Driving forces and structural determinants of steric zipper peptide oligomer formation elucidated by atomistic simulations

Understanding the structural and energetic requirements of non-fibrillar oligomer formation harbors the potential to decipher an important yet still elusive part of amyloidogenic peptide and protein aggregation. Low-molecular-weight oligomers are described to be transient and polymorphic intermediates in the nucleated self-assembly process to highly ordered amyloid fibers and were additionally found to exhibit a profound cytotoxicity. However, detailed structural information on the oligomeric species involved in the nucleation cannot be readily inferred from experiments. Here, we study the spontaneous assembly of steric zipper peptides from the tau protein, insulin and α-synuclein with atomistic molecular dynamics simulations on the microsecond timescale. Detailed analysis of the forces driving the oligomerization reveals a common two-step process akin to a general condensation-ordering mechanism and thus provides a rational understanding of the molecular basis of peptide self-assembly. Our results suggest that the initial formation of partially ordered peptide oligomers is governed by the solvation free energy, whereas the dynamical ordering and emergence of β-sheets are mainly driven by optimized inter-peptide interactions in the collapsed state. A novel mapping technique based on collective coordinates is employed to highlight similarities and differences in the conformational ensemble of small oligomer structures. Elucidating the dynamical and polymorphic β-sheet oligomer conformations at atomistic detail furthermore suggests complementary sheet packing characteristics similar to steric zipper structures, but with a larger heterogeneity in the strand alignment pattern and sheet-to-sheet arrangements compared to the cross-β motif found in the fibrillar or crystalline states.     

Investigating the effects of N-terminal acetylation on KFE8 self-assembly with 2D IR spectroscopy

Peptide self-assembly is an exciting and robust approach to create novel nanoscale materials for biomedical applications. However, the complex interplay between intra- and intermolecular interactions in peptide aggregation means that minor changes in peptide sequence can yield dramatic changes in supramolecular structure. Here, we use two-dimensional infrared spectroscopy to study a model amphiphilic peptide, KFE8, and its N-terminal acetylated counterpart, AcKFE8. Two-dimensional infrared spectra of isotope-labeled peptides reveal that AcKFE8 aggregates comprise two distinct β-sheet structures although KFE8 aggregates comprise only one of these structures. Using an excitonic Hamiltonian to simulate the vibrational spectra of model β-sheets, we determine that the spectra are consistent with antiparallel β-sheets with different strand alignments, specifically a two-residue shift in the register of the β-strands. These findings bring forth new insights into how N-terminal acetylation may subtly impact secondary structure, leading to larger effects on overall aggregate morphology. In addition, these results highlight the importance of understanding the residue-level structural differences that result from changes in peptide sequence to facilitate the rational design of peptide materials.     

Inhibition of amyloid fibril formation and disassembly of pre-formed fibrils by natural polyphenol rottlerin

Natural polyphenols, curcumin, rottlerin and EGCG were selected for initial computational modeling of protein-ligand interaction patterns. The docking calculations demonstrated that these polyphenols can easily adjust their conformational shape to fit well into the binding sites of amyloidogenic proteins. The experimental part of the study focused on the effect of rottlerin on fibrillation of three distinct amyloidogenic proteins, namely insulin, lysozyme and Aβ_1–40 peptide. Different experimental protocols such as fluorescence spectroscopy, circular dichroism and atomic force microscopy, demonstrated that amyloid fibril formation of any of the three proteins is inhibited by low micromolar rottlerin concentrations. Most likely, the inhibition of amyloid formation proceeded via interaction of rottlerin with amyloidogenic regions of the studied proteins. Moreover, rottlerin was also effective in pre-formed fibrils disassembly, suggesting that interactions of rottlerin with fibrils were capable to interrupt the fibril-stabilizing bonds of β-sheets. The apparent IC₅₀ and DC₅₀ values were calculated in the range of 1.3–36.4 μM and 15.6–25.8 μM, respectively. The strongest inhibiting/disassembling effect of rottlerin was observed on Aβ_1–40 peptide. The cytotoxicity assay performed on the Neuro 2a cells indicated time-dependent cell morphology changes but rottlerin affected the cell viability only at concentration above 50 μM. The results of this study suggest that chemical modifications on rottlerin could be tested in the future as a promising strategy for the modulation of amyloidogenic proteins aggregation.     

