than wild-type hIAPP. Rat amylin (rIAPP) does not have aggregation propensity or cytotoxicity. Mounting evidence suggests that soluble low-molecular-weight amyloid oligomers formed during early aggregation are more cytotoxic than mature fibrils. The self-assembly dynamics and oligomeric conformations remain unknown because the oligomers are heterogeneous and transient. The molecular mechanism of sequence-variation rendering dramatically different aggregation propensity and cytotoxicity is also elusive. Here, we investigate the oligomerization dynamics and conformations of amyloidogenic hIAPP, hIAPP(S20G), and non-amyloidogenic rIAPP using atomistic discrete molecular dynamics (DMD) simulations. Our simulation results demonstrated that all three monomeric amylin peptides mainly adopted an unstructured formation with partial dynamical helices near the N-terminus. Relatively transient ß-hairpins were more abundant in hIAPP and hIAPP(S20G) than in rIAPP. The S20G-substituting mutant of hIAPP altered the turn region of the ß-hairpin motif, resulting in more hydrophobic residue-pairwise contacts within the ß-hairpin. Oligomerization dynamic investigation revealed that all three peptides spontaneously accumulated into helix-populated oligomers. The conformational conversion to form ß-sheet-rich oligomers was only observed in hIAPP and hIAPP(S20G). The population of high-ß-sheet-content oligomers was enhanced by S20G substitution. Interestingly, both hIAPP and hIAPP(S20G) could form ß-barrel formations, and the ß-barrel propensity of hIAPP(S20G) was three times larger than that of hIAPP. No ß-sheet-rich or ß-barrel formations were observed in rIAPP. Our direct observation of the correlation between ß-barrel oligomer formation and cytotoxicity suggests that ß-barrels might play a critically important role in the cytotoxicity of amyloidosis.

The destructive mechanism of Aß1-42 protofibrils by norepinephrine revealed via molecular dynamics simulations.

Amyloid-ß (Aß) fibrillary plaques represent the main hallmarks of Alzheimer's disease (AD), in addition to tau neurofibrillary tangles. Disrupting early-formed Aß protofibrils is considered to be one of the primary therapeutic strategies to interfere with AD. Our previous work showed that norepinephrine (NE), an important neurotransmitter in the brain, can effectively inhibit the aggregation of the Aß1-42 peptide. However, whether and how NE molecules disassemble Aß1-42 protofibrils remains to be elucidated. Herein we investigate the influence of NE (in protonated and deprotonated states) on the recently cryo-EM solved LS-shaped Aß1-42 protofibrils and the underlying molecular mechanism by performing all-atom molecular dynamics simulations. Our simulations showed that protonated and deprotonated NE exhibited distinct disruptive mechanisms on Aß1-42 protofibrils. Protonated NE could significantly disrupt the N-terminal (residues D1-H14) structure of Aß1-42 protofibrils and destabilize the global structure of the protofibril. It preferentially bound with N-terminal residues of Aß1-42 protofibrils and formed hydrogen bonds with E3, D7, E11, Q15, E22, and D23 residues and π-π stackings with H6, H13, and F20 residues, and thus destroyed the hydrogen bonds between H6 and E11 and increased the kink angle around Y10. Compared to protonated NE, deprotonated NE displayed a higher disruptive capability on Aß1-42 protofibrils, and stronger hydrophobic and π-π stacking interactions with the protofibril structure. This study revealed the molecular mechanism of NE in the destruction of Aß1-42 protofibrils, which may be helpful in the design of potent drug candidates against AD.

Expanding the Structural Diversity and Functional Scope of Diphenylalanine-Based Peptide Architectures by Hierarchical Coassembly.

Modulation of the structural diversity of diphenylalanine-based assemblies by molecular modification and solvent alteration has been extensively explored for bio- and nanotechnology. However, regulation of the structural transition of assemblies based on this minimal building block into tunable supramolecular nanostructures and further construction of smart supramolecular materials with multiple responsiveness are still an unmet need. Coassembly, the tactic employed by natural systems to expand the architectural space, has been rarely explored. Herein, we present a coassembly approach to investigate the morphology manipulation of assemblies formed by N-terminally capped diphenylalanine by mixing with various bipyridine derivatives through intermolecular hydrogen bonding. The coassembly-induced structural diversity is fully studied by a set of biophysical techniques and computational simulations. Moreover, multiple-responsive two-component supramolecular gels are constructed through the incorporation of functional bipyridine molecules into the coassemblies. This study not only depicts the coassembly strategy to manipulate the hierarchical nanoarchitecture and morphology transition of diphenylalanine-based assemblies by supramolecular interactions but also promotes the rational design and development of smart hydrogel-based biomaterials responsive to various external stimuli.

Molecular mechanisms of resveratrol and EGCG in the inhibition of Aß42 aggregation and disruption of Aß42 protofibril: similarities and differences.

