The formation of straight and twisted filaments from short tau peptides

We studied fibril formation in a family of peptides based on PHF6 (VQIVYK), a short peptide segment found in the microtubule binding region of tau protein. N-Acetylated peptides AcVYK-amide (AcVYK), AcIVYK-amide (AcPHF4), AcQIVYK-amide (AcPHF5), and AcV-QIVYK-amide (AcPHF6) rapidly formed straight filaments in the presence of 0.15 m NaCl, each composed of two laterally aligned protofilaments approximately 5 nm in width. X-ray fiber diffraction showed the omnipresent sharp 4.7-A reflection indicating that the scattering objects are likely elongated along the hydrogen-bonding direction in a cross-beta conformation, and Fourier transform IR suggested the peptide chains were in a parallel (AcVYK, AcPHF6) or antiparallel (AcPHF4, AcPHF5) beta-sheet configuration. The dipeptide N-acetyl-YK-amide (AcYK) formed globular structures approximately 200 nm to 1 microm in diameter. The polymerization rate, as measured by thioflavin S binding, increased with the length of the peptide going from AcYK --> AcPHF6, and peptides that aggregated most rapidly displayed CD spectra consistent with beta-sheet structure. There was a 3-fold decrease in rate when Val was substituted for Ile or Gln, nearly a 10-fold decrease when Ala was substituted for Tyr, and an increase in polymerization rate when Glu was substituted for Lys. Twisted filaments, composed of four laterally aligned protofilaments (9-19 nm width, approximately 90 nm half-periodicity), were formed by mixing AcPHF6 with AcVYK. Taken together these results suggest that the core of PHF6 is localized at VYK, and the interaction between small amphiphilic segments of tau may initiate nucleation and lead to filaments displaying paired helical filament morphology.     

Secondary nucleating sequences affect kinetics and thermodynamics of tau aggregation

Tau protein was scanned for highly amyloidogenic sequences in amphiphilic motifs (X)(n)Z, Z(X)(n)Z (n ≥ 2), or (XZ)(n) (n ≥ 2), where X is a hydrophobic residue and Z is a charged or polar residue. N-Acetyl peptides homologous to these sequences were used to study aggregation. Transmission electron microscopy (TEM) showed seven peptides, in addition to well-known primary nucleating sequences Ac(275)VQIINK (AcPHF6*) and Ac(306)VQIVYK (AcPHF6), formed fibers, tubes, ribbons, or rolled sheets. Of the peptides shown by TEM to form amyloid, Ac(10)VME, AcPHF6*, Ac(375)KLTFR, and Ac(393)VYK were found to enhance the fraction of β-structure of AcPHF6 formed at equilibrium, and Ac(375)KLTFR was found to inhibit AcPHF6 and AcPHF6* aggregation kinetics in a dose-dependent manner, consistent with its participation in a hybrid steric zipper model. Single site mutants were generated which transformed predicted amyloidogenic sequences in tau into non-amyloidogenic ones. A M11K mutant had fewer filaments and showed a decrease in aggregation kinetics and an increased lag time compared to wild-type tau, while a F378K mutant showed significantly more filaments. Our results infer that sequences throughout tau, in addition to PHF6 and PHF6*, can seed amyloid formation or affect aggregation kinetics or thermodynamics.     

Differential effects of phe19 and phe20 on fibril formation by amyloidogenic peptide Aβ16–22 (Ac‐KLVFFAE‐NH2)

The sequence KLVFFAE (Aβ16–22) in Alzheimer's β‐amyloid is thought to be a core β‐structure that could act as a template for folding other parts of the polypeptide or molecules into fibrillar assemblies rich in β‐sheet. To elucidate the mechanism of the initial folding process, we undertook combined X‐ray fiber/powder diffraction and infrared (IR) spectroscopy to analyze lyophilized Aβ16–22 and solubilized/dried peptide containing nitrile probes at F19 and/or F20. Solubilized/dried wild‐type (WT) Aβ16–22 and the peptide containing cyanophenylalanine at F19 (19CN) or at F20 (20CN) gave fiber patterns consistent with slab‐like β‐crystallites that were cylindrically averaged around the axis parallel to the polypeptide chain direction. The WT and 19CN assemblies showed 30‐Å period arrays arising from the stacking of the slabs along the peptide chain direction, whereas the 20CN assemblies lacked any such stacking. The electron density projection along the peptide chain direction indicated similar side‐chain dispositions for WT and 20CN, but not for 19CN. These X‐ray results and modeling imply that in the assembly of WT Aβ16–22 the F19 side chain is localized within the intersheet space and is involved in hydrophobic contact with amino acids across the intersheet space, whereas the F20 side chain localized near the slab surface is less important for the intersheet interaction, but involved in slab stacking. IR observations for the same peptides in dilute solution showed a greater degree of hydrogen bonding for the nitrile groups in 20CN than in 19CN, supporting this interpretation. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Structure of beta-crystallite assemblies formed by Alzheimer beta-amyloid protein analogues: analysis by x-ray diffraction.

