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INTRODUCTION |
The polymerization of the amyloid
-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, A
1 has been
shown to exist in an antiparallel
-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
A
11-28 by homology modeling, using the structure of
-keratin. The resulting electron density map suggested a hydrophobic
core of A
17-20 (LVFF). A high-resolution model has not
been possible because A
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 A
34-42,
indicating a pleated antiparallel
-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 A
(A
16-20) are essential for A
polymerization, which
is prevented by the substitution of these residues (6, 10-13).
A
16-20 binds to the homologous region,
A
17-21 or/and A
18-22, in A
and
forms an antiparallel
-sheet structure (7). Peptides containing the
A
16-20 motif, and peptides binding to this motif,
prevent A
-fibril formation (7). However, residues 16-20 are not
sufficient for polymerization. When incubated under conditions allowing
polymerization of full-length A
, A
16-20 forms
amorphous aggregates but not fibrils (6).
In the present study, we have focused on A
-fragments
containing A
16-20 and identified the shortest
fibril-forming A
-fragment containing this sequence as
A
14-23. Based on substitution studies, electron
microscopy (EM), molecular modeling, and the correlations of side-chain
pairs found in
-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 A
1-40-fibril formation and should thus be of
relevance also for fibrils formed from full-length A
. In support of
this contention, deletion of the A
14-23 motif from
A
1-42 rendered a peptide incapable of forming fibrils.
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EXPERIMENTAL PROCEDURES |
Materials--
Synthetic A
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
-sheet conformations generated by the Biopolymer
module of the program suite.
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RESULTS |
Identification of the Shortest Fibril-forming Sequence in
A
--
A set of peptides with the A
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 A
.
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 (A
13-23)
was the shortest peptide in this series capable of forming amyloid fibrils similar to those of full-length A
(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 A
peptide sequences containing the A
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.
A 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 A . The micrograph shows fibrils formed by the 11-residue
peptide HHQKLVFFAED. Bar = 100 nm.
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One N- and one C-terminally truncated variant of A
13-23
were synthesized and studied under the same conditions as above. The
N-terminal truncated variant (A
14-23) bound more ThT
than the C-terminally truncated variant (A
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 A
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 A
14-23. No fibrils were formed when
A
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
A 13-23. Truncated variants
of A 13-23 were subjected to the ThT assay as described
in the legend to Fig. 1. The nonapeptide A 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
A 14-23 (HQKLVFFAED). B, only a few fibrils,
ultrastructurally different from the fibrils formed by
A 14-23 and full-length A , were observed after
incubation of the decapeptide A 13-22 (HHQKLVFFAE). The
nonapeptide A 15-23 (QKLVFFAED) and the octapeptide
A 16-23 (KLVFFAED) (C) formed thin flakes.
Shorter peptides did not form ordered structures. Bars = 100 nm.
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In addition to the symmetric extensions around the
A
16-20 motif described above, we also examined a few
peptides that extend at either end of this sequence, viz.
A
11-20, A
12-21, and
A
16-25. None of these peptides formed fibrils.
A
11-20 and A
12-21 gave rise to rigid
rods (up to about 200 × 20 nm), whereas A
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 A
14-23 is the minimum
A
sequence giving rise to amyloid fibrils.
To investigate further the mechanism of fibril formation, substituted
and truncated variants of A
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
A
-fibrils (Fig. 6). To investigate
whether the identified central minimum fibril-forming motif is
necessary for A
-fibril formation, a peptide corresponding to
A
1-42 without the A
14-23 sequence,
A
1-42
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 A
14-23 sequence is not only sufficient but also
necessary for A
-fibril formation.

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Fig. 5.
ThT binding to variants of
A 14-23 and to
A 1-42 devoid of the
A 14-23 sequence. Substituted
and truncated variants of the shortest fibril-forming sequence,
A 14-23, and a variant of A 1-42 in which
residues 14-23 were deleted (A 1-42 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
A 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
A 14-23 in
A 1-42 inhibits fibril
formation. A 1-42 14-23 was incubated and
examined with EM as described in the legend to Fig. 2.
Bar = 100 nm.
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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 A
-fragments containing the KLVFF or LVFFA
motif. From this observation and the above-mentioned fact that
full-length A
adopts an antiparallel
-sheet conformation in
fibrils, it is natural to posit an antiparallel
-sheet conformation
also for the oligomeric decamers A
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 A
-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,
or
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
-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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Fig. 8.
Molecular modeling of
A 14-23 and
A 14-42. A, two
strands of the fibril-forming decamer A 14-23 were
aligned as an antiparallel -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
A 14-42. The hydrophobic C terminus of A forms an
intramolecular -sheet that folds over a core consisting of
A 14-23. His14 and Asp23 are
highlighted in blue and red, respectively. The N
terminus of A , 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
A is often truncated in plaques.
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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 A
10-35
indicating a turn-strand-turn motif in residues 13-24 (18), and
assuming that the hydrophobic C terminus of A
forms an
intramolecular
-sheet, we propose a model for A
-fibril formation
(Fig. 8D). The N terminus, considered not to be necessary
for amyloidogenesis because N-terminal truncated variants of A
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 A
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.
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DISCUSSION |
The KLVFF-sequence (A
16-20) is the region in A
that most efficiently binds to A
, and this sequence is necessary for
fibril formation (6). A
16-20 binds to the homologous
region (A
17-21 or/and A
18-22) in A
in an antiparallel manner (7), and it is likely that this interaction
exists also in the fibrils. The pentapeptide A
16-20
does not form fibrils, and thus, also amino acid residues flanking this
region are important for A
-fibril formation. To delineate a
structural model for A
-fibril formation, we identified the shortest
fibril-forming A
-sequence containing A
16-20. EM
examination of systematically selected peptides showed this sequence to
be HQKLVFFAED (A
14-23). Molecular models of a dimer, a
tetramer, and an oligomer of A
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 A
exists as a stable dimer even at low concentrations, and circular dichroism studies show a conformational change from random coil to
-sheet in association with fibril formation (22, 23). NMR studies of
the solution structure of A
have been complicated by the fact that
A
readily forms aggregates at the concentrations necessary for the
use of this technique. However, the A
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
-turn in the
region of amino acids 25-29 of A
. These observations are both in
agreement with the proposed model (Fig. 8D).
X-ray studies of fibrils formed by A
11-28 suggest that
residues 17-20 (LVFF) form a hydrophobic core in these fibrils (8), and Phe-Phe interactions are favored in
-sheets. In accordance with
the proposed model, Asp-His pairs, and especially Glu-Lys pairs, are
frequently found in antiparallel
-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 A
-fibril formation (17). The extreme stability of the
-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
A
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 A
is an important kinetic
determinant for polymerization. A
1-42 polymerizes
faster than A
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
-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 A
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 A
peptides not containing the whole
A
14-23 sequence have previously been shown to form
fibrils. However, in all cases except for A
15-28 (17),
these fibrils were morphologically distinct from those formed by the
full-length peptide and by A
14-23. Fibrils formed by
A
22-35 (29) and A
26-33 (30) were thin
and flexible, whereas A
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
-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 A
(32). Short sequences from several such
proteins have been shown to form fibrils. Examples include the
decapeptide SNNFGAILSS derived from pancreatic
-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 A
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.