Báo cáo khoa học: High-resolution NMR studies of the zinc-binding site of the Alzheimer’s amyloid b-peptide pdf

High-resolution NMR studies of the zinc-binding site
of the Alzheimer’s amyloid b-peptide
Jens Danielsson
1
, Roberta Pierattelli
2
, Lucia Banci
2
and Astrid Gra
¨
slund
1
1 Department of Biochemistry and Biophysics, Stockholm University, Sweden
2 Department of Chemistry and Magnetic Resonance Center, Universita
`
di Firenze, Sesto Fiorentino, Italy
The amyloid b-peptide (Ab) is the major component
of senile plaques and soluble oligomers, amyloid
b-derived diffusible ligands, which are considered to
play an important role in Alzheimer’s disease (AD)
pathology. Ab is the product of cleavage of amyloid
precursor protein (APP) [1] at position(s) 39–42, which
creates a soluble monomer.
There is evidence that APP is involved in copper
homeostasis. APP has a selective metal-binding site
and is able to reduce bound Cu(II) to Cu(I). It also
participates in the regulation of copper levels, and its
expression is affected by copper concentration. Also
other metal ions, such as Zn
2+
and Fe

2+
, are known
to interact with APP [2–6].
The Ab peptide mainly appears as a random coil
in aqueous solution, but contains some secondary
structure elements: A poly(proline II) helix (PII) in
the N-terminus, and two b-strands in the central
part and in the C-terminus [7–9]. The soluble mono-
meric peptide has a high tendency to form Ab oligo-
mers, which eventually produce Ab fibrils. Although
the monomeric peptide is not neurotoxic and neither
are the fibrils, Ab oligomers have been shown to
induce cognitive loss due to neurodegeneration, via
Keywords
aggregation; Alzheimer amyloid b-peptide;
copper binding; NMR; zinc binding
Correspondence
A. Gra
¨
slund, Department of Biochemistry
and Biophysics, Stockholm University,
S-106 91 Stockholm, Sweden
Fax: +46 8 155597
Tel: +46 8 162450
E-mail:
(Received 22 June 2006, revised 20 October
2006, accepted 27 October 2006)
doi:10.1111/j.1742-4658.2006.05563.x
Metal binding to the amyloid b-peptide is suggested to be involved in the
pathogenesis of Alzheimer’s disease. We used high-resolution NMR to

study zinc binding to amyloid b-peptide 1–40 at physiologic pH. Metal
binding induces a structural change in the peptide, which is in chemical
exchange on an intermediate rate, between the apo-form and the holo-form,
with respect to the NMR timescale. This causes loss of NMR signals in the
resonances affected by the binding. Heteronuclear correlation experiments,
15
N-relaxation and amide proton exchange experiments on amyloid b-pep-
tide 1–40 revealed that zinc binding involves the three histidines (residues 6,
13 and 14) and the N-terminus, similar to a previously proposed copper-
binding site [Syme CD, Nadal RC, Rigby SE, Viles JH (2004) J Biol Chem
279, 18169–18177]. Fluorescence experiments show that zinc shares a com-
mon binding site with copper and that the metals have similar affinities for
amyloid b-peptide. The dissociation constant K
d
of zinc for the fragment a-
myloid b-peptide 1–28 was measured by fluorescence, using competitive
binding studies, and that for amyloid b-peptide 1–40 was measured by
NMR. Both methods gave K
d
values in the micromolar range at pH 7.2
and 286 K. Zinc also has a second, weaker binding site involving residues
between 23 and 28. At high metal ion concentrations, the metal-induced
aggregation should mainly have an electrostatic origin from decreased
repulsion between peptides. At low metal ion concentrations, on the other
hand, the metal-induced structure of the peptide counteracts aggregation.
Abbreviations
Ab, amyloid b-peptide; AD, Alzheimer’s disease; APP, Alzheimer precursor protein; HSQC, heteronuclear single quantum coherence; LMCT,
ligand to metal charge transfer.
46 FEBS Journal 274 (2007) 46–59 ª 2006 The Authors Journal compilation ª 2006 FEBS
pathophysiologic mechanisms that are not completely