To elucidate the relation between amyloid fibril formation in Alzheimer disease and the primary structure of the beta/A4 protein, which is the major component of the amyloid, we have been investigating the ability of peptides sharing sequences with beta/A4 to form fibrils in vitro. In previous studies we focused on the macroscopic morphology of the assemblies formed by synthetic peptides corresponding in sequence to different regions of this protein. In the present study we analyze the x-ray diffraction patterns obtained from these assemblies. All specimens showed wide angle reflections that could be indexed by an orthogonal lattice of beta-crystallites having unit cell dimensions a = 9.4 A, b = 7 A, and c = 10 A, where a refers to hydrogen bonding direction, b to polypeptide chain direction, and c to intersheet direction. Given the amino acid sequence of beta/A4 as NH2-DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIAT-COOH, we found that, based on their orientation and assembly, the analogues could be classified into three groups: Group A, residues 19-28, 13-28, 12-28, 11-28, 9-28, 1-28, 1-38, 1-40, 6-25, 11-25 and 34-42; Group B, residues 18-28, 17-28, and 15-28; and Group C, residues 22-35 and 26-33. For Groups A and C, the sharpest reflections were (h00), indicating that the assemblies were fibrillar, i.e., elongated in a single direction. Lateral alignment of the crystallites in Group A account for its cross-beta pattern, in which the hydrogen bonding (H-bonding) direction is the fiber (rotation) axis. By comparison, the beta-crystallites of Group C had no preferential orientation, thus giving circular scattering. For Group B, the sharpest reflections were (h0l) on the meridian, indicating that the assemblies were plate-like, i.e., extended in two directions. A series of equatorial Bragg reflections having a 40 A period indicated regular stacking of the plates, and the rotation axis was normal to the surface of the plates. Of the Group A peptides, the analogues 11-28 and 6-25 showed intensity maxima on the equator as well as on higher layer lines, indicating that the beta-crystallites are highly ordered relative to one another in the axial, H-bonding direction. This sampling of the layer lines by a larger period (60 A) suggests that the beta-crystallites are arrayed either in cylindrical or small restricted crystalline lattices. Consistent with its electron microscopic images, we modeled the structure as a tube with five or six f,-crystallites constituting the wall and with the individual crystallite, which either rotates freely or is restricted, made of five or fewer beta-pleated sheets. For the Group B peptides, the electron density projection along the b-axis was calculated from the observed intensities using phase combinations from fl-keratin.Amino acid side-chain positions were apparent and, when refined as 4-A-diameter spheres, led to a substantial decrease in the R-factors.For peptide 18-28 the electron density peaks, which are thought to correspond to side chains, were centered 3.3 A from the peptide backbone, whereas for peptides 17-28 and 15-28, these peaks were centered 1 A or more further from the backbone. Peaks having high electron density faced peaks having lower density, suggesting a favorable stereochemical arrangement of the residues. Thus, our analysis of the fiber x-ray patterns from beta/A4 peptides shows the organization of the beta-crystallites that form the wall of the amyloid fibrils as well as possible side-chain interactions.     