The aggregation of amyloid-ß protein (Aß) into fibrillary deposits is implicated in Alzheimer's disease (AD), and inhibiting Aß aggregation and clearing Aß fibrils are considered as promising strategies to treat AD. It has been reported that resveratrol (RSV) and epigallocatechin-3-gallate (EGCG), two of the most extensively studied natural polyphenols, are able to inhibit Aß fibrillization and remodel the preformed fibrillary aggregates into amorphous, non-toxic species. However, the mechanisms by which RSV inhibits Aß42 aggregation and disrupts Aß42 protofibril, as well as the inhibitory/disruptive mechanistic similarities and differences between RSV and EGCG, remain mostly elusive. Herein, we performed extensive all-atom molecular dynamics (MD) simulations on Aß42 dimers (the early aggregation state of Aß42) and protofibrils (the intermediate of Aß42 fibril formation and elongation) in the absence/presence of RSV or EGCG molecules. Our simulations show that both RSV and EGCG can bind with Aß42 monomers and inhibit the dimerization of Aß42. The binding of RSV with Aß42 peptide is mostly viaπ-π stacking interactions, while the binding of EGCG with Aß42 is mainly through hydrophobic, π-π stacking, and hydrogen-bonding interactions. Moreover, both RSV and EGCG disrupt the ß-sheet structure and K28-A42 salt bridges, leading to a disruption of Aß42 protofibril structure. RSV mainly binds with residues whose side-chains point inwards from the surface of the protofibril, while EGCG mostly binds with residues whose side-chains point outwards from the surface of the protofibril. Furthermore, RSV interacts with Aß42 protofibrils mostly viaπ-π stacking interactions, while EGCG interacts with Aß42 protofibrils mainly via hydrogen-bonding and hydrophobic interactions. For comparison, we also explore the effects of RSV/EGCG molecules on the aggregation inhibition and protofibril disruption of the Iowa mutant (D23N) Aß. Our findings may pave the way for the design of more effective drug candidates as well as the utilization of cocktail therapy using RSV and EGCG for the treatment of AD.

Heparin remodels the microtubule-binding repeat R3 of Tau protein towards fibril-prone conformations.

Abnormal aggregation of proteins into pathological amyloid fibrils is implicated in a wide range of devastating human neurodegenerative diseases. Intracellular fibrillary inclusions formed by Tau protein are characterized as the hallmark of tauopathies, including Alzheimer's disease and frontotemporal dementia. Heparin has been often used to trigger Tau aggregation in in vitro studies. However, the conformational changes induced by heparin and the underlying mechanism of promotion of Tau aggregation by heparin are not well understood. Structural characterization of Tau oligomers in the early stage of fibrillation is of great importance but remains challenging due to their dynamic and heterogeneous nature. R3, the third microtubule-binding repeat of Tau, contains the fibril-nucleating core (PHF6) and is crucial for Tau aggregation. In this study, utilizing extensive all-atom replica-exchange molecular dynamic simulations, we explored the conformational ensembles of R3 monomer/dimer in the absence and presence of heparin. Our results show that without heparin, both monomeric and dimeric R3 preferentially adopt collapsed ß-sheet-containing conformations and PHF6 plays an important role in the formation of interchain ß-sheet structures, while in the presence of heparin, R3 can populate relatively extended disordered states where chain dimension is similar to that of R3 in Tau filaments. Through electrostatic, hydrogen-bonding and hydrophobic interactions, heparin has a preference for interacting with residues V306/Q307/K317/K321/H329/H330/K331 which distribute throughout the entire sequence of R3, in turn acting as a template to extend R3 conformations. More importantly, heparin alters intramolecular/intermolecular interaction patterns of R3 and increases the intermolecular contact regions. Our results suggest that heparin remodels the conformations of R3 towards fibril-prone structures by increasing chain dimension and intermolecular contact regions, which may shed light on the atomic mechanism of heparin-induced amyloid fibrillization of Tau protein.

Conformational stability and dynamics of the cancer-associated isoform Δ133p53ß are modulated by p53 peptides and p53-specific DNA.

p53 is a tumor suppressor protein that maintains genome stability, but its Δ133p53ß and Δ160p53ß isoforms promote breast cancer cell invasion. The sequence truncations in the p53 core domain raise key questions related to their physicochemical properties, including structural stabilities, interaction mechanisms, and DNA-binding abilities. Herein, we investigated the conformational dynamics of Δ133p53ß and Δ160p53ß with and without binding to p53-specific DNA by using molecular dynamics simulations. We observed that the core domains of the 2 truncated isoforms are much less stable than wild-type (wt) p53ß, and the increased solvent exposure of their aggregation-triggering segment indicates their higher aggregation propensities than wt p53. We also found that Δ133p53ß stability is modulable by peptide or DNA interactions. Adding a p53 peptide (derived from truncated p53 sequence 107-129) may help stabilize Δ133p53. Most importantly, our simulations of p53 isomer-DNA complexes indicate that Δ133p53ß dimer, but not Δ160p53ß dimer, could form a stable complex with p53-specific DNA, which is consistent with recent experiments. This study provides physicochemical insight into Δ133p53ß, Δ133p53ß-DNA complexes, Δ133p53ß's pathologic mechanism, and peptide-based inhibitor design against p53-related cancers.-Lei, J., Qi, R., Tang, Y., Wang, W., Wei, G., Nussinov, R., Ma, B. Conformational stability and dynamics of the cancer-associated isoform Δ133p53ß are modulated by p53 peptides and p53-specific DNA.