understood. The Ab oligomers are thus more neuro-
toxic than the Ab fibrils [10–15]. The detailed struc-
ture of these Ab oligomers has yet to be resolved,
although they seem to be similar to other amyloid-
forming peptide oligomers [16].
The aggregation process is accompanied by a signifi-
cant change in structure, whereby monomeric, mostly
unstructured A b folds to form oligomeric b-sheet-rich
forms. The detailed mechanism of this transition is not
known.
Increased metal concentrations (mainly copper, iron
and zinc) have been found in the brains of AD
patients, both in the amyloid plaques (copper and zinc)
and in the cortical tissue (zinc) [17,18]. Interactions of
copper and zinc with Ab induce peptide aggregation if
the metal concentration is high enough, i.e. > 1 : 1
metal ⁄ peptide ratio [19–21]. The formed aggregate is
suggested to be amorphous, and thus high concentra-
tions of copper and zinc prevent fibril formation by
promoting the formation of nonfibril aggregates
[20,22–24]. The N-terminal fragment Ab(1–28) has,
however, been shown to undergo increased fibril
formation upon interaction with zinc [25]. Another
N-terminal fragment of Ab,Ab(1–16), forms stable
oligomers in the presence of both copper and zinc [26].
On the other hand, binding of low concentrations of
copper or zinc ions to the full-length Ab(1–40) at less
than 1 : 5 metal ⁄ peptide ratio reduces the Ab oligo-
meric stability and prevents aggregation, whether
amorphous or fibril forming [27,28]. The effect of

metals on Ab is thus dependent on the experimental
conditions, such as pH, salt concentration and, most
important, metal concentrations [21,29]. In addition,
Ab-bound Cu(II) may be reduced to Cu(I), and the
complex may produce hydrogen peroxide, which has
been suggested to be neurotoxic in AD [30].
Soluble monomeric Ab has a high-affinity copper-
binding site in the N-terminus, and the metal ion is
suggested to coordinate in a planar configuration with
the three histidines (6, 13 and 14) and the N-terminus
or Tyr10. N-terminal deletions alter the binding affin-
ity, suggesting that the N-terminal amide participates
in copper coordination and that Tyr10 does not [31–
34]. The metal-binding site, involving the three histi-
dines, is also able to bind a zinc ion. The fourth ligand
for zinc binding has been suggested to be either Tyr10,
Glu11, Arg5 or the N-terminus [35–37].
Both copper and zinc have been suggested to have a
second binding site. The ligands of this hypothetical
weaker binding site are unknown [31,38,39].
The binding affinity of the metal ions to Ab is
pH-dependent: copper has a higher affinity under mild
acidic conditions, whereas the affinity for zinc is less
pH-dependent over a range of pH values between 6.5
and 7.5 [29]. At pH below 6, when the histidines are
mainly protonated, no zinc binding occurs [21]. Under
physiologic conditions, Ab has a higher propensity to
bind zinc, whereas under mildly acidic conditions, as
in physiologic acidosis following an inflammatory pro-
cess, copper is preferentially bound [40]. This differ-

ence between copper and zinc affinity at different pH
values has been suggested to be a pathogenic mechan-
ism of Ab: under normal conditions, zinc protects
against copper-induced Ab toxicity, which is induced
by physiologic acidosis [22,29,40].
The present study shows for the first time the
detailed molecular effects on the full-length Ab of zinc
binding. Thanks to the combined use of heteronuclear
NMR, fluorescence and CD spectroscopy, a molecular
model for the zinc interaction in solution is obtained.
We show that the high-affinity zinc-binding site is
formed by three histidines and the N-terminus,
whereas a second, low-affinity binding site comprises
residues 23–28. The results of these studies are dis-
cussed within the context of the general mechanism of
the onset of pathologic states upon Ab aggregation.
Results
NMR spectroscopy
Interaction with zinc ) heterocorrelation experiments
Ab(1–40) in aqueous solution has been extensively
characterized by NMR spectroscopy, mainly through
two-dimensional
1
H–
1
H NMR and
1
H–
15
N-heteronuc-

lear single quantum correlation (HSQC) experiments.
In
1
H–
15
N correlation experiments, some signals are
affected by an exchange process with the solvent and
are missing in the spectra. Thus, despite the good reso-
lution, not all resonances are visible in the
1
H–
15
N
HSQC spectra. In contrast,
13
C-bound protons are not
affected by an exchange process and show a suitable
signal dispersion, which allows high-resolution charac-
terization. Therefore, the combination of these two sets
of data (
1
H–
15
N and
1
H–
13
C) provides a detailed char-
acterization of metal binding to Ab. As the metal ions
could be sequestered from solution by complexation