Alternative Conformations of the Tau Repeat Domain in Complex with an Engineered Binding Protein

The aggregation of Tau into paired helical filaments is involved in the pathogenesis of several neurodegenerative diseases, including Alzheimer disease. The aggregation reaction is characterized by conformational conversion of the repeat domain, which partially adopts a cross-β-structure in the resulting amyloid-like fibrils. Here, we report the selection and characterization of an engineered binding protein, β-wrapin TP4, targeting the Tau repeat domain. TP4 was obtained by phage display using the four-repeat Tau construct K18ΔK280 as a target. TP4 binds K18ΔK280 as well as the longest isoform of human Tau, hTau40, with nanomolar affinity. NMR spectroscopy identified two alternative TP4-binding sites in the four-repeat domain, with each including two hexapeptide motifs with high β-sheet propensity. Both binding sites contain the aggregation-determining PHF6 hexapeptide within repeat 3. In addition, one binding site includes the PHF6* hexapeptide within repeat 2, whereas the other includes the corresponding hexapeptide Tau(337–342) within repeat 4, denoted PHF6**. Comparison of TP4-binding with Tau aggregation reveals that the same regions of Tau are involved in both processes. TP4 inhibits Tau aggregation at substoichiometric concentration, demonstrating that it interferes with aggregation nucleation. This study provides residue-level insight into the interaction of Tau with an aggregation inhibitor and highlights the structural flexibility of Tau.     

Structural transitions in tau k18 on micelle binding suggest a hierarchy in the efficacy of individual microtubule‐binding repeats in filament nucleation

The protein tau is found in an aggregated filamentous state in the intraneuronal paired helical filament deposits characteristic of Alzheimer's disease and other related dementias and mutations in tau protein and mRNA cause frontotemproal dementia. Tau isoforms include a microtubule‐binding domain containing either three or four imperfect tandem microtubule binding repeats that also form the core of tau filaments and contain hexapaptide motifs that are critical for tau aggregation. The tau microtubule‐binding domain can also engage in direct interactions with detergents, fatty acids, or membranes, which can greatly facilitate tau aggregation and may also mediate some tau functions. Here, we show that the alternatively spliced second microtubule‐binding repeat exhibits significantly different structural characteristics compared with the other three repeats in the context of the intact repeat domain. Most notably, the PHF6* hexapeptide motif located at the N‐terminus of repeat 2 has a lower propensity to form strand‐like structure than the corresponding PHF6 motif in repeat 3, and unlike PHF6 converts to partially helical structure in the micelle‐bound state. Interestingly, the behavior of the Module‐B motif, located at the beginning of repeat 4, resembles that of PHF6* rather than PHF6. Our observations, combined with previous results showing that PHF6* and Module‐B are both less effective than PHF6 in nucleating tau aggregation, suggest a hierarchy in the efficacy of these motifs in nucleating tau aggregation that originates in differences in their intrinsic propensities for extended strand‐like structure and the resistance of these propensities to changes in tau's environment.     

Assemblies of Alzheimer's peptides A beta 25-35 and A beta 31-35: reverse-turn conformation and side-chain interactions revealed by X-ray diffraction

Alzheimer's beta amyloid protein (A beta) is a 39 to 43 amino acid peptide that is a major component in the neuritic plaques of Alzheimer's disease (AD). The assemblies constituted from residues 25-35 (A beta 25-35), which is a sequence homologous to the tachykinin or neurokinin class of neuropeptides, are neurotoxic. We used X-ray diffraction and electron microscopy to investigate the structure of the assemblies formed by A beta 25-35 peptides and of various length sequences therein, and of tachykinin-like analogues. Most solubilized peptides after subsequent drying produced diffraction patterns characteristic of beta-sheet structure. Moreover, the peptides A beta 31-35 (Ile-Ile-Gly-Leu-Met) and tachykinin analogue A beta(Phe(31))31-35 (Phe-Ile-Gly-Leu-Met) gave powder diffraction patterns to 2.8A Bragg spacing. The observed reflections were indexed by an orthogonal unit cell having dimensions of a=9.36 A, b=15.83 A, and c=20.10 A for the native A beta 31-35 peptide, and a=9.46 A, b=16.22 A, and c=11.06 A for the peptide having the Ile31Phe substitution. The initial model was a beta strand where the hydrogen bonding, chain, and intersheet directions were placed along the a, b, and c axes. An atomic model was fit to the electron density distribution, and subsequent refinement resulted in R factors of 0.27 and 0.26, respectively. Both peptides showed a reverse turn at Gly33 which results in intramolecular hydrogen bonding between the antiparallel chains. Based on previous reports that antagonists for the tachykinin substance P require a reverse turn, and that A beta is cytotoxic when it is oligomeric or fibrillar, we propose that the tachykinin-like A beta 31-35 domain is a turn exposed at the A beta oligomer surface where it could interact with the ligand-binding site of the tachykinin G-protein-coupled receptor.     

