A Molecular Model of Alzheimer Amyloid beta -Peptide Fibril Formation*

Lars O. TjernbergDagger §, David J. E. Callaway, Agneta Tjernbergparallel , Solveig HahneDagger , Christina LilliehöökDagger , Lars TereniusDagger , Johan Thyberg**, and Christer NordstedtDagger Dagger Dagger

From the Dagger  Laboratory of Biochemistry and Molecular Pharmacology, Section of Drug Dependence Research, Department of Clinical Neuroscience, CMM L8:01, Karolinska Hospital, S-171 76 Stockholm, Sweden, the  Picower Institute for Medical Research, Manhasset, New York 11030, the parallel  Mass Spectrometric Section, Department of Structural Chemistry, Pharmacia & Upjohn, S-112 87 Stockholm, Sweden, and the ** Department of Cell and Molecular Biology, Medical Nobel Institute, Karolinska Institutet, S-171 77 Stockholm, Sweden

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Polymerization of the amyloid beta (Abeta ) peptide into protease-resistant fibrils is a significant step in the pathogenesis of Alzheimer's disease. It has not been possible to obtain detailed structural information about this process with conventional techniques because the peptide has limited solubility and does not form crystals. In this work, we present experimental results leading to a molecular level model for fibril formation. Systematically selected Abeta -fragments containing the Abeta 16-20 sequence, previously shown essential for Abeta -Abeta binding, were incubated in a physiological buffer. Electron microscopy revealed that the shortest fibril-forming sequence was Abeta 14-23. Substitutions in this decapeptide impaired fibril formation and deletion of the decapeptide from Abeta 1-42 inhibited fibril formation completely. All studied peptides that formed fibrils also formed stable dimers and/or tetramers. Molecular modeling of Abeta 14-23 oligomers in an antiparallel beta -sheet conformation displayed favorable hydrophobic interactions stabilized by salt bridges between all charged residues. We propose that this decapeptide sequence forms the core of Abeta -fibrils, with the hydrophobic C terminus folding over this core. The identification of this fundamental sequence and the implied molecular model could facilitate the design of potential inhibitors of amyloidogenesis.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The polymerization of the amyloid beta -peptide into protease-resistant fibrillar deposits in the brain parenchyma and vasculature is a significant step in the pathogenesis of Alzheimer's disease (1). Compounds capable of interfering with the polymerization process thus may lead to potent therapeutics (2-7). To design such compounds, a detailed knowledge of the polymerization process at the molecular level is essential. Conventional experimental techniques alone have not been able to yield this detail. In the present work, we therefore employ both theoretical and experimental techniques to evolve a molecular model of fibril formation.

In fibrils, Abeta 1 has been shown to exist in an antiparallel beta -sheet conformation by x-ray diffraction and Fourier-transformed infrared spectroscopy (FTIR). Inouye and Kirschner (8) have refined their x-ray data from fibrils of Abeta 11-28 by homology modeling, using the structure of beta -keratin. The resulting electron density map suggested a hydrophobic core of Abeta 17-20 (LVFF). A high-resolution model has not been possible because Abeta does not form crystals that are necessary for x-ray crystallography. Solid state NMR, not requiring crystals, has been used to study polymers formed by Abeta 34-42, indicating a pleated antiparallel beta -sheet (9). Unfortunately, solid state NMR is not suited for studies of longer peptides.

It has previously been shown that amino acid residues 16-20 in Abeta (Abeta 16-20) are essential for Abeta polymerization, which is prevented by the substitution of these residues (6, 10-13). Abeta 16-20 binds to the homologous region, Abeta 17-21 or/and Abeta 18-22, in Abeta and forms an antiparallel beta -sheet structure (7). Peptides containing the Abeta 16-20 motif, and peptides binding to this motif, prevent Abeta -fibril formation (7). However, residues 16-20 are not sufficient for polymerization. When incubated under conditions allowing polymerization of full-length Abeta , Abeta 16-20 forms amorphous aggregates but not fibrils (6).

In the present study, we have focused on Abeta -fragments containing Abeta 16-20 and identified the shortest fibril-forming Abeta -fragment containing this sequence as Abeta 14-23. Based on substitution studies, electron microscopy (EM), molecular modeling, and the correlations of side-chain pairs found in beta -sheets (14), we present a detailed structural model of fibrils formed by this sequence. This model includes interactions between sequences previously shown to interact in and to be necessary for Abeta 1-40-fibril formation and should thus be of relevance also for fibrils formed from full-length Abeta . In support of this contention, deletion of the Abeta 14-23 motif from Abeta 1-42 rendered a peptide incapable of forming fibrils.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- Synthetic Abeta 1-40 was obtained from Dr. David Teplow, the Biopolymer Laboratory at Harvard University, MA. All other peptides were purchased from Research Genetics, Huntsville, AL. The peptides were purified on a Polymer Laboratories (Church Stretton, UK) PLRP-S column (150 × 25 mm or 150 × 7.5 mm; Polymer Labs.), using a water-acetonitrile gradient with 0.1% trifluoroacetic acid. Identity and purity were verified with electrospray mass spectrometry using a Quattro triple quadropole (Micromass, Altrincham, UK). After purification, the peptides were lyophilized and stored at -80 °C.

Incubation of Peptides-- The peptides were dissolved at 200 µM in 50 mM Tris-buffered saline (TBS) (150 mM NaCl), pH 7.4 and incubated at 37 °C for 3 days. In control experiments, the peptides were initially dissolved in 2 volumes of 50 mM Tris, pH 10, to ensure starting solutions free from possible seeds. After 3 min, 1 volume of 50 mM Tris-HCl containing 450 mM NaCl was added to give a final pH of 7.4 and a concentration of 150 mM NaCl. No differences in the results were observed using this protocol.

Thioflavine T Fluorescence Assay-- The incubated samples were vortex mixed and 40 µl aliquots were withdrawn and mixed with 960 µl of 10 µM thioflavine T (ThT) in 10 mM phosphate-buffered saline, pH 6.0. The samples were analyzed in a Perkin Elmer (Beaconsfield, UK) LS 50 luminescence spectrometer with excitation at 437 nm and emission at 485 nm. Slit widths were set to 5 nm.

Electron Microscopy-- The incubated samples were centrifuged at 20,000 × g for 20 min, and the supernatants were aspirated. The pellets were sonicated for 5 s in 100 µl of water and 8 µl of these suspensions were placed on grids covered by a carbon-stabilized formvar film. Excess fluid was withdrawn after 30 s, and the grids were negatively stained with 3% uranyl acetate in water. The stained grids were then examined and photographed in a JEOL 100CX at 60 kV.

Gel Electrophoresis-- Polyacrylamide gradient gels, 5-18%, were used in a Tris-Tricine buffer system. Supernatants (20,000 × g) from the incubated peptides were mixed with Laemmli sample buffer and loaded, without boiling, onto the gel. Rainbow markers (Amersham, Little Chalfont, UK) were used to estimate molecular weights. After 4 h at 70 mA, the gels were stained with Coomassie Brilliant Blue or were silver stained.

Molecular Modeling-- The molecular simulations were performed using the Insight/Discover 2.9.7 program suite (Biosym/MSI, San Diego, CA). The simulations were performed in vacuum, with the dielectric constant set to unity. Default values were used for all other parameters. Steepest-descent and conjugate gradient minimization schemes were used to optimize the putative structures, with the backbones fixed in beta -sheet conformations generated by the Biopolymer module of the program suite.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Identification of the Shortest Fibril-forming Sequence in Abeta -- A set of peptides with the Abeta 16-20 sequence systematically extended at both ends, were synthesized (Fig. 1). The peptides were incubated for 3 days under conditions allowing polymerization of full-length Abeta . Following incubation, an aliquot of the samples was mixed with 10 µM ThT, and the fluorescence was measured (Fig. 1). ThT shifts excitation and emission maxima upon binding to amyloid fibrils and has therefore been used to monitor amyloid fibril formation (15, 16). The incubates were then centrifuged at 20,000 × g, and the sedimented material was negatively stained and examined by EM. The undecapeptide HHQKLVFFAED (Abeta 13-23) was the shortest peptide in this series capable of forming amyloid fibrils similar to those of full-length Abeta (Fig. 2). Moreover, all peptides longer than 11 residues formed similar fibrils (not shown). The shorter peptides bound ThT considerably less efficiently than the longer, fibril-forming peptides (Fig. 1). When supernatants from the samples were analyzed by gel electrophoresis, all fibril-forming peptides displayed bands corresponding to dimers and/or tetramers, whereas no such species were detected in samples containing nonfibril-forming peptides.


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Fig. 1.   ThT binding to Abeta peptide sequences containing the Abeta binding motif KLVFF. Peptides with the indicated sequences were incubated at a concentration of 200 µM in TBS, pH 7.4, for 3 days. The samples were vortex mixed, aliquots were withdrawn and mixed with 10 µM ThT, and the fluorescence was measured. The binding capacity correlated to fibril formation; all peptides except the three shortest were found to form fibrils as detected by EM (Fig. 2).


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Fig. 2.   Abeta 13-23 forms fibrils. After 3 days of incubation, the peptides shown in Fig. 1 were centrifuged at 20,000 × g, and the supernatants were aspirated. The pellets were resuspended in water, placed on grids and negatively stained with 3% uranyl acetate. Examination of the stained samples revealed, in all cases except for the three shortest peptides, fibrils similar to those formed by full-length Abeta . The micrograph shows fibrils formed by the 11-residue peptide HHQKLVFFAED. Bar = 100 nm.

One N- and one C-terminally truncated variant of Abeta 13-23 were synthesized and studied under the same conditions as above. The N-terminal truncated variant (Abeta 14-23) bound more ThT than the C-terminally truncated variant (Abeta 13-22) did (Fig. 3). EM showed that deletion of the N-terminal His did not affect the tendency to form fibrils. However, the deletion of the C-terminal Asp impaired fibril formation (Fig. 4, A and B). Gel electrophoretic analysis of the supernatants revealed in both cases a band at 4-5 kDa, suggesting that the peptides formed stable tetramers. To investigate whether the Abeta 14-23 sequence could be further truncated in the N terminus and still form fibrils, we analyzed nona- to hexapeptides with an intact C terminus as above. The nonapeptide bound ThT most efficiently, the octapeptide showed intermediate binding, whereas the hepta- and hexapeptide displayed only weak ThT binding (Fig. 3). Examination by EM revealed no ordered structures for hexa- and heptapeptides, the octa- and nonapeptide formed thin flakes (Fig. 4C), but none formed fibrils. Hence, the minimum sequence that was able to form amyloid fibrils was Abeta 14-23. No fibrils were formed when Abeta 14-23 was incubated in TBS, pH 9, indicating that protonation of His was necessary for fibril formation.


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Fig. 3.   ThT binding to truncated variants of Abeta 13-23. Truncated variants of Abeta 13-23 were subjected to the ThT assay as described in the legend to Fig. 1. The nonapeptide Abeta 15-23 (QKLVFFAED) was found to be the most potent ThT binder. Interestingly, this peptide formed thin flakes but no fibrils as detected by EM.


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Fig. 4.   A decapeptide is the shortest fibril-forming peptide. N- and C-terminal truncated variants of the shortest fibril-forming peptide found in the first experiment were incubated and examined by EM as described in the legend to Fig. 2. A, the shortest fibril-forming sequence was found to be the decapeptide Abeta 14-23 (HQKLVFFAED). B, only a few fibrils, ultrastructurally different from the fibrils formed by Abeta 14-23 and full-length Abeta , were observed after incubation of the decapeptide Abeta 13-22 (HHQKLVFFAE). The nonapeptide Abeta 15-23 (QKLVFFAED) and the octapeptide Abeta 16-23 (KLVFFAED) (C) formed thin flakes. Shorter peptides did not form ordered structures. Bars = 100 nm.

In addition to the symmetric extensions around the Abeta 16-20 motif described above, we also examined a few peptides that extend at either end of this sequence, viz. Abeta 11-20, Abeta 12-21, and Abeta 16-25. None of these peptides formed fibrils. Abeta 11-20 and Abeta 12-21 gave rise to rigid rods (up to about 200 × 20 nm), whereas Abeta 16-25 mainly produced larger diffuse flakes (up to about 500 × 100 nm) and short fibrillar fragments (usually <50 nm in length). These findings confirm the notion that Abeta 14-23 is the minimum Abeta sequence giving rise to amyloid fibrils.

To investigate further the mechanism of fibril formation, substituted and truncated variants of Abeta 14-23 were synthesized (Fig. 5). Although several of the peptides were found to bind ThT, some even more than the nonsubstituted decapeptide (Fig. 5), only AQKLVFFAEA formed fibrils with any similarities to Abeta -fibrils (Fig. 6). To investigate whether the identified central minimum fibril-forming motif is necessary for Abeta -fibril formation, a peptide corresponding to Abeta 1-42 without the Abeta 14-23 sequence, Abeta 1-42Delta 14-23, was synthesized and incubated as above. This peptide bound ThT (Fig. 5) and formed small aggregates but no fibrils (Fig. 7). Hence, we conclude that the Abeta 14-23 sequence is not only sufficient but also necessary for Abeta -fibril formation.


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Fig. 5.   ThT binding to variants of Abeta 14-23 and to Abeta 1-42 devoid of the Abeta 14-23 sequence. Substituted and truncated variants of the shortest fibril-forming sequence, Abeta 14-23, and a variant of Abeta 1-42 in which residues 14-23 were deleted (Abeta 1-42Delta 14-23) were assayed for ThT binding as described in the legend to Fig. 1.


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Fig. 6.   Substitutions and deletions in Abeta 14-23 have a distinct influence on polymer morphology. The peptides shown in Fig. 5 were incubated and examined by EM as described in the legend to Fig. 2. A, DQKLVFFAEH; B, DEKLVFFAQH; C, AQKLVFFAED; D, HQKLVFFAEA; E, AQKLVFFAEA; F, HKLVFFAED; G, HQKLVFFAD; H, HKLVFFAD. Bars = 100 nm.


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Fig. 7.   Deletion of Abeta 14-23 in Abeta 1-42 inhibits fibril formation. Abeta 1-42Delta 14-23 was incubated and examined with EM as described in the legend to Fig. 2. Bar = 100 nm.

It is of interest to note that ThT binds not only to amyloid-like fibrils, but also to other peptide aggregates (Figs. 4C, 6, E-F, and 7). Thus, when the ThT assay is used to monitor fibril formation, other techniques such as EM are a valuable complement to confirm the nature of the aggregates formed.

Molecular Modeling-- It is known (7) that radiolabeled KKLVFFA binds efficiently only to Abeta -fragments containing the KLVFF or LVFFA motif. From this observation and the above-mentioned fact that full-length Abeta adopts an antiparallel beta -sheet conformation in fibrils, it is natural to posit an antiparallel beta -sheet conformation also for the oligomeric decamers Abeta 14-23, with the LVFF residues paired with hydrophobic groups. Additionally, the strength of a salt bridge is increased by a factor of approximately 80 (the dielectric constant of water) when water is driven out of a hydrophobic environment. In a water-free environment like the interior of a fibril, salt bridges are thus energetically comparable with covalent bonds. The correct model should therefore involve a maximum of salt bridges and hydrogen bonds. Additional evidence for the importance of salt bridges arises from the observation of structural transitions in Abeta -fibril assembly as pH is varied, suggesting a strong dependence upon electrostatic interactions (17). In this latter study, fibrils were formed only at pH values where the His and Asp/Glu side chains are ionized. The twin requirements of hydrophobic overlap and maximal salt bridges limit the allowed pairings to two possibilities,


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Dimer I

or



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Dimer II
or a combination of these. Dimer "I" is formed with a maximum of intermolecular hydrogen bonds and salt bridges between Asp and His, whereas there is maximal interaction between hydrophobic side chains and salt bridges between Glu and Lys in dimer "II." We subjected Dimer I to molecular modeling and energy minimization (Fig. 8A). Adding these dimers to each other in a beta -sheet conformation would give a fibril with unpaired lysines on one side and unpaired glutamates on the other side. However, if the dimers are added to each other with an offset of one, Lys and Glu could form ion pairs (note that sequences 2 and 3 are aligned as in Dimer II).


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Tetramer I



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Fig. 8.   Molecular modeling of Abeta 14-23 and Abeta 14-42. A, two strands of the fibril-forming decamer Abeta 14-23 were aligned as an antiparallel beta -sheet and subjected to energy minimization schemes. Note that His and Asp form ion pairs at both ends of the dimer. B, two dimers were aligned. The resulting tetramer was energy minimized, and two ion pairs, Lys (blue) and Glu (red), were formed between the dimers. C, two tetramers were aligned to form an octamer and were energy minimized. The fibril axis is in the plane of the paper and perpendicular to the peptide chains, and the Lys/Glu pairs are alternatively above and below the plane. D, a model of Abeta 14-42. The hydrophobic C terminus of Abeta forms an intramolecular beta -sheet that folds over a core consisting of Abeta 14-23. His14 and Asp23 are highlighted in blue and red, respectively. The N terminus of Abeta , not always present in amyloid deposits, is not shown. It contains many charged residues, all except one at odd positions (1, 3, 5, 7, 11, 13, and 8). Most of these may be exposed to water, while the noncharged residues make hydrophobic interactions with the fibril. Alternatively, the N terminus could be totally exposed to water and thus also to proteases, offering an explanation as to why this part of Abeta is often truncated in plaques.

An energy-minimized model of such a tetramer is shown in Fig. 8B. Dimer after dimer (or tetramer after tetramer) could be added in this way, building a protofibril (Fig. 8C). On the basis of this model and NMR data from studies of Abeta 10-35 indicating a turn-strand-turn motif in residues 13-24 (18), and assuming that the hydrophobic C terminus of Abeta forms an intramolecular beta -sheet, we propose a model for Abeta -fibril formation (Fig. 8D). The N terminus, considered not to be necessary for amyloidogenesis because N-terminal truncated variants of Abeta are found in amyloid plaques (19), is not shown. In our model the core is made up from the fibril-forming decapeptide, and the C terminus of Abeta folds alternately above and below this core, minimizing the hydrophobic surface exposed to water. The diameter of the protofibril in our model is 4 nm, consistent with the data obtained with atomic force microscopy (20). Two or more such protofibrils could then twist around each other to exclude hydrophobic surfaces and form an amyloid fibril.

    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The KLVFF-sequence (Abeta 16-20) is the region in Abeta that most efficiently binds to Abeta , and this sequence is necessary for fibril formation (6). Abeta 16-20 binds to the homologous region (Abeta 17-21 or/and Abeta 18-22) in Abeta in an antiparallel manner (7), and it is likely that this interaction exists also in the fibrils. The pentapeptide Abeta 16-20 does not form fibrils, and thus, also amino acid residues flanking this region are important for Abeta -fibril formation. To delineate a structural model for Abeta -fibril formation, we identified the shortest fibril-forming Abeta -sequence containing Abeta 16-20. EM examination of systematically selected peptides showed this sequence to be HQKLVFFAED (Abeta 14-23). Molecular models of a dimer, a tetramer, and an oligomer of Abeta 14-23, where the charged residues form ion pairs and the hydrophobic residues form a hydrophobic core, were energy-minimized in a molecular modeling program (Fig. 8, A-D).

We suggest that the polymerization starts with the formation of dimers (Fig. 8A) which in turn form tetramers (Fig. 8B) and that dimers and/or tetramers are added to form oligomers (Fig. 8C). The existence of dimers and tetramers (modeled in Fig. 8, A and B) is supported by gel electrophoresis which showed that all fibril-forming peptides also formed dimers and/or tetramers. Likewise, a recent study by Garcon-Rodriguez (21) shows that Abeta exists as a stable dimer even at low concentrations, and circular dichroism studies show a conformational change from random coil to beta -sheet in association with fibril formation (22, 23). NMR studies of the solution structure of Abeta have been complicated by the fact that Abeta readily forms aggregates at the concentrations necessary for the use of this technique. However, the Abeta 10-35 fragment has been studied by NMR in a solution free from detergents and organic solvents. This study shows a turn-strand-turn motif for residues 13-24 (18), and Chou and Fasman (24) analysis indicates a beta -turn in the region of amino acids 25-29 of Abeta . These observations are both in agreement with the proposed model (Fig. 8D).

X-ray studies of fibrils formed by Abeta 11-28 suggest that residues 17-20 (LVFF) form a hydrophobic core in these fibrils (8), and Phe-Phe interactions are favored in beta -sheets. In accordance with the proposed model, Asp-His pairs, and especially Glu-Lys pairs, are frequently found in antiparallel beta -sheets (14). Moreover, the pH-dependence of fibril formation shows the importance of salt bridges, and interactions between His and Glu or Asp have been suggested to be involved in Abeta -fibril formation (17). The extreme stability of the beta -sheet structure formed by a peptide with the sequence AEAEAKAKAEAEAKAK (25) and thermodynamic studies (26) also demonstrate the strength of charge complementarity and salt bridges between Glu and Lys. The polar Gln is located in a polar environment between the Asp-His and Glu-Lys pairs in our model. Although Gln does not seem to form any strong interactions with these pairs, no fibrils but high concentrations of dimers were formed when this residue was deleted from Abeta 14-23. In this case, both the Asp-His and the Glu-Lys pairs can be formed within one dimer, offering an explanation to the absence of fibrils.

The length of the C terminus of Abeta is an important kinetic determinant for polymerization. Abeta 1-42 polymerizes faster than Abeta 1-40 (27), but amino acids 41 (Ile) and 42 (Ala) are (in contrast to residues 16-20) not necessary for fibril formation. The increased polymerization rate can be explained by increased hydrophobic interaction, first by the formation of a longer intramolecular beta -sheet in the C terminus and second by the folding of this sheet over the core of the dimer (Fig. 8D). Another example of how increased hydrophobicity leads to a higher rate of fibril formation is the Glu-22 to Gln mutation in Abeta found in families with hereditary cerebral hemorrhage with amyloidosis, Dutch type (HCHWA-D) (28). Gln, as well as Glu, interacts favorably with Lys, and the loss of a salt-bridge can be compensated by increased hydrophobic interactions.

A few short Abeta peptides not containing the whole Abeta 14-23 sequence have previously been shown to form fibrils. However, in all cases except for Abeta 15-28 (17), these fibrils were morphologically distinct from those formed by the full-length peptide and by Abeta 14-23. Fibrils formed by Abeta 22-35 (29) and Abeta 26-33 (30) were thin and flexible, whereas Abeta 34-42 (30) displayed a thicker, rope-like structure similar to the alanine-substituted decapeptide shown in Fig. 6E.

Although a model by Kirschner et al. (31) is superficially similar to the one proposed here, the specific residue pairings differ. In particular, residue His-13 is important in their model, whereas it proved inessential for fibril formation in the present study and is not included in our fundamental decapeptide. Their model also involves only a single residue pairing, rather than the alternating alignments (Dimers I and II) of the present structure. Moreover, the Lys-Phe pairing of the Kirschner model is significantly less frequent in beta -sheets than the Lys-Glu pairing incorporated into our model (14).

A large variety of proteins form amyloid fibrils structurally similar to those formed by Abeta (32). Short sequences from several such proteins have been shown to form fibrils. Examples include the decapeptide SNNFGAILSS derived from pancreatic beta -cell islet amyloid polypeptide (33), the nonapeptide (FNNGNCFIL) derived from gelsolin (34), and the octapeptide (AGAAAAGA) derived from the prion protein (35). Neither these fragments nor Abeta has a net charge, and it is possible that exposed hydrophobic side chains and appropriate pairing of extant charges and dipoles in a sequence of about 10 residues are sufficient for fibril assembly. The present model may be of general relevance for fibril formation, and the strategy we used to identify functionally important sequences could be applied to delineate the fibril-forming motifs in other amyloidogenic proteins.

    ACKNOWLEDGEMENTS

We are grateful to Karin Blomgren for expert technical assistance.

    FOOTNOTES

* This work was supported by The Swedish Medical Research Council (to L. T. and J. T.), The Swedish Heart Lung Foundation (to J. T.) and The King Gustaf V 80th Birthday Fund (to J. T.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed: Tel.: 46-8-517 739 19; Fax: 46-8-517 761 80; E-mail: Lars.Tjernberg{at}cmm.ki.se.

Dagger Dagger Recipient of fellowships from The Swedish Medical Research Council, The Berth von Kantzow Foundation, The Swedish Society for Medical Research, The Axel and Margret Ax:son Johnson Foundation, and The Nicholson Foundation.

    ABBREVIATIONS

The abbreviations used are: Abeta , Alzheimer amyloid beta -peptide; EM, electron microscopy; TBS, Tris-buffered saline; ThT, thioflavine T.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
  1. Selkoe, D. J. (1996) J. Biol. Chem. 271, 18295-18298[Free Full Text]
  2. Camilleri, P., Haskins, N. J., and Howlett, D. R. (1994) FEBS Lett. 341, 256-258[CrossRef][Medline] [Order article via Infotrieve]
  3. Lorenzo, A., and Yankner, B. A. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 12243-12247[Abstract/Free Full Text]
  4. Tomiyama, T., Asano, S., Suwa, Y., Morita, T., Kataoka, K., Mori, H., and Endo, N. (1994) Biochem. Biophys. Res. Commun. 204, 76-83[CrossRef][Medline] [Order article via Infotrieve]
  5. Ghanta, J., Shen, C.-H., Kiessling, L. L., and Murphy, R. M. (1996) J. Biol. Chem. 271, 29525-29528[Abstract/Free Full Text]
  6. Tjernberg, L. O., Näslund, J., Lindqvist, F., Johansson, J., Karlström, A. R., Thyberg, J., Terenius, L., and Nordstedt, C. (1996) J. Biol. Chem. 271, 8545-8548[Abstract/Free Full Text]
  7. Tjernberg, L. O., Lilliehöök, C., Callaway, D. J. E., Näslund, J., Hahne, S., Thyberg, J., Terenius, L., and Nordstedt, C. (1997) J. Biol. Chem. 272, 12601-12605[Abstract/Free Full Text]
  8. Inouye, H., and Kirschner, D. A. (1996) The Nature and Origin of Amyloid Fibrils, pp. 22-39, Wiley, Chichester, United Kingdom
  9. Lansbury, P. T. J., Costa, P. C., Griffiths, J. M., Simon, E. J., Auger, M., Halverson, K. J., Kocisko, D. A., Hendsch, Z. S., Ashburn, T. T., Spencer, R. G. S., Tidor, B., and Griffin, R. G. (1995) Nat. Struct. Biol. 2, 990-998[Medline] [Order article via Infotrieve]
  10. Hilbich, C., Kisters-Woike, B., Reed, J., Masters, C. L., and Beyreuther, K. (1992) J. Mol. Biol. 228, 460-473[Medline] [Order article via Infotrieve]
  11. Hilbich, C., Kisters-Woike, B., Reed, J., Masters, C. L., and Beyreuther, K. (1991) J. Mol. Biol. 218, 149-163[Medline] [Order article via Infotrieve]
  12. Wood, S. J., Wetzel, R., Martin, J. D., and Hurle, M. R. (1995) Biochemistry 34, 724-730[Medline] [Order article via Infotrieve]
  13. Hughes, S. T., Goyal, S., Sun, J. E., Gonzalez-DeWhitt, P., Fortes, M. A., Riedel, N. G., and Sahasrabudhe, S. R. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 2065-2070[Abstract/Free Full Text]
  14. Wouters, M. A., and Curmi, P. M. G. (1995) Proteins 22, 119-131[Medline] [Order article via Infotrieve]
  15. LeVine, H., III. (1993) Protein Sci. 2, 404-410[Abstract/Free Full Text]
  16. Naiki, H., and Nakakuki, K. (1996) Lab. Invest. 74, 374-383[Medline] [Order article via Infotrieve]
  17. Fraser, P. E., Nguyen, J. T., Surewicz, W. K., and Kirschner, D. A. (1991) Biophys. J. 60, 1190-1201[Abstract]
  18. Lee, J. P., Stimson, E. R., Ghilardi, J. R., Mantyh, P. W., Lu, Y. A., Felix, A. M., Llanos, W., Behbin, A., Cummings, M., Van Criekinge, M., Timms, W., and Maggio, J. E. (1995) Biochemistry 34, 5191-5200[Medline] [Order article via Infotrieve]
  19. Näslund, J., Schierhorn, A., Hellman, U., Lannfelt, L., Roses, A. D., Tjernberg, L. O., Silberring, J., Gandy, S. E., Winblad, B., Greengard, P., Nordstedt, C., and Terenius, L. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 8378-8382[Abstract]
  20. Harper, J. D., Wong, S., Lieber, C. M., and Lansbury, P. T., Jr. (1997) Chem. Biol. (Lond.) 4, 119-125[Medline] [Order article via Infotrieve]
  21. Garzon-Rodriguez, W., Sepulveda-Becerra, M., Milton, S., and Glabe, C. G. (1997) J. Biol. Chem. 272, 21037-21044[Abstract/Free Full Text]
  22. Simmons, L. K., May, P. C., Tomaselli, K. J., Rydel, R. E., Fuson, K. S., Brigham, E. F., Wright, S., Lieberburg, I., Becker, G. W., Brems, D. N., and Li, W. Y. (1994) Mol. Pharmacol. 45, 373-379[Abstract]
  23. Terzi, E., Hölzemann, G., and Seelig, J. (1995) J. Mol. Biol. 252, 633-642[CrossRef][Medline] [Order article via Infotrieve]
  24. Chou, P., and Fasman, G. (1978) Annu. Rev. Biochem. 47, 251-276[CrossRef][Medline] [Order article via Infotrieve]
  25. Zhang, S., Lockshin, C., Cook, R., and Rich, A. (1994) Biopolymers 34, 663-672[Medline] [Order article via Infotrieve]
  26. Smith, C. K., and Regan, L. (1995) Science 270, 980-982[Abstract]
  27. Jarrett, J. T., Berger, E. P., and Lansbury, P. T., Jr. (1993) Biochemistry 32, 4693-4697[Medline] [Order article via Infotrieve]
  28. Clements, A., Walsh, D. M., Williams, C. H., and Allsop, D. (1993) Neurosci. Lett. 161, 17-20[Medline] [Order article via Infotrieve]
  29. Fraser, P. E., Duffy, L. K., O'Malley, M. B., Nguyen, J., Inouye, H., and Kirschner, D. A. (1991) J. Neurosci. Res. 28, 474-485[Medline] [Order article via Infotrieve]
  30. Halverson, K., Fraser, P. E., Kirschner, D. A., and Lansbury, P. T., Jr. (1990) Biochemistry 29, 2639-2644[Medline] [Order article via Infotrieve]
  31. Kirschner, D. A., Inouye, H., Duffy, L. K., Sinclair, A., Lind, M., and Selkoe, D. J. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 6953-6957[Abstract]
  32. Sunde, M., Serpell, L. C., Bartlam, M., Fraser, P. E., Pepys, M. B., and Blake, C. C. F. (1997) J. Mol. Biol. 273, 729-739[CrossRef][Medline] [Order article via Infotrieve]
  33. Westermark, P., Engström, U., Johnson, K. H., Westermark, G. T., and Betsholtz, C. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 5036-5040[Abstract]
  34. Maury, C. P. J., Nurmiaho-Lassila, E.-L., and Rossi, H. (1994) Lab. Invest. 70, 558-564[Medline] [Order article via Infotrieve]
  35. Lundberg, K. M., Stenland, C. J., Cohen, F. E., Prusiner, S. B., and Millhauser, G. L. (1997) Chem. Biol. (Lond.) 4, 345-355[CrossRef][Medline] [Order article via Infotrieve]


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