with the buffer, we can only obtain an estimate of the
order of magnitude of the dissociation constants. On
the other hand, from NMR we obtain an unambigu-
ous description of the binding.
When zinc ions are added to a solution of Ab(1–40),
all the signals in
1
H–
15
N and
1
H–
13
C HSQC experi-
ments are reduced in intensity. At one equimolar zinc
concentration, the remaining intensity fraction for the
J. Danielsson et al. Zinc-binding site of the amyloid b -peptide
FEBS Journal 274 (2007) 46–59 ª 2006 The Authors Journal compilation ª 2006 FEBS 47
CH
a
signals of the residues in the C-terminal region is
0.69 ± 0.04, whereas His13 has a relative intensity of
0.15 ± 0.02, and His6 and His14 have relative intensi-
ties of 0.3 ± 0.01. This observation confirms the
involvement of the histidines as metal ligands. Asp1
shows a 0.60 residual signal intensity, whereas Val12
has a residual signal intensity of 0.52 ± 0.06. The sig-
nal reduction of Asp1 is equal to the reduction seen
for other residues located in the N-terminal region,
such as Ser8 and Arg5. Asp1, His6, Val12, His13 and

His14 also show small but significant chemical shift
changes (0.010–0.015 p.p.m. in the proton dimension)
upon zinc binding (Fig. 1). The chemical shift varia-
tions are probably the result of conformational chan-
ges of the backbone upon metal binding. For other
residues, no chemical shift changes are observed. The
C
b
region shows the same signal intensity reduction
pattern but with no or very small induced chemical
shift changes. The selective spectral changes observed
upon zinc addition for Asp1, His6, His13 and His14
clearly indicate the involvement of these residues in
metal coordination. In particular, the specific changes
in the Asp1 chemical shift provide a direct indication
of the involvement of the N-terminus in zinc binding.
The chemical shift change observed for Val12 is prob-
ably due to its proximity to the histidine ligands.
V12
D1
Y10
H6/H14
H13
Fig. 1.
1
H–
13
C HSQC spectra of the C
a
region of 50 lM Ab(1–40) in 10 mM phosphate buffer at pH 7.4 and 286 K without (black) and with

(red) 30 l
M zinc as chloride. All resonance peaks show reduced intensity, but some specific reduction is present for the histidines, Asp1,
Tyr10 and Val12. The resonances of His6, His13, His14, Asp1 and Val12 exhibit induced chemical shift changes upon zinc binding. The inset
shows the broadening and chemical shift changes of Asp1 upon zinc titration with 0, 20 and 50 l
M zinc, indicating the induced chemical shift
changes.
Zinc-binding site of the amyloid b -peptide J. Danielsson et al.
48 FEBS Journal 274 (2007) 46–59 ª 2006 The Authors Journal compilation ª 2006 FEBS
The aromatic region of the
1
H–
13
C HSQC spectrum
is particularly informative about the effects of Zn
2+
(Fig. 2). The Tyr10 aromatic resonances are unchanged
upon zinc addition, indicating that the aromatic ring of
Tyr10 is not involved in binding of the metal. Instead,
the e and d resonances of the histidines show significant
intensity losses, of > 80%. The d-proton signals of His6
and His13 show some induced chemical shift changes,
whereas His14 only exhibits intensity loss for the d-pro-
ton signal (Fig. 2A). Furthermore, there is a total loss of
the e-protons of the His6 and His14 signals and a weak-
ening (> 80% reduction) of His13 resonance (Fig. 2B).
The zinc binding can thus be envisaged as coordination
of the metal ion by the nitrogens of the imidazole rings
of the histidines and the amino group in the N-terminus.
Use of the signal intensity reduction of the
1

H–
13
C
HSQC crosspeaks to estimate the dissociation constant
of zinc yields K
d
 1 lm at pH 7.2, in phosphate buf-
fer, at 286 K.
Also, the
1
H–
15
N HSQC spectrum shows that the
reduction in intensity does not affect all the signals to
the same extent. This signal reduction, where the signal
is lost due to addition of zinc, could as a first approxi-
mation be interpreted in terms of a first-order binding
process. An apparent dissociation constant
app
K
d
* can
be calculated; the star indicates that the dissociation
Y10
Y10
A
B
H6
H14
H13