Morphology of self‐assembled structures formed by short peptides from the amyloidogenic protein tau depends on the solvent in which the peptides are dissolved

The aggregation behavior of peptides Ac‐VQIVYK‐amide (AcPHF6) and Ac‐QIVYK‐amide (AcPHF5) from the amyloidogenic protein tau was examined by atomic force microscopy (AFM) and fluorescence microscopy. Although AcPHF5 did not show enhancement of thioflavin T (ThT) fluorescence in aqueous buffer, distinct aggregates were discernible when peptide was dissolved in organic solvents such as methanol (MeOH), trifluoroethanol (TFE), and hexafluoroisopropanol (HFIP) dried on mica and examined by AFM. Self‐association was evident even though the peptide did not have the propensity to form secondary structures in the organic solvents. In dried films, the peptide adopts predominantly β‐conformation which results in the formation of distinct aggregates. ThT fluorescence spectra and fluorescence images indicate the formation of fibrils when AcPHF6 solutions in organic solvents were diluted into buffer. AcPHF6 had the ability to organize into fibrillar structures when AFM samples were prepared from peptide dissolved in MeOH, TFE, HFIP, and also when diluted into buffer. AcPHF6 showed propensity for β‐structure in aqueous buffer. In MeOH and TFE, AcPHF6 showed helical and β‐structure. Morphology of the fibrils was dependent on peptide conformation in the organic solvents. The structures observed for AcPHF6 are formed rapidly and long incubation periods in the solvents are not necessary. The structures with varying morphologies observed for AcPHF5 and AcPHF6 appear to be mediated by surfaces such as mica and the organic solvents used for dissolution of the peptides. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Characterization of two VQIXXK motifs for tau fibrillization in vitro

Tau proteins are building blocks of the filaments that form neurofibrillary tangles of Alzheimer's disease (AD) and related neurodegenerative tauopathies. It was recently reported that two VQIXXK motifs in the microtubule (MT) binding region, named PHF6 and PHF6*, are responsible for tau fibrillization. However, the exact role each of these motifs plays in this process has not been analyzed in detail. Using a recombinant human tau fragment containing only the four MT-binding repeats (K18), we show that deletion of either PHF6 or PHF6* affected tau assembly but only PHF6 is essential for filament formation, suggesting a critical role of this motif. To determine the amino acid residues within PHF6 that are required for tau fibrillization, a series of deletion and mutation constructs targeting this motif were generated. Deletion of VQI in either PHF6 or PHF6* lessened but did not eliminate K18 fibrillization. However, removal of the single K311 residue from PHF6 completely abrogated the fibril formation of K18. K311D mutation of K18 inhibited tau filament formation, while K311A and K311R mutations had no effect. These data imply that charge change at position 311 is important in tau fibril formation. A similar requirement of nonnegative charge at this position for fibrillization was observed with the full-length human tau isoform (T40), and data from these studies indicate that the formation of fibrils by T40K311D and T40K311P mutants is repressed at the nucleation phase. These findings provide important insights into the mechanisms of tau fibrillization and suggest targets for AD drug discovery to ameliorate neurodegeneration mediated by filamentous tau pathologies.     

Mutations of tau protein in frontotemporal dementia promote aggregation of paired helical filaments by enhancing local beta-structure

The microtubule-associated protein tau is a natively unfolded protein in solution, yet it is able to polymerize into the ordered paired helical filaments (PHF) of Alzheimer's disease. In the splice isoforms lacking exon 10, this process is facilitated by the formation of beta-structure around the hexapeptide motif PHF6 ((306)VQIVYK(311)) encoded by exon 11. We have investigated the structural requirements for PHF polymerization in the context of adult tau isoforms containing four repeats (including exon 10). In addition to the PHF6 motif there exists a related PHF6* motif ((275)VQIINK(280)) in the repeat encoded by the alternatively spliced exon 10. We show that this PHF6* motif also promotes aggregation by the formation of beta-structure and that there is a cross-talk between the two hexapeptide motifs during PHF aggregation. We also show that two of the tau mutations found in hereditary frontotemporal dementias, DeltaK280 and P301L, have a much stronger tendency for PHF aggregation which correlates with their high propensity for beta-structure around the hexapeptide motifs.     

Prediction of nucleating sequences from amyloidogenic propensities of tau-related peptides

Physical properties, including amyloid morphology, FTIR and CD spectra, enhancement of Congo red absorbance, polymerization rate, critical monomer concentration, free energy of stabilization, hydrophobicity, and the partition coefficient between soluble and amyloid states, were measured for the tau-related peptide Ac-VQIVYK amide (AcPHF6) and its single site mutants Ac-VQIVXK amide (X not equal Cys). Transmission electron microscopy showed that 15 out of the 19 peptides formed amyloid in buffer, with morphologies ranging from straight and twisted filaments to sheets and rolled sheets. Using principal component analysis (PCA), measured properties were treated in a comprehensive manner, and scores along the most significant principal components were used to define individual amino acid amyloidogenic propensities. Quantitative structure-activity modeling (QSAM) showed that residues with greater size and hydrophobicity made the largest contributions to the propensity of peptides to form amyloid. Using individual amino acid propensities, sequences within tau with high amyloid-forming potential were estimated and found to include 226VAVVR230 in the proline-rich region, 275VQIINK280 (PHF6) and 306VQIVYK311 (PHF6) within the microtubule binding region, and 392IVYK395 in the C-tail region of the protein. The results suggest that regions outside the microtubule-binding region may play important roles in tau aggregation kinetics or paired helical filament structure.     

Self Assembly of Short Aromatic Peptides into Amyloid Fibrils and Related Nanostructures

The formation of amyloid fibrils is the hallmark of more than twenty human disorders of unrelated etiology. In all these cases, ordered fibrillar protein assemblies with a diameter of 7–10 nm are being observed. In spite of the great clinical important of amyloidassociated diseases, the molecular recognition and self-assembly processes that lead to the formation of the fibrils are not fully understood. One direction to decipher the mechanism of amyloid formation is the use of short peptides fragments as model systems. Short peptide fragments, as short as pentapeptides, were shown to form typical amyloid assemblies in vitro that have ultrastructural, biophysical, and cytotoxic properties, as those of assemblies that are being formed by full length polypeptides. When we analyzed such short fragments, we identified the central role of aromatic moieties in the ability to aggregate into ordered nano-fibrillar structures. This notion allowed us to discover additional very short amyloidogenic peptides as well as other aromatic peptide motifs, which can form various assemblies at the nano-scale (including nanotubes, nanospheres, and macroscopic hydrogels with nano-scale order). Other practical utilization of this concept, together with novel β breakage methods, is their use for the development of novel classes of amyloid formation inhibitors.Key Words: Alzheimer''s disease, amyloid disease, molecular recognition, nanostructures, protein aggregation, protein misfolding, self-assembly, type II diabetes     

Fluorescence-coupled CD conformational monitoring of filament formation of tau microtubule-binding repeat domain

To clarify the contribution of the three- or four-repeated peptide moiety in tau microtubule-binding domain (MBD) to paired helical filament (PHF) formation, conformational transition accompanied by heparin-induced filament formation was investigated stepwise for four repeat peptides (R1-R4), one three-repeated R1-R3-R4 peptide (3RMBD), and one four-repeated R1-R2-R3-R4 peptide (4RMBD) using a combination of thioflavin S fluorescence and circular dichroism (CD) measurements in a neutral buffer (pH 7.6). The comparison of the fluorescence profile of each repeat peptide with those of 3RMBD and 4RMBD showed the synergistic contribution of R1-R4 to PHF formation of MBD. The CD spectrum measured as a function of filament formation time indicates that: (i) two conformational transitions occur for the filament formations of R3 (from the random structure to the beta-sheet structure) and 3RMBD (from the random structure to the alpha-helix structure), (ii) the filament formations of R2 and 4RMBD proceed via the synchronized conformational transitions of the alpha-helix and random structures, and (iii) the filament formation of 4RMBD is dependent on the aggregation behavior of R2. These data are useful for elucidating the MBD conformational transition in tau PHF formation.     

Structural changes in a hydrophobic domain of the prion protein induced by hydration and by ala-->Val and pro-->Leu substitutions

X-ray diffraction was used to study the structure of assemblies formed by synthetic peptide fragments of the prion protein (PrP) that include the hydrophobic domain implicated in the Gerstmann-Str?ussler-Scheinker (GSS) mutation (P102L). The effects of hydration on polypeptide assembly and of Ala-->Val substitutions in the hydrophobic domain were characterized. Synthetic peptides included: (i) Syrian hamster (SHa) hydrophobic core, SHa106-122 (KTNMKHMAGAAAAGAVV); (ii) SHa104-122(3A-V), with A-->V mutations at 113, 115 and 118 (KPKTNMKHMVGVAAVGAVV); (iii) mouse (Mo) wild-type sequence of the N-terminal hydrophobic domain, Mo89-143WT; and (iv) the same mouse sequence with leucine substitution for proline at residue number 101, Mo89-143(P101L). Samples of SHa106-122 that formed assemblies while drying under ambient conditions showed X-ray patterns indicative of 33 A thick slab-like structures having extensive H-bonding and intersheet stacking. By contrast, lyophilized peptide that was equilibrated against 100 % relative humidity showed assemblies with only a few layers of beta-sheets. The Ala-->Val substitutions in SHa104-122 and Mo89-143(P101L) resulted in the formation of 40 A wide, cross-beta fibrils. Observation of similar size beta-sheet fibrils formed by peptides SHa104-122(3A-V) and the longer Mo89-143(P101L) supports the notion that the hydrophobic sequence forms a template or core that promotes the beta-folding of the longer peptide. The substitution of amino acids in the mutants, e.g. 3A-->V and P101L, enhances the folding of the peptide into compact structural units, significantly enhancing the formation of the extensive beta-sheet fibrils.     

The role of Phe in the formation of well-ordered oligomers of amyloidogenic hexapeptide (NFGAIL) observed in molecular dynamics simulations with explicit solvent

We observed fast aggregation of partially ordered oligomers in an earlier simulation study of an amyloidogenic hexapeptide NFGAIL. In this work, the nucleation of highly ordered oligomers was further investigated by a combined total of 960 ns molecular dynamics simulations with explicit solvent on NFGAIL and its nonamyloidogenic mutant NAGAIL. In these simulations, four dimer subunits that each was constrained by harmonic forces as a two-strand beta-sheet were used to enhance the rate of formation. It was found that a critical role played by the aromatic residue Phe was to direct the stacking of beta-sheets to form ordered multilayer aggregates. We also found that many molecular arrangements of the peptide satisfied the "cross-beta-structure", a hallmark of amyloid fibrils. The tendency for the peptide to form either parallel or antiparallel beta-sheet was comparable, as was the tendency for the beta-sheets to stack either in parallel or antiparallel orientation. Overall, approximately 85% of the native hexapeptide formed octamers. The fact that only 8% of the octamers were well-ordered species suggests that the dissociation of the disordered oligomers be the rate-limiting step in the formation of highly ordered oligomers. Among the well-ordered subunit pairs, about half was formed by the beta-sheet extension along the main-chain hydrogen-bond direction, whereas the other half was formed by the beta-sheet stacking. Hence, a delicate balance between intersheet and intrasheet interactions appeared to be crucial in the formation of a highly ordered nucleus of amyloid fibrils. The disordered oligomers were mainly stabilized by nonspecific hydrophobic interactions, whereas the well-ordered oligomers were further stabilized by cross-strand hydrogen bonds and favorable side-chain stacking.     

Aromatic di-alanine repeats (AdAR) are structural motifs characteristic of the soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) family

The aromatic di-alanine repeat is a novel 12-amino acid-long motif constituting alternate small and large hydrophobic residues that mediate the close packing of alpha-helices. A hidden Markov model profile was constructed from the motifs initially described in Soluble N-ethyl maleimide-sensitive factor attachment proteins (SNAP), a family of soluble proteins involved in intracellular membrane fusion. Scanning different sets of protein sequences showed unambiguously that this profile defines a structural motif independent of the tetratrico peptide repeat, another widespread alpha-helical motif. In addition to SNAP, aromatic di-alanine repeats are found in selective LIM homeodomain binding proteins (SLB) and in proteins from the Pyrococcus and Archaeoglobus prokaryotes.     

Possible role of each repeat structure of the microtubule-binding domain of the tau protein in in vitro aggregation

Although one of the priorities in Alzheimer's research is to clarify the filament formation mechanism of the tau protein, it is currently unclear how it is transformed from a normal structure in a neuron. To examine which part and what structural change in the tau protein are involved in its transformation into a pathological entity, the initial in vitro self-aggregation features of each repeat peptide (R1-R4) constituting a three- or four-repeat microtubule-binding domain (3RMBD or 4RMBD) in the tau protein was investigated by measuring both the fluorescence and light scattering (LS) spectra on the same instrument, because these MBD domains constitute the core moiety of the tau paired helical filament (PHF) structure. The conformational features of the R1 and R4 peptides in trifluoroethanol were also investigated by (1)H-NMR and molecular modeling analyses and compared with those of the R2 and R3 peptides. The analyses of the LS spectra clarified (i) the self-aggregation rates of R1-R4, 3RMBD and 4RMBD at a fixed concentration (15 mM), (ii) their minimum concentrations for starting filament extension, and (iii) the concentration dependence of their self-aggregations. The fluorescence analyses showed that the R2 and R3 peptides have high self-aggregation abilities at the extension and nucleation steps, respectively, in their filament formation processes. It was shown that the R2 repeat exhibits a positive synergistic effect on the aggregation of 4RMBD. The R1 and R4 repeats, despite their weak self-aggregation abilities, are necessary for the intact PHF formation of tau MBD, whereas they exerted a negative effect on the R3-driven aggregation of 3RMBD. The conformational analyses showed the importance of the amphipathic conformational features of the R1 to R4 peptides, and the intermolecular disulfide bonding abilities of the R2 and R3 peptides for the PHF formation. On the basis of the present spectral and conformational results, the possible role of each repeat structure in the dimeric formation of MBD at the initial in vitro aggregation stage is discussed.     

Molecular organization of amyloid protofilament-like assembly of betabellin 15D: helical array of beta-sandwiches

Betabellin is a 32-residue peptide engineered to fold into a four-stranded antiparallel beta-sheet protein. Upon air oxidation, the betabellin peptides can fold and assemble into a disulfide-bridged homodimer, or beta-sandwich, of 64 residues. Recent biophysical and ultrastructural studies indicate that betabellin 15D (B15D) (a homodimer of HSLTAKIpkLTFSIAphTYTCAVpkYTAKVSH, where p = DPro, k = DLys, and h = DHis) forms unbranched, 35-A wide assemblies that resemble the protofilaments of amyloid fibers. In the present study, we have analyzed in detail the X-ray diffraction patterns of B15D prepared from acetonitrile. The fiber diffraction analysis indicated that the B15D fibril was composed of a double helix defined by the selection rule l = n + 7m (where l is even, and n and m are any integers), and having a 199-A period and pitch, 28-A rise per unit, and 10-A radius. This helical model is equivalent to a reverse-handed, single helix with half the period and defined by the selection rule l = -3n + 7m (where l is any integer). The asymmetric unit is the single B15D beta-sandwich molecule. These results suggest that the betabellin assembly that models the protofilaments of amyloid fibers is made up of discrete subunits on a helical array. Multiple intersheet hydrogen bonds in the axial direction and intersandwich polar interactions in the lateral direction stabilize the array.     

Charge-pairing mechanism of phosphorylation effect upon amyloid fibrillation of human tau core peptide

Phosphorylation of a fibrillogenic protein, human tau, is believed to play crucial roles in the pathogenesis of Alzheimer's disease. For elucidating molecular mechanisms of the phosphorylation effect on tau fibrillation, we synthesized a peptide, VQIVY 310K (PHF6) and its phosphorylated derivative (PHF6pY). PHF6 is a partial peptide surrounding a plausible in vivo phosphorylation site Tyr310 and forms amyloid-type fibrils similar to those generated by full-length tau. Fibrillation of PHF6 and PHF6pY were studied by spectroscopic and microscopic methods, and the critical concentration of the fibrillation was determined for comparing the fibril stability. The results showed that the phosphorylation strongly influenced the fibrillation propensity of PHF6 by changing its dependency on pH and ionic strength. On the basis of the observations, we suggested that charged sites on the phosphate group and its electrostatic pairing with the neighboring charged residues were physical origins of the phosphorylation effect. To verify this charge-pairing mechanism, we conducted experiments using a series of PHF6 derivatives with non-native charge distributions. The electrostatic interaction in an intermolecular mode was also demonstrated by the system composed of two different peptide species, which found that fibrillation of nonphosphorylated PHF6 was drastically enhanced when a trace amount of phosphorylated PHF6 molecules coexisted. A simulation analysis utilizing crystal coordinates of the PHF6 fibril was also performed for interpreting the experimental results in a molecular level. The present study using the model peptide system gave us a microscopically insightful view on the roles of tau phosphorylation in amyloid-related diseases.