Intrinsic origin of amyloid aggregation: Behavior of histidine (εεε) and (δδδ) tautomer homodimers of Aβ (1–40)

Amyloid-beta protein (Aβ) accumulation in the brain, which is influenced by several factors, is a hallmark of Alzheimer's disease (AD). Despite the important role of histidine in stabilizing the fibrillar structure of the Aβ peptide at neutral pH, the effect of histidine tautomerism on Aβ peptide aggregation is still largely unknown. Histidine is in equilibrium between δ and ε tautomers and there are three histidine residues (H6, H13, and H14) in the Aβ(1–40) peptide. We performed molecular dynamics simulation on (δδδ) and (εεε) histidine tautomers with different initial homodimeric configurations to elucidate structural and aggregation features. Results indicate that (εεε) homodimers have very low propensity or almost no tendency to form β-sheets, whereas (δδδ) dimers predominantly form β-sheets due to interactions between central hydrophobic core (CHC) residues and C-terminal residues. β-sheet formation occurred in the same regions of each dimer chain at the CHC and C-/N- terminals for different configurations of (δδδ). These results suggest that (δδδ) has an important role in AD progression. Our study provides deeper insight into the effect of tautomerism of histidine residues in Aβ(1–40) on amyloid aggregation.     

Solid state self-assembly mechanism of RADA16-I designer peptide

We report that synthetic RADA16-I peptide transforms to β-strand secondary structure and develops intermolecular organization into β-sheets when stored in the solid state at room temperature. Secondary structural changes were probed using solid state nuclear magnetic resonance spectroscopy (ssNMR) and Fourier transform infrared spectroscopy (FTIR). Intermolecular organization was analyzed via wide-angle X-ray diffraction (WAXD). Observed changes in molecular structure and organization occurred on the time scale of weeks during sample storage at room temperature. We observed structural changes on faster time scales by heating samples above room temperature or by addition of water. Analysis of hydration effects indicates that water can enhance the ability of the peptide to convert to β-strand secondary structure and assemble into β-sheets. However, temperature dependent FTIR and time dependent WAXD data indicate that bound water may hinder the assembly of β-strands into β-sheets. We suggest that secondary structural transformation and intermolecular organization together produce a water-insoluble state. These results reveal insights into the role of water in self-assembly of polypeptides with hydrophilic side chains, and have implications on future optimization of RADA16-I nanofiber production.     

Molecular dynamics simulations reveal the disruption mechanism of a 2,4-thiazolidinedione derivative C30 against tau hexapeptide (PHF6) oligomer

Derivatives of 2,4-thiazolidinedione have been reported to inhibit the aggregation of tau protein, in which compound 30 (C30) not only inhibit 80% of paired helical filament 6 (PHF6) aggregation, but also inhibit K18 and full-length tau aggregation. However, its inhibitory mechanism is unclear. In this study, to investigate the effect of C30 on tau protein, all-atom molecular dynamics simulation was performed on the PHF6 oligomer with and without C30. The results show that C30 can cause significant conformational changes in the PHF6 oligomer. The nematic order parameter P2 and secondary structure analyses show that C30 destroys the ordered structure of PHF6 oligomer, reduces the content of β-sheet structure, and transforms β-sheet into random coil structure. By clustering analysis, it was found that C30 has four possible binding sites on the PFH6 oligomer, and the binding ability order is S1 > S2 > S4 > S3. Following a more in-depth analyses of each site, it was determined that the S1 site is the most possible binding site mainly located between layers of L1 and L3. The hydrophobic interaction is the driving force for the binding of C30 to PHF6 oligomer. In addition, L1P4_Y310, L1P5_Y310, L3P1_V309, and L3P2_V309 are key residues for C30 binding to oligomer. Moreover, π-π interaction formed by L1P4_Y310 and L1P5_Y310 with C30 and the hydrogen bonding interaction formed by C30 with L3P3_Q307 are beneficial to the combination of C30 and oligomer. The fully understanding disrupt the mechanism of 2,4-thiazolidinedione derivative on PHF6 oligomer and the identification of binding sites will help design and discover new AD inhibitors in the future.     