H13
H14
H6
A
B
Fig. 2.
1
H–
13
C HSQC spectra of the aroma-
tic regions of 50 l
M Ab(1–40) in 10 mM
phosphate buffer at pH 7.4 and 286 K with-
out (black) and with (red) 30 l
M zinc as
chloride (red). The d and e resonances of
the histidines shown in (A) and (B), respect-
ively, show significant signal intensity los-
ses. All three histidines are affected. In (A),
it is shown that H6 and H13 show some
induced shift but H14 only exhibits signal
intensity loss for the d-protons. The e-pro-
tons shown in (B) exhibit a total loss of H6
and H14 resonances, and a weak H13 res-
onance remains at the same chemical shift.
The tyrosine aromatic crosspeaks are unaf-
fected upon zinc addition.
J. Danielsson et al. Zinc-binding site of the amyloid b -peptide
FEBS Journal 274 (2007) 46–59 ª 2006 The Authors Journal compilation ª 2006 FEBS 49
constant calculated here is not a quantitative measure

of binding of zinc by the molecule, but rather a meas-
ure of which residues are most involved in the binding.
Such an apparent dissociation constant was calculated
for all individual resonances (Fig. 3A). The apparent
dissociation constant varies along the peptide chain,
indicating that this is a generalized description of
binding, which in turn reflects the induced changes in
the peptide upon binding. The C-terminus residues 29–40,
which are not directly affected by the zinc interaction,
show a fairly constant apparent dissociation constant,
but four signals, originating from residues 23, 24, 26
and 29, are significantly more affected. In the N-termi-
nus, the regions corresponding to the binding site show
a very high apparent affinity constant. With a simpler
approach, the decrease in intensity of the crosspeaks
can be studied, and using this approach a similar pat-
tern is observed (data not shown). It can be seen that,
in addition to a reduction over the entire polypeptide
sequence, a more dramatic effect is observed for the
first 15 residues. This behavior is similar to that
observed with
13
C HSQC, and can be interpreted in
the same terms as specific binding of the metal in the
N-terminal part of the peptide.
These observations confirm that the zinc-binding site
is in the N-terminus, and are consistent with three his-
tidines participating in the binding, possibly with the
formation of a bend or a turn in region 7–12 and one
app

K
d
/µM
R
2
Difference
Amide Proton Stability
Change
A
B
C
10
0
0
-4
-8
0.4
0.2
0
0.4
0.2
0
residue
Relative Signal Intensity
D
** * **
**
***
**
***

**
*
**
11020 30 40
Fig. 3. (A)
1
H–
15
N HSQC signal intensity of
50 l
M Ab(1–40) in 10 mM phosphate buffer
(pH 7.2) at 286 K. The calculated apparent
dissociation constant,
app
K
d
*, of zinc for Ab.
A second interaction site is suggested in
residues 23, 24, 26 and 28. The residues
indicated with an asterisk (*) were not
observed, due to rapid exchange with sol-
vent water. (B) Change in transverse relaxa-
tion rate, R
2
, upon addition of zinc under the
same conditions as in (A). The increase in
R
2
upon addition of zinc (negative differ-
ence) suggests greater rigidity at the binding

site. (C) The amide proton exchange vari-
ation with temperature measured as des-
cribed in the text. The amide proton stability
is increased along the whole peptide. How-
ever, the stability increase is more promin-
ent close to the binding site. This approach
shows a good correlation with relaxation
data. (D) Signal intensity decrease upon cop-
per binding. The fractional remaining inten-
sity after addition of 20 l
M copper to 50 lM
Ab. Copper binding shows the same pattern
as zinc binding.
Zinc-binding site of the amyloid b -peptide J. Danielsson et al.
50 FEBS Journal 274 (2007) 46–59 ª 2006 The Authors Journal compilation ª 2006 FEBS
in region 2–5. The
1
H–
13
C HSQC and
1
H–
15
N HSQC
results show that Tyr10 does not participate in zinc
binding, supporting previous proposals for copper
binding [25,31,32]. The presence of a second binding
site for zinc is suggested by the selective effect for
some residues in the C-terminal region.
The general signal intensity reduction in the

1
H–
15
N
HSQC spectra due to zinc binding could have multiple
origins, such as aggregation, the presence of chemical
exchange rate between conformations in an intermedi-
ate regime with respect to the NMR timescale,
increased amide proton exchange rate in the binding
region, and ⁄ or increased relaxation rates due to
decreased dynamics in the binding region. We favor
aggregation as the main explanation for the general
intensity loss, as this should give signal reduction due
to the line-broadening of the large aggregates.
In the
1
H–
13
C HSQC spectrum, there is some cross-
peak intensity left at high zinc concentrations, whereas
there is no residual crosspeak intensity in the amide pro-
ton region of the
1
H–
15
N HSQC spectra. This different
feature suggests that in the
1
H–
15

N HSQC case, there
are further mechanisms (in addition to aggregation and
metal-binding effects) that lead to specific reductions in
signal intensity, e.g. relaxation effects and proton
exchange effects. In order to vary the chemical exchange
rate and to be able to detect additional resonances,
a
1
H–
15
N HSQC spectrum was recorded at a lower
temperature (278 K). As shown in Fig. 4, four new
crosspeaks appeared, suggesting that some additional
residues exhibit slower exchange at lower temperatures.
The intensity of the new peaks was too low to attempt
the assignment of the newly identified resonances.
Relaxation and diffusion measurements
The local mobility changes upon zinc binding to Ab
were studied by NMR relaxation measurements.
Amide H
N
R
1
and R
2
were measured. The average R
1
value for the peptide with no zinc bound is
2.16 ± 0.40 s
)1

and is unchanged upon zinc binding.
This suggests that the peptide remains monomeric
upon zinc binding. This was also confirmed using
pulsed field gradient-NMR diffusion studies. Ab(1–40)’s
diffusion coefficient is 1.04 ± 0.02 · 10
)10
m
2
Æs
)1
at
286 K, which is in fairly good agreement with the
expected value of 1.09 · 10
)10
m
2
Æs
)1
, with the visco-
sity changes accounted for by an empirical function
[8,41]. Upon zinc titration, no significant change of dif-
fusion coefficient was measured (data not shown). All
changes were within the range of experimental error,
suggesting that no stable small soluble oligomers of
the peptide were induced by zinc binding. The zinc-
induced peptide aggregates immediately grow to sizes
where the linewidth in the NMR spectra broadens
beyond detectable limits, so the relaxation and diffu-
sion properties of the aggregate cannot be measured
with NMR.

G38
G29
G9
G25
G37
G33
S8
S26
N27
E3
E22
I31
V24
V39
V12
V18
L34
Y10
A30
R5
F19
L17
F20
V36
F4
D7K28
M35
D23
Q15
K16

E11
I32
A21
V40
Fig. 4.
1
H–
15
N HSQC spectra of 75 lM
Ab(1–40) in 10 mM phosphate buffer at
pH 7.4 (278 K) and 50 l
M ZnCl
2
. The assign-
ment is shown. Four new crosspeaks
appear when the temperature is lowered
and the exchange rate is therefore slowed.
The new crosspeaks are highlighted in
circles.
J. Danielsson et al. Zinc-binding site of the amyloid b -peptide
FEBS Journal 274 (2007) 46–59 ª 2006 The Authors Journal compilation ª 2006 FEBS 51
The mean R
2
value of the peptide changes upon zinc
addition from 4.85 ± 1.40 s
)1
to 7.12 ± 6.44 s
)1
.
Figure 3B shows the changes in R

2
upon zinc binding
for all the residues. The C-terminal residues show no
or small changes in R
2
, whereas the residues close to
the binding site show increased R
2
upon zinc binding,
as expected because of the presence of a zinc-induced
structure in this region. To confirm that the R
2
chan-
ges are due to decreased local mobility, amide proton
exchange stability was measured. As shown in Fig. 3C,
the amide protons show slightly increased stability,
which is most prominent close to the binding site. We
conclude that zinc binding induces increased order in
the N-terminus of the peptide. This is reflected by the
increased R
2
in this region, and also by the increased
amide proton stability, which is most likely due to
increased protection of the amide protons from the
solvent water.
Interaction with copper
To obtain some complementary information, the inter-
action of copper was investigated using
1
H–

15
N HSQC
experiments and the paramagnetic broadening effect
expected from bound copper. Figure 3D shows that
copper addition produced a significant decrease in the
crosspeak intensities for residues 3, 5, 8, 9, 10, 11, 12,
16 and 17, and a total loss of the Phe4 signal when
40% of the peptide is bound to copper. The signals of
residues 7 and 15 also show a signal intensity decrease,
but not as prominent as for the others. Crosspeaks for
residