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INTRODUCTION |
The amyloid
peptide
(A
)1 is a normally soluble
4.3-kDa peptide found in all biological fluids, but it accumulates as
the major constituent of the extracellular deposits that are the
pathologic hallmarks of Alzheimer's disease (AD) (1). Genetic evidence from early onset cases of AD indicates that A
metabolism is linked to the disease (2). A
peptides are neurotoxic (3, 4), but the
mechanism of toxicity and the species of A
responsible have not been
clearly defined.
There is mounting evidence that oxidative stress causing cellular
damage is central to the neurodegeneration of AD (5, 6). There is an
increase in oxidation of proteins as well as nuclear and mitochondrial
DNA in AD brains (7-9). A
has the ability to enhance the generation
of reactive oxygen species in cells of neural origin as well as
in cell-free media (10-12). A
in vitro binds
metal ions, including Zn2+, Cu2+, and
Fe3+, inducing peptide aggregation that may be reversed by
treatment with chelators such as EDTA (13, 14). Furthermore, extensive redox chemical reactions take place when A
binds Cu2+
and Fe3+, reducing the oxidation state of both metals and
producing H2O2 from O2 in a
catalytic manner (12). Because elevated levels of copper (400 µM), zinc (1 mM), and iron (1 mM)
are found in amyloid deposits in AD-affected brains (15, 16), the
oxidative stress observed in AD may be related to the production of
reactive oxygen species by metal-bound forms of A
. This hypothesis
is supported by the recent observation that senile plaques and
neurofibrillary tangles isolated from AD brains were capable of
generating reactive oxygen species and that copper and iron were
essential (17). Moreover, our studies (18) have shown that the
solubilization of A
from post-mortem brain tissue of AD patients was
increased in the presence of metal chelators such as
N,N,N',N'-tetrakis(2-pyridyl-methyl) ethylene diamine and bathocuproine. Recently, the dramatic inhibition of amyloid deposition in transgenic mice treated orally with a Cu2+/Zn2+-selective chelator has been reported
(19).
The discovery that metal binding to A
may be responsible for some of
the pathological effects of AD makes characterization of the
metal-binding site of interest as a potential therapeutic target. The
aim of the present study was to characterize the structural consequences of A
binding to Cu2+ and Zn2+
in solution and to identify amino acid residues involved in metal binding.
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MATERIALS AND METHODS |
Peptides were synthesized as described previously (20) or
obtained from Auspep (Melbourne, Australia) and from the W. M. Keck Laboratory (Yale University, CT). 2H2O,
TFE-2H3, NaO2H,
SDS-2H25, and 2HCl were obtained
from Cambridge Isotope Laboratories (Andover, MA). The spin-labeled
zwitterionic phospholipid 1-palmitoyl-2-(16-doxyl stearoyl)
phosphatidyl choline was obtained from Avanti Polar Lipids Inc.
(Pelham, AL). The acidic phospholipid spin label
1-palmitoyl-2-(16-doxyl stearoyl) phosphatidyl serine was synthesized
according to Hubbell and McConnell (21). Both spin probes were checked
for purity and to ensure that the number of spins/mol were
>90% of theory (22). Synthetic palmitoyloleoyl phosphatidyl choline
(POPC) was purchased from Sigma, and palmitoyloleoyl phosphatidyl
serine (POPS) was purchased from Avanti Polar Lipids Inc. LUV
were prepared by the method described by Mayer et al. (23).
Peptides were added to the desired concentration to a suspension of LUV
in PBS, and the mixture was vortexed under N2 for 10 min at
305 K. Negatively charged LUV were made with 50% POPS and 50% POPC.
Zwitterionic LUV were made with 100% POPC. Because the longer A
peptides are prone to aggregation in solution, all experiments were
carried out with freshly prepared samples.
The samples for NMR varied depending on the solution conditions used;
in aqueous PBS with 10% 2H2O added, samples
containing A
28 had a peptide concentration of 1 mM,
whereas those containing A
40 were run at 0.3 mM. When the peptide is made up fresh in metal-free conditions and the undissolved aggregates are spun out before use, the peptide aggregates only very slowly (days). Some studies were performed in SDS solution (50 mM phosphate, 200 mM
SDS-2H25, pH 5.3, 10%
2H2O) where all peptide concentrations were 1.5 mM. NMR spectra were recorded on Bruker DRX-600 and AMX-500
spectrometers as described previously (24). Ultracentrifugation
measurements were carried out on A
28 and A
40 in PBS at 1 and 0.3 mM, respectively, as described previously (24).
EPR Spectroscopy--
X-band continuous wave EPR spectra of the
Cu2+-peptide complexes were obtained using a Bruker EC106
spectrometer. Samples were loaded into hematocrit capillary tubes and
inserted reproducibly into the cavity using a Kornberg holder (22). The
sample temperature was maintained at 110 K using a flow-through
cryostat. The microwave frequency was measured using a Bruker EIP 548B
frequency counter, and the magnetic field was calibrated with an
,
'-diphenyl-betapicryl hydrazyl sample. The exact peptide
concentrations were determined by amino acid analysis of total copper
by inductively coupled plasma mass spectrometry. EPR of
peptide/spin-labeled LUV was carried out as above, except that the
temperature was kept at 305 K, above the liquid crystal/gel transition
temperature of the lipids. Analysis of EPR spectra of peptide/lipid
mixtures was carried out using the spectral subtraction and addition
methods described by Marsh (25). To check the validity of these
procedures, the lipid spin label spectra were simulated using modified
Bloch equations, as described in model I of Davoust and Devaux
(26).
CD Spectroscopy--
The CD spectra of peptides in the LUV were
obtained using a CD spectropolarimeter model 62DS (AVIV,
Lakewood, NJ). The peptide concentrations were determined using the
molar extinction of the UV absorption from the tyrosine residue. CD was
obtained for each solution both neat and after a 1:4 dilution with
MilliQ® water. CD spectra were obtained using a 1-mm-pathlength quartz
cell, acquired at 297 K in 0.5-nm steps over a 185-250-nm-wavelength range. The base line acquired in the absence of peptide was subtracted, and the resulting spectra were smoothed and analyzed for the
percentages of
-helix,
-strand, and disordered structures using
the K2d Kohonen neural network program (27, 28).
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RESULTS |
NMR Spectroscopy--
Sedimentation equilibrium
measurements in aqueous solution at pH 6.9 (100 mM NaCl, 50 mM phosphate, PBS) indicated that under the conditions used
for our NMR experiments, all peptides were monomeric (Table
I). Tseng et al. (29) also
report that synthetic A
40 freshly dissolved in aqueous solution is
monomeric. In aqueous solution, there was little chemical shift
difference between the backbone amide and C
H resonances
of A
28 (A
1-28) and those of the corresponding residues of A
40
(A
1-40), suggesting that both peptides were in a similar conformation. Indeed, A
28 and A
40 backbone chemical shifts
deviated little from random coil values. This, coupled with the lack of both inter- and intra-residue nuclear Overhauser enhancement
connectivities in the nuclear Overhauser enhancement spectroscopy
spectra, indicated that both peptides were undergoing significant
conformational exchange in aqueous solution. NMR spectra were also
recorded for A
28 where the N
2 nitrogens
of the imidazole ring of the His residues 6, 13, and 14 were
methylated, hereafter referred to as Me-A
28. This peptide (purchased
from Auspep) was prepared by incorporating histidine residues that were
already methylated at the N
2 nitrogens of
the imidazole ring into the synthesis of A
28, and its identity was
verified by mass spectrometry and NMR. The spectra of Me-A
28 were
virtually identical to A
28, the only significant differences being
three strong singlets in the 1H spectrum at 3.80, 3.82, and
3.83 ppm from the methyl groups attached to the His imidazole
rings.
Metal Binding--
When Zn2+ was added to the
solutions of A
28 or A
40 in PBS, a precipitate formed. NMR spectra
of the supernatant of A
28 treated with Zn2+ showed that
peaks assigned to C2H and C4H of His6, His13,
and His14 of A
28 had broadened significantly. However,
there was little or no change in the rest of the spectrum compared with
A
28 prior to the addition of Zn2+ (Fig.
1). This broadening of the NMR peaks
caused by histidine residues is the result of the interaction of these
residues with Zn2+. The histidyl side chain is a well
established ligand of zinc in proteins and peptides (30), and this
result suggested that three of the ligands bound to Zn2+
were most likely the imidazole rings of the histidine residues. The
broadening of these peaks is the result of chemical exchange between
free and metal-bound states or among different metal-bound states. The
broadening of peaks is indicative of intermediate exchange that on the
NMR time scale suggests that the metal binding affinity is in the
micromolar range, in agreement with the low affinity site described by
Bush et al. (31). The absence of any change in the rest of
the spectrum suggests that the metal-bound form of the peptide is
monomeric and that there is little or no significant amount of soluble
oligomer in solution, because higher order aggregates would result in
significantly broadened resonances. When Cu2+ or
Fe3+ was titrated into an aqueous solution of A
28,
similar changes were observed in the 1H spectrum, with the
peaks assigned to the C2H and C4H of His6,
His13, and His14 disappearing from the
spectrum. A slight broadening of all peaks in the spectrum (associated
with the paramagnetism of Cu2+ and Fe3+) was
also observed, but there were no other major changes following the
addition of Cu2+ or Fe3+. The metal-induced
precipitation prevented the addition of enough metal to saturate the
metal-binding site. Addition of Zn2+ or Cu2+ to
an aqueous solution of A
40 (0.3 mM) at pH 6.9 caused the formation of large amounts of precipitate, as previously observed (13,
14, 31). The precipitate made the observation of NMR spectra
problematic, and few conclusions could be drawn from spectra of the
peptide that remained in solution. When Cu2+ was added to
an aqueous solution of Me-A
28, the changes observed in the spectrum
were identical to those observed for Cu2+ added to A
28,
but there was no visible precipitate.

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Fig. 1.
Amide and aromatic region of the 600 MHz
1H NMR spectra of A 28 in aqueous
PBS solution (A) and following addition of one
equivalent of Zn2+ (B). Peaks caused
by the C2H (marked with an asterisk) and C4H (marked with #)
of histidines 6, 13, and 14 have been broadened because of binding to
the zinc (approximately micromolar affinity). The rest of the
spectrum was unaffected by zinc binding. Similar spectra were observed
when Cu2+ or Fe3+ was added to A 28.
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Copper ions also induced aggregation in the rat A
28 peptide in
aqueous solution. Rat A
28 differs from human A
by three substitutions, with Arg5, Tyr10, and
His13 of human A
becoming Gly5,
Phe10, and Arg13 (32). In vitro it
has been shown that, compared with human A
, rat A
binds
Zn2+ and Cu2+ less avidly (14, 33), that the
coordination of Cu2+ or Fe3+ does not induce
redox chemical reactions, and that limited reactive oxygen species are
generated (11). 1H NMR spectra of the supernatant showed
that peaks from His6 and His14 had broadened
beyond detection, indicating that these residues were involved in
copper binding. Apart from some general broadening associated with
paramagnetic Cu2+, no other significant changes
were observed in the spectra.
We next studied metal binding to A
28 and A
40 in
SDS-micelle solution to investigate the metal binding properties
of A
in membrane-mimicking environments. Chemical shift differences
between A
28 and A
40 were very small, again suggesting that both
peptides adopt similar conformations in solution. In SDS-micelle
solution we determined that A
adopted a well defined
-helical
structure from residue 15 to the C terminus. For A
40 the helix was
kinked near residue 31. These results are similar to those recently
described (34-36).
At pH <6.5 no interaction was observed between Zn2+ and
A
in SDS-micelles. The addition of Zn2+ to A
40 in
SDS-micelles at pH 6.5 broadened resonances caused by C2H and C4H of
all three histidine residues such that they were not observed,
indicating that the zinc was in exchange with these residues, but
precipitation was not observed. Addition of more Zn2+
(~10-fold) in an attempt to saturate binding did not make the resonances observable. Raising the pH to 7 sharpened the resonances slightly, although they were still broad, suggesting that
Zn2+ binding was slightly stronger at this pH. Raising the
pH further did not measurably increase the affinity for
Zn2+ by the peptide. Further addition of large quantities
of Zn2+ (up to 200 equivalents) failed to produce sharper
resonances attributable to the histidine residues, suggesting that
binding was not saturated and is weak under these conditions.
When Cu2+ was added to A
40 in SDS-micelles at pH 5.5, resonances caused by residues in the 6-14 region were broadened as a result of their proximity to bound paramagnetic copper. This region of
the peptide contains the three histidine residues previously implicated
in copper binding, and peaks attributable to the side chains of these
residues disappear completely from the spectrum. This region also
contains residues Asp7, Tyr10, and
Glu11 that could act as ligands for Cu2+, and
peaks caused by the side chains of these residues were also broadened
beyond detection. The rest of the spectrum was largely unaffected,
suggesting that there is no major conformational change by the peptide
in SDS-micelles upon copper binding and that the helical C terminus is
unaffected. Precipitation was not observed when Cu2+ bound
to A
40 in SDS-micelle solution.
EPR Spectroscopy: Cu2+ Studies--
The EPR spectra of
Cu2+ complexed with A
28 peptide in PBS over a
metal/peptide molar fraction range of 0.2-1.0 are shown in Fig.
2. By the criteria of Peisach and
Blumberg (37), the A
(15.9 millikaisers)
and g
(2.295) values for spectrum 2A suggest a square planar configuration for Cu2+ with, most
probably, a 3N1O coordination sphere about the metal. Spectra of
Cu2+ complexed at a 0.2 molar fraction with A
42 peptide
in 35 mM SDS in PBS were identical for those of A
28 in
PBS at the same Cu2+ molar fraction. On the other hand, the
A
(15.8 millikaisers) and
g
(2.341) for the rat A
28 peptide in
PBS, which has one histidine less, fell clearly within the criteria for
a 2N2O coordination sphere. The concentration of Cu2+ in
each sample was determined by double integration of the spectra of the
complexes and of a pure standard solution of CuCl2. The concentration of Cu2+ determined in this way in each sample
was within 95% of the total copper determined by inductively coupled
plasma mass spectrometry, suggesting that, within the limits of
experimental error, reduction of Cu2+ to the EPR-silent
Cu+ had not occurred in the presence of A
28 or rat
A
28, in agreement with the findings of Huang et al. (11),
nor did reduction occur for A
42 bound to Cu2+ in
SDS-micelles.

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Fig. 2.
X-band EPR spectra of increasing
concentrations of Cu2+ added to
A 28 in PBS at pH 6.9. 0.2 mole fraction
Cu2+ (Spectrum A) was increased by 0.1 mole
fraction steps (spectra B-I) to 1 mole fraction. Frequency
was 9.485 GHz, modulation was 50 KHz, and power 2mW. Temperature was
110 K.
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When the Cu2+/peptide molar fraction was increased above
0.2, line broadening was observed with A
28 that was attributed to Heisenberg exchange effects brought about by the metal beginning to
occupy sites adjacent to those initially occupied at lower molar
ratios. As can be seen from Fig. 2, the broadening was more pronounced
with the increasing molar fraction until the spectrum was apparently
completely broadened at 1.0 mole equivalent of Cu2+, giving
a line identical to that found by Ohtsu et al. (38) for the
exchange broadened spectrum in Cu2+-bridged imidazolate
complexes. The amount of broadened line in each spectrum was determined
by subtracting from it the spectrum obtained at the 0.1 molar fraction
until the remaining line was identical to that of spectrum I
in Fig. 2. The integral of each broadened Cu2+ line was
plotted against added Cu2+ (Fig.
3, curve A) to give a
nonlinear curve, suggesting that metal binding to the
exchange-broadened sites was cooperative in nature. A Hill coefficient
of 5.9 suggested that copper binding to A
exhibits strong positive
cooperativity. When the same experiment was repeated in the presence of
excess Zn2+ (molar fraction 4.0), the exchange-broadened
curve shifted to the right (Fig. 3, curve B) and
concomitantly decreased the amount of maximal exchanged
Cu2+ by 50%. This indicates that the Zn2+
selectively competed for the Cu2+-binding sites but that
the Cu2+ binding remained cooperative. When
Cu2+ was added to Me-A
28 peptide, no Heisenberg exchange
broadening was observed in the EPR spectra up to a 0.9 molar fraction,
indicating that the peptide was not forming multimers. It appears that
metal ions have to coordinate both nitrogens on the imidazole ring of the His residues before aggregation can occur.

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Fig. 3.
Plots of Heisenberg-exchanged
Cu2+ against mole fraction Cu2+ added to
A 28 (A) or
A 28 (B) in the presence of
4.0 molar fraction Zn2+, both in PBS at pH 6.9.
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EPR Studies of the Interaction of A
Peptide with Spin-labeled
LUV--
Fig. 4 shows spectra of
1-palmitoyl-2-(16-doxyl stearoyl) phosphatidyl serine in the negatively
charged LUV in the absence (spectrum A) and the presence
(spectrum B) of a 0.05 mole fraction of A
42, coordinated
by 0.3 mole equivalents of Cu2+. Spectrum C was
obtained by subtracting the broad Cu2+ line from
spectrum B and shows that there is a relatively immobilized component in the spectra. Increasing amounts of spectrum A of the
control LUV preparation were subtracted from spectrum C
until a spectrum with a clear end point was obtained (Fig. 4,
spectrum D). Attempts were made to determine whether the
presence of the peptide extensively perturbed the bulk lipid, as
suggested by McIntyre et al. (39), by subtracting the
spectrum of the control LUV recorded at 300 K. This assumes that the
lower temperature spectrum would simulate that of bulk lipid, the
motion of which had been restricted by long range effects arising from
the presence of the peptide. Using the lower temperature spectrum did
not lead to a clear end point, and it was impossible to obtain a well
behaved first integral with no negative excursions below the base line (40). It was concluded, therefore, that long range effects of the type
described by McIntyre et al. (39) were absent in our system.
It was also possible to simulate the spectrum as described by Davoust
and Devaux (26). The proportion of the slow motion component in
spectrum C was therefore calculated by double integration of
spectrum D. Spectra were then run over a range of
Cu2+A
/lipid ratios, from 0.025 to 0.15 mole fraction,
and the proportion of slow motion component at each mole fraction is
plotted in Fig. 4 (bottom panel). The relationship between
the mole fraction and proportion of slow component was linear,
suggesting that even at a fraction of 15%, all of the peptide was
associated with the lipid and that the aggregation of the peptide did
not increase with the increasing mole fraction. The fact that this
ratio did not change when peptide:lipid ratios or peptide:metal ratios
changed suggested that the structure that penetrated the lipid membrane was well defined. The lipid:peptide ratio of ~4:1 is much lower than
the value of 10:1 usually associated with a single
-helix penetrating the hydrophobic region of the membrane yet higher than the
expected ratio for
-sheet conformation, which is 1-2 lipids/strand,
depending on the tilt of the structure in the bilayer (41).

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Fig. 4.
X-band EPR spectra of negatively charged LUV
made from 50% POPS and 50% POPC containing the negatively charged
spin probe 1-palmitoyl-2-(16-doxyl stearoyl) phosphatidyl serine
(probe/lipid 1/300) (spectrum A) and X-band spectra of
system A at the same temperature after the addition of
Cu2+A 42 (peptide/lipid 1/50)
(spectrum B). The curved base line of
spectrum B is due to the broad copper resonance at high
temperature (296 K). Spectrum C, spectrum B with
the broad Cu2+ line subtracted, showing a shoulder to the
left of the low field line. This is typical of peptide
penetration into the bilayer core. Spectrum D,
difference spectrum obtained when spectrum A is subtracted
from spectrum C. This spectrum represents the motionally
restricted lipid in the boundary layer of the peptide. All spectra were
recorded at 305 K. The bottom panel is plot of integrals of
spectrum C, giving the percentage of boundary lipid
obtained at increasing concentrations of added Cu2+ bound
A 42.
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The Cu2+ A
42 peptide/negatively charged phospholipid
mixtures were examined at 110 K to determine the nature of the
coordination sphere about the copper bound to A
. They gave spectra
characteristic of those illustrated in Fig. 2, except that a small
amount of broadening was evident at the high field end of the line
caused by the contribution of frozen phospholipid spin label. The
copper spectra taken at a 0.3 mole fraction of Cu2+ showed
no trace of exchange broadening, but at Cu2+ mole fractions
above 0.5 there was exchange broadening characteristic of the spectra
shown in Fig. 2. Measurement of the parameters of the copper EPR signal
at a 0.3 mole fraction showed that the coordination sphere about the
copper ions in each case was 3N1O.
A similar relatively immobilized component was found when
Zn2+ coordinated to A
42 was added to the negatively
charged LUV, except that much higher concentrations of metal (up to a
molar ratio of 4) were required. This reflects the lower affinity of zinc for the metal-binding sites of A
(31, 42, 43), suggesting that
the metal is playing a structural role and that redox chemistry is
not involved in this process.
These experiments were repeated in the presence of the chelating agent
diethylenetriaminepentaacetic acid at a 2:1 molar ratio relative to
Cu2+. No sign of peptide penetration of the bilayer was
then evident in the EPR spectra, establishing that A
42 penetration
of the membrane was a consequence of metal binding. No bilayer
penetration was observed over a range of mole fractions from 0.025 to
0.1 for Cu2+A
28. Similarly, no penetration over this
mole fraction range was observed with the zwitterionic phospholipid
DOPC LUV and Cu2+A
42 probed with 1-palmitoyl-2-(16-doxyl
stearoyl) phosphatidyl choline, indicating that A
penetration only
occurs with negatively charged membranes.
CD Spectroscopy--
CD spectra were obtained on freshly
prepared solutions of A
42 with one molar equivalent of
CuCl2, in the presence of negatively charged LUV so that
the peptide:LUV mole fraction was 0.15. Spectra were also obtained of
0.15 mole fraction A
42 in 1:1 DOPC:DPPS LUV and 1 mM
DTPA. In the absence of Cu2+ in a membrane environment
(Fig. 5, curve A), A
42 was
mainly
-sheet, as indicated by the minimum at ~220 nm. There is a
small inflection in the spectrum at 228 nm. This has been observed
previously in CD spectra of lipid-bound peptides and was believed an
artifact caused by anomalous light scattering. The structures of these peptides were confirmed as
-sheets by Fourier transform infrared spectroscopy in the same lipid (44).

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Fig. 5.
Spectrum A, CD spectra of A 42 bound
to negatively charged LUV. The minimum at 220 nm is diagnostic of a
-sheet structure. Spectrum B, following addition of
Cu2+, the double minima at 208 and 222 nm are classic
indicators of -helix structure. Spectra were recorded at 298 K, pH
6.9.
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A
42 in the presence of Cu2+ was significantly more
-helical, as indicated by a double minimum at 208 and 222 nm (Fig.
5, curve B). The helix content was calculated to be 57% for
the Cu2+-containing system and 8% in the absence of the
metal, indicating that it is involved in converting A
42 from
predominantly
-sheet to predominantly
-helix in this
membrane-mimetic environment. This is consistent with Cu2+
promoting A
42 membrane insertion as helical multimers.
Reduction of Cu2+ in the Presence of
Phospholipids--
Double integration of the copper EPR spectrum of
aliquots taken at 30-s intervals of the Cu2+ A
1-42
peptide/acidic phospholipid mixtures at 110 K determined the amount of
the Cu2+ in each sample. We assumed that the diminution in
the EPR signal was due to Cu2+ reduction to Cu+
and not due to the formation of antiferromagnetically coupled (S = 0)
dicopper because it has already been shown by other assays that A
42
reduces Cu2+ (11, 12). As shown in Fig.
6A, there was a 25% reduction of Cu2+ to Cu+ by A
42 in the presence of
acidic phospholipid, as compared with <10% for the peptide in SDS and
almost 100% reduction in aqueous buffer. In neutral lipids there was
almost 90% reduction of Cu2+.

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Fig. 6.
A, plot of the reduction of
Cu2+ versus time in the presence of A 42 in
SDS ( ), LUV ( ), and aqueous PBS, pH 6.9 ( ). B,
reduction of Cu2+ by A peptides in the presence of two
mole equivalents of methionine in various solvents. , A 28 in PBS;
, A 28 in SDS + Met; , A 42 in SDS + Met; , A 42 in
POPS/POPC LUV + Met; , A 42 in PBS.
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Role of Methionine in the Reduction of Cu2+ by
A
--
The decrease in the reduction of Cu2+ to
Cu+ by A
42 in the presence of acidic phospholipids and
SDS might have been due to the seclusion of the hydrophobic
Met35 residue in the hydrocarbon core lipid bilayer of the
LUV or the interior of the SDS-micelles. Therefore, we studied the
effect of adding soluble methionine to the metal-peptide mixtures.
Cu2+A
28 was also studied, because it lacks the
hydrophobic region containing the Met residue. As shown in Fig.
6B, adding methionine (mole fraction 2.0) to the
metal-peptides dramatically increased the rate of reduction of
Cu2+ in each case, supporting a critical role for
Met35 in the redox activity of A
.
 |
DISCUSSION |
The Structure of the Zn2+ and Cu2+-binding
Sites on A
Resembles Superoxide Dismutase 1--
NMR and EPR
evidence (Figs. 1 and 2) revealed that the Zn2+ and
Cu2+ coordinate to the three His residues of A
28. Zinc
and copper are normally tetravalent, but the NMR spectra give no
definitive indication as to what the fourth ligand might be. The EPR
spectra of human A
28 and A
42 plus Cu2+ suggest an
oxygen ligand that could come from one of the carboxyl or hydroxyl side
chains, water/hydroxo, or phosphate from the buffer. The evidence of
Miura et al. (45) suggests that for copper the oxygen ligand
is the hydroxyl group from the side chain of Tyr10. The
fourth ligand for Zn2+ has not been identified, but the
lack of changes observed in the rest of the NMR spectrum upon the
addition of Zn2+ favor the water/hydroxo or phosphate
options. Fig. 7A shows a model
of the initial and Cu2+-binding sites of A
incorporating
His6, His13, His14, and
Tyr10.

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Fig. 7.
Models of the coordination site(s) of
Cu2+ bound to A .
A, the initial coordination site of Cu2+ on A
in solution (His6, His13, His14,
and Tyr10) as determined by NMR and EPR spectroscopy.
B, a proposed model explaining the aggregation, cooperative
binding, and redox properties of metal bound A peptides. The
imidazole ring of His6 is shown forming a bridge between
copper atoms to form a dimeric species; this residue is used as an
example, and other histidine residues could form similar bridges and
therefore lead to aggregation. The coordination sphere about the metal
ions is similar to that observed in the active site of SOD. The models
were generated by binding Cu2+ to the appropriate residues
of A 28 with the coordination sphere about the copper atoms
constrained to a square planar configuration; the rest of the molecule
was not constrained. Structures were then energy minimized
in vacuo with a distance-dependent dielectric
using the esff force field within the Discover module of
Insight98.
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The NMR evidence indicates that initial metal binding does not cause a
significant structural change in the peptide, indicating that
metal-induced aggregation is not mediated by metal-induced conformational changes. One possibility is that the metal could bridge
between histidine residues of different peptides as proposed by Miura
et al. (45). Another possibility involves histidine ligands
acting as bridges between metal centers on different peptides. When a
metal ion binds to the N
1 of a histidine
residue, it reduces the pKa of
N
2 NH, making this nitrogen more available
for metal coordination (46). This results in a histidine residue that
is capable of bridging metal ions, the best known example being
His63 at the active site of superoxide dismutase (SOD)
(47). Similar bridging histidine residues have been proposed within the
octarepeat region of the prion protein when this protein binds
Cu2+ (48). Miura et al. (45) reported that
complexes containing bridging histidine residues were formed between
Zn2+ and A
but were not observed for Cu2+
and A
. It is important to note that the Heisenberg exchange broadening observed at Cu2+ mole fractions >0.3 with A
was eliminated when the N
2 of three
imidazole rings of the His residues were methylated, thereby preventing
the formation of histidine-bridged complexes. This evidence provides
definitive proof that the imidazole rings of the His residues of A
are able to bridge between metal centers under the conditions studied
here. Fig. 7B shows how the histidine could act as a bridge
between metal centers; the distance between copper ions in this model
is 6 Å, and exchange phenomena would be evident in the EPR of such a
complex, matching those observed (Fig. 2). It is the bridging histidine
that is probably responsible for the reversible metal-induced
aggregation that is observed when A
is metallated with
Cu2+ and Zn2+. Bridging histidine residues
would also explain the multiple metal-binding sites observed for each
peptide (43) and the high degree of cooperativity evident for
subsequent metal binding (Fig. 3). With three histidines bound to the
metal center there are several potential sites for further coordination
of metal ions such that a large scope exists for metal-mediated
cross-linking of the peptides leading to aggregation, which will be
reversible when the metal is removed by chelation. This type of metal
binding with bridging histidine residues would result in complexes very similar to the active site of SOD. The occurrence of exchange broadening in the Cu2+ EPR spectra of A
28 and its
modulation by Zn2+, which has also been observed in model
SOD imidazolate-bridged dinuclear complexes (38), further suggests the
occurrence of structured complexes that are not merely random
aggregates of peptide.
Cu2+ and Zn2+ Induce A
to Form
Allosterically Ordered Multimers That Penetrate Lipid
Membranes--
Zn2+ and Cu2+ bound to the same
site on A
in SDS-micelles as in aqueous solution, although there
were significant differences in the effects of metal binding.
Metal-induced aggregation and reduction of Cu2+ to
Cu+, as observed for A
in aqueous solution (11, 12, 14),
were not observed when copper bound to A
40/42 in SDS-micelles or
LUVs. One of the effects of SDS is to drive the C-terminal part of the peptide (residues 15-42) into an
-helical conformation, preventing the formation of aggregated
-sheet structures. The
-helical region of the peptide starts just after the metal-binding site. This
result suggests that the peptide may need to be in a
-sheet form
before it becomes redox-active, which would be consistent with previous
observations that the neurotoxicity associated with A
also requires
the presence of aggregated
-sheet structures (49-51). However,
results with the addition of exogenous methionine that are discussed
below show that
-sheet/aggregation are not necessary for Cu/A
to
produce redox-active species.
We found that as a consequence of Zn2+ and Cu2+
binding, A
42 forms allosterically ordered,
-helical structures
that penetrate negatively charged membranes. These results suggest that
the A
42- and A
43-induced decrease in fluidity in the hydrocarbon
core of human cortex membranes as measured by fluorescence polarization (52) was most likely due to association of the membrane lipids with the
hydrophobic face of the rigid peptide. There have also been reports of
cation channel formation (53, 54) by A
40 and A
42 that indicate
penetration of the bilayer. Many previous studies have indicated that
A
peptides form
-sheet structures in lipid environments (55-57),
although the structure appears to be dependent on the composition of
the lipid. For example, it was shown that A
1-40 was
-helical in
phosphatidyl glycerol vesicles but adopted a
-sheet conformation
when GM1 ganglioside was added (58). Further, the physical state of the
lipid systems used varied widely, with small unilamellar vesicles being
the most common. In many cases, the temperature at which the
measurements were made was not reported, leaving some doubt as to
whether the lipids were in the liquid crystal or gel state. It is
highly probable that the A
peptides are pleiomorphic, able to adopt
different conformations in different lipid environments. Because the
cell membrane is a mosaic of different lipid environments, it is even possible that the peptides will exhibit different structures with different properties in different parts of the mosaic.
We found that approximately four lipids are associated with each
membrane-penetrating peptide subunit (Fig. 4, bottom panel), which is a low ratio for lipid association with a single
-helix and
suggests the presence of oligomers penetrating the lipid. Preliminary
modeling showed that 24 lipid molecules would fit around an oligomer of
six helices. Taken with the value of 5.9 for the Hill coefficient
calculated from the data in Fig. 3 (curve A), the modeling
suggests that A
42 forms an allosterically induced hexamer in the
presence of metal ions. The metal ions do not penetrate the membrane
but form structures very similar to the active site of SOD on the
surface of the membrane.
Earlier studies of A
peptide-membrane interaction were not
controlled for the presence of metal ions, ubiquitous trace
contaminants, the concentration of which was minimized in our controls
by the use of excess chelator. It is possible, therefore, that the
other reported results of A
penetration into the membrane, measured by sensitive methods, might have been initiated by the peptide binding
trace Cu2+ or Zn2+.
The failure of Cu2+:A
28 to perturb the vesicle bilayer
shows that, as is the case with the redox activity of the peptide (11, 12), the hydrophobic C terminus is essential for the interaction. The
absence of line broadening, which is characteristic of Heisenberg exchange between free radicals and transition metals (59), in the
phospholipid nitroxide label spectrum in the LUV interacting with the
Cu2+/A
42 also indicates that the
Cu2+-binding site is outside the bilayer. The role of
Cu2+ in the interaction may indicate that the peptide must
be oligomerized for it to penetrate the bilayer. It has been suggested
recently (60) that aggregation of A
peptides might be a prerequisite for penetration of the lipid bilayer.
The Role of Methionine in A
Redox Reactions--
It has been
reported that Met35 is essential for the toxicity and
induced oxidative stress of A
(61, 62). The lack of redox activity
associated with the copper interactions with A
28 suggested that the
Met of A
42 incubated in SDS or negatively charged LUV was buried in
the hydrophobic core of the micelles and vesicle bilayers and therefore
unavailable as a cofactor in the metal reduction. Addition of exogenous
methionine restored redox activity, albeit with a much slower rate of
reaction. These results suggest that Met35 participates in
the reduction of copper by A
. Although A
28 and A
42 in LUV with
bound copper have multiple copper-binding sites resulting in
peptide aggregation, A
42 in SDS is monomeric in the presence of
bound Cu2+ with a single metal-binding site as determined
by the lack of Heisenberg interactions in the EPR spectra. The
coordination sphere of Cu2+ under these conditions includes
the three histidine residues and an oxygen ligand, probably
Tyr10, and this is the initial copper-binding site of A
.
The effects of the addition of methionine (Fig. 6) showed that this is
potentially a redox-active site.
Pathological versus Functional Metal Binding to A
: a Model for
Alzheimer's Disease--
We have shown previously that
Cu2+ and Zn2+ binding to A
modulate the
toxicity of the peptide through the generation of
H2O2 by electron transfer to O2 (9,
12). Cu2+ and Zn2+ binding to A
also induces
the precipitation of the peptide (13, 14, 31, 42). Increased binding of
these metals to A
is evident in AD (18), and we have found recently
that the metal-mediated redox activity and aggregation of A
, as well
as amyloid deposition in APP2576 transgenic mice, are inhibited by
treatment with a bioavailable zinc/copper-selective chelator,
clioquinol (19). Therefore, the structural characterization of the
Cu2+- and Zn2+-binding sites on A
may be
essential for elucidating the pathogenesis of AD, as well as for
developing new therapeutics.
In light of our current findings that Cu2+ and
Zn2+ binding induce monomeric A
to form allosterically
ordered SOD-like metallopeptide complexes that insert into negatively
charged membranes, it is interesting to speculate on the likelihood
that this redox-inert A
assembly may be biologically relevant and
subsume some function. We have recently reported that
Cu2+/Zn2+-bound A
possesses significant SOD
catalytic activity (69). Our current structural data support the
possibility of such SOD-like activity. Physiologically, the combination
of A
with a lipid vesicle occurs in high density lipoproteins
(HDLs), which are found in plasma and cerebral spinal fluid (63, 64).
HDLs possess antioxidant properties (65-67), and we hypothesize that
copper/zinc-A
complexes inserted into HDL membranes may play a role
in superoxide clearance by HDLs. Supporting a role for HDLs in
nullifying the adverse redox activity of soluble A
, HDLs are able to
decrease A
toxicity in cortical cell culture (68). In the heuristic model that we contemplate, the perturbation of Zn2+ or
Cu2+ homeostasis that is associated with AD (15) may
interfere with A
insertion into membranes, liberating increased
amounts of neurotoxic, redox-active A
.