Stability of single sheet GNNQQNY aggregates analyzed by replica exchange molecular dynamics: antiparallel versus parallel association

Protein and peptide aggregation into amyloid plaques is associated with a large variety of neurodegenerative diseases. The definition of the molecular bases of these pathologies is hampered by the transient nature of pre-fibrillar small-oligomers that are considered the toxic species. The ability of the peptide GNNQQNY to form amyloid-like structures makes it a good model to investigate the complex processes involved into amyloid fiber formation. By employing full atomistic replica exchange molecular dynamics simulations, we constructed the free energy surface of small assemblies of GNNQQNY to gain novel insights into the fiber formation process. The calculations suggest that the peptide exhibits a remarkable tendency to form both parallel and antiparallel β-sheets. The data show that GNNQQNY preference for parallel or antiparallel β-sheets is governed by a subtle balance of factors including assemblies’ size, sidechain-sidechain interactions and pH. The samplings analysis provides a rationale to the observed trends.     

Positional effects of phosphorylation on the stability and morphology of tau-related amyloid fibrils

Hyperphosphorylated forms of tau protein are the main component of paired helical filaments (PHFs) of neurofibrillary tangles in the brain of Alzheimer's disease patients. To understand the effect of phosphorylation on the fibrillation of tau, we utilized tau-derived phosphorylated peptides. The V(306)QIVYK(311) sequence (PHF6) in the microtubule-binding domain is known to play a key role in the fibrillation of tau, and the short peptide corresponding to the PHF6 sequence forms amyloid-type fibrils similar to those generated by full-length tau. We focused on the amino acid residue located at the N-terminus of the PHF6 sequence, serine or lysine in the native isoform of tau, and synthesized the PHF6 derivative peptides with serine or lysine at the N-terminus of PHF6. Peptides phosphorylated at serine and/or tyrosine were synthesized to mimic the possible phosphorylation at these positions. The critical concentrations of the fibrillation of peptides were determined to quantitatively assess fibril stability. The peptide with the net charge of near zero tended to form stable fibrils. Interestingly, the peptide phosphorylated at the N-terminal serine residue exhibited remarkably low fibrillation propensity as compared to the peptide possessing the same net charge. Transmission electron microscopy measurements of the fibrils visualized the paired helical or straight fibers and segregated masses of the fibers or heterogeneous rodlike fibers depending on the phosphorylation status. Further analyses of the fibrils by the X-ray fiber diffraction method and Fourier transform infrared spectroscopic measurements indicated that all the peptides shared a common cross-β structure. In addition, the phosphoserine-containing peptides showed the characteristics of β-sandwiches that could interact with both faces of the β-sheet. On the basis of these observations, possible protofilament models with four β-sheets were constructed to consider the positional effects of the serine and/or tyrosine phosphorylations. The electrostatic intersheet interaction between phosphate groups and the amino group of lysine enhanced the lateral association between β-sheets to compensate for the excess charge. In addition to the previously postulated net charge of the peptide, the position of the charged residue plays a critical role in the amyloid fibrillation of tau.     

Characteristics of piraltin, a polyphenol concentrate, produced by freeze-drying of red wine

Moderate consumption of red wine is associated with a decreased risk for coronary heart disease. Apart from alcohol, an additive role for wine polyphenols has been suggested. However, the real contribution of these compounds can only be studied when available without the alcohol component. The objective of the study was to prepare a wine polyphenol concentrate not containing alcohol and to compare the quantitative and qualitative properties of this concentrate with those of the original wine from which the concentrate is made. This polyphenol concentrate, called piraltin, was made out of red wine by a freeze-drying technique. Both piraltin and the original red wine were analyzed quantitatively for the main polyphenols present: gallic acid, catechin, epicatechin and quercetin. The qualitative comparison comprised the inhibitory effect of the two products on LDL oxidation in vitro. In the process of freeze-drying recovery of the four determined flavonoids of red wine is fairly constant (average 68 +/- 7%). In a copper induced LDL oxidation assay both red wine and piraltin prolonged lag-times over 300% compared to controls without a significant difference between the two products. The freeze-dried polyphenol concentrate piraltin contains about 70% of the total polyphenol content of the original wine. This preparation technique does not cause a loss of antioxidative properties of the phenols. Piraltin creates the possibility to study the effects of wine polyphenols separately without the influence of alcohol both in vitro and in vivo.     

Temporary secondary structures in tau,an intrinsically disordered protein

The tau protein belongs to the category of intrinsically disordered proteins, which in their native state do not have an average stable structure and fluctuate between many conformations. In its physiological state, tau helps nucleating and stabilising the microtubules in the axons of the neurons. On the other hand, the same tau is involved in the development of Alzheimer disease, when it aggregates in paired helical filaments forming fibrils, which form insoluble tangles. The beginning of the pathological aggregation of tau has been attributed to a local transition of protein portions from random coil to a β-sheet. These structures would very likely be transient; therefore, we performed a molecular dynamics simulation of tau to gather information on the existence of segments of tau endowed with a secondary structure. We combined the results of our simulation with small-angle X-ray scattering experimental data to extract from the dynamics a set of most probable conformations of tau. The analysis of these conformations highlights the presence of transient secondary structures such as turns, β-bridges, β-sheets and α-helices. It also shows that a large segment of the N-terminal region is found near the repeats domain in a globular-like shape.     

Synergistic influence of phosphorylation and metal ions on tau oligomer formation and coaggregation with alpha-synuclein at the single molecule level

ABSTRACT: BACKGROUND: Fibrillar amyloid-like deposits and co-deposits of tau and alpha-synuclein are found in several common neurodegenerative diseases. Recent evidence indicates that small oligomers are the most relevant toxic aggregate species. While tau fibril formation is well-characterized, factors influencing tau oligomerization and molecular interactions of tau and alpha-synuclein are not well understood. RESULTS: We used a novel approach applying confocal single-particle fluorescence to investigate the influence of tau phosphorylation and metal ions on tau oligomer formation and its coaggregation with alpha-synuclein at the level of individual oligomers. We show that Al3+ at physiologically relevant concentrations and tau phosphorylation by GSK-3beta exert synergistic effects on the formation of a distinct SDS-resistant tau oligomer species even at nanomolar protein concentration. Moreover, tau phosphorylation and Al3+ as well as Fe3+ enhanced both formation of mixed oligomers and recruitment of alpha-synuclein in pre-formed tau oligomers. CONCLUSIONS: Our findings provide a new perspective on interactions of tau phosphorylation, metal ions, and the formation of potentially toxic oligomer species, and elucidate molecular crosstalks between different aggregation pathways involved in neurodegeneration.     

Role of peptide primary sequence in polyphenol–protein recognition: An example with neurotensin

Polyphenols are known for their impact on health and one of their major properties is the formation of complexes with proteins. To investigate the involvement of polyphenol–protein complexes in health, the interactions between bioactive polyphenols and neurotensin were examined by structural NMR and molecular modeling. Neurotensin is a linear bioactive tridecapeptide and polyphenols seem to affect the NT metabolism. We studied the polyphenols resveratrol and its glucoside the piceid in order to observe the possible role of glucose group and the penta-O-galloyl-d-glucopyranose (PGG). NMR data and molecular modeling showed that interaction occurred with the three polyphenols involving hydrophobic stacking and hydrogen bonds. Moreover, the peptide primary sequence plays a role in the specificity of complex formation.     

Molecular dynamics simulations to investigate the stability and aggregation behaviour of the amyloid-forming peptide VQIVYK from tau protein

The formation of paired helical filaments arising from the short hexapeptide in the third repeat of tau protein, ³⁰⁶VQIVYK³¹¹, is critical for tau polymerisation. The atomic structure of the VQIVYK oligomer has revealed a dry, tightly self-complementing structure between the neighbouring β-sheet layers, termed as ‘steric zipper’. In this study, several molecular dynamics simulations with all-atom explicit water were conducted to investigate the structural stability and aggregation behaviour of the VQIVYK peptide with various sizes and its single alanine replacement mutations. Our results indicate that the van der Waals interaction between side chains of Q2, the π–π stacking interaction between aromatic rings of Y5, and the electrostatic interaction between K6 and the C-terminus play an important role in stabilising the VQIVYK oligomers within the same β-sheet layer, while hydrophobic steric zipper involving V1, I3 and Y5 is responsible for holding the neighbouring β-sheet layers together. The twisted angles of the VQIVYK oligomers were also analysed and shown to be size dependent. The present results not only provide atomic insights into amyloid formation, but are also helpful for designing new or modified capping peptides and inhibitors to prevent fibril formation of the VQIVYK peptide from tau protein.     

Nucleation of β-rich oligomers and β-barrels in the early aggregation of human islet amyloid polypeptide

The self-assembly of human islet amyloid polypeptide (hIAPP) into β-sheet rich amyloid aggregates is associated with pancreatic β-cell death in type 2 diabetes (T2D). Prior experimental studies of hIAPP aggregation reported the early accumulation of α-helical intermediates before the rapid conversion into β-sheet rich amyloid fibrils, as also corroborated by our experimental characterizations with transmission electron microscopy and Fourier transform infrared spectroscopy. Although increasing evidence suggests that small oligomers populating early hIAPP aggregation play crucial roles in cytotoxicity, structures of these oligomer intermediates and their conformational conversions remain unknown, hindering our understanding of T2D disease mechanism and therapeutic design targeting these early aggregation species. We further applied large-scale discrete molecule dynamics simulations to investigate the oligomerization of full-length hIAPP, employing multiple molecular systems of increasing number of peptides. We found that the oligomerization process was dynamic, involving frequent inter-oligomeric exchanges. On average, oligomers had more α-helices than β-sheets, consistent with ensemble-based experimental measurements. However, in ~4–6% independent simulations, β-rich oligomers expected as the fibrillization intermediates were observed, especially in the pentamer and hexamer simulations. These β-rich oligomers could adopt β-barrel conformations, recently postulated to be the toxic oligomer species but only observed computationally in the aggregates of short amyloid protein fragments. Free-energy analysis revealed high energies of these β-rich oligomers, supporting the nucleated conformational changes of oligomers in amyloid aggregation. β-barrel oligomers of full-length hIAPP with well-defined three-dimensional structures may play an important pathological role in T2D etiology and may be a therapeutic target for the disease.     

Molecular Dynamics Simulations to Investigate the Structural Stability and Aggregation Behavior of the GGVVIA Oligomers Derived from Amyloid β Peptide

Abstract

Several neurodegenerative diseases, such as Alzheimer's, Parkinson's, and Huntington's dis-eases, are associated with amyloid fibrils formed by different polypeptides. Recently, the atomic structure of the amyloid-forming peptide GGVVIA from the C-terminal hydrophobic segment of amyloid-β (Aβ) peptide has been determined and revealed a dry, tightly self-com-plementing structure between two β-sheets, termed as “steric zipper”. In this study, several all-atom molecular dynamics simulations with explicit water were conducted to investigate the structural stability and aggregation behavior of the GGVVIA oligomers with various sizes. The results of our single-layer models suggested that the structural stability of the GGVVIA oligomers increases remarkably with increasing the numbers of β-strands. We fur-ther identified that SH2-ST2 may act as a stable seed in prompting amyloid fibril formations. Our results also demonstrated that hydrophobic interaction is the principle driving force to stabilize and associate the GGVVIA oligomers between β-strands; while the hydrophobic steric zipper formed via the side chains of V3, V4, and I5 plays a critical role in holding the two neighboring β-sheets together. Single glycine substitution at V3, V4, and I5 directly disrupted the hydrophobic steric zipper between these two β-sheets, resulting in the destabili-zation of the oligomers. Our simulation results provided detailed insights into understanding the aggregation behavior of the GGVVIA oligomers in the atomic level. It may also be help-ful for designing new inhibitors able to prevent the fibril formation of Aβ peptide.     

Direct observations of shifts in the β-sheet register of a protein-peptide complex using explicit solvent simulations

Using explicit solvent molecular dynamics simulations, we were able to obtain direct observations of shifts in the hydrogen-bonding register of an intermolecular β-sheet protein-peptide complex. The β-sheet is formed between the FHA domain of cancer marker protein Ki67 (Ki67FHA) and a peptide fragment of the hNIFK signaling protein. Potential encounter complexes of the Ki67FHA receptor and hNIFK peptide are misregistered states of the β-sheet. Rearrangements of one of these misregistered states to the native state were captured in three independent simulations. All three rearrangements occurred by a common mechanism: an aromatic residue of the peptide (F263) anchors into a transient hydrophobic pocket of the receptor to facilitate the formation of native hydrogen bonds. To our knowledge, these simulations provide the first atomically detailed visualizations of a mechanism by which nature might correct for errors in the alignment of intermolecular β-sheets.     

Copper binding properties of a tau peptide associated with Alzheimer's disease studied by CD, NMR, and MALDI-TOF MS

We have previously reported the copper binding properties of R3 peptide (residues 318-335: VTSKCGSLGNIHHKPGGG, according to the longest tau protein) derived from the third repeat microtubule-binding domain of water-soluble tau protein. In this work, we have investigated copper binding properties of R2 peptide (residues 287-304: VQSKCGSKDNIKHVPGGG) derived from the second repeat region of tau protein. Similar to R3 peptide, R2 peptide also plays an important role in the formation of neurofibrillary tangles (NFTs) which is one of the two main biological characteristics of Alzheimer's disease (AD). Based on the copper binding properties of R2 peptide, the possible influences of the binding on the formation of NFTs were investigated. Results from circular dichroism (CD) spectra, nuclear magnetic resonance (NMR) spectroscopy, and matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) suggest that the binding is pH-dependent and stoichiometry-determined. In addition, these results also reveal that R2 peptide adopts a monomeric alpha-helical structure in aqueous solutions at physiological pH after the addition of 1 mol equiv. of Cu2+. Since alpha-helix structure is responsible for the formation of paired helical filaments (PHFs) which aggregate into NFTs, it is hypothesized that Cu2+ induces R2 peptide to self-assemble into a PHFs-like structure. Hence, it is postulated that Cu2+ plays an important role in the aggregation of R2 peptide and tau protein and that copper binding to R2 peptide may be another possible involvement in AD.     

Molecular Simulation to Aid in the Understanding of the Aβ(1–42) Peptide of Alzheimer's Disease

Abstract

The Aβ(1–42) peptide of Alzheimer's disease was studied by molecular modeling. The coordinates of the peptide were experimentally generated from solution-NMR spectroscopy, and the conformations were energy minimized using a combination of connectivity-based iterative partial equalization of orbital electronegativity with the MM + force field.

There is a central folded domain in the Aβ peptide. This part is an apolar α-helix. The remaining residues form β-sheets. Aggregation requires that β-sheets interact by noncovalent bonding forces. The unsoluble, aggregated complexes are energetically stable and have ordered structures.

A perspective in drug research is to design compounds that inhibit the hydrophobic cores of the individual Aβ peptides, blocking so the associations between the β-strains.     

基于厚壳贻贝Mytilin-1的抗菌肽设计、固相合成及抗菌谱分析

贻贝抗菌肽Mytilin是贻贝免疫系统的重要组成部分,对其结构与功能的研究表明,其序列中连接两段β-折叠的发夹区域是其抗菌功能的关键所在。为验证该区域是否具有抗菌活性,通过对厚壳贻贝Mytilus coruscus抗菌肽Mytilin进行空间结构模拟,选取其中β-发夹部分肽段,采用了固相化学合成的方法合成了两条10肽,分别命名为Mytilin Derived Peptide-1(MDP-1)和Mytilin Derived Peptide-2(MDP-2)。高效液相色谱以及质谱检测结果表明,合成是成功的。抗菌谱研究表明,MDP-1和MDP-2对革兰氏阳性菌、阴性菌以及真菌均具有明显的抑制作用,同时,合成的MDP由于序列短且有两对二硫键,因此对于温度及人血浆均表现出很强的稳定性。上述研究结果为深入了解厚壳贻贝抗菌肽Mytilin的抗菌机制以及在此基础上开发具有应用价值的新型抗菌肽奠定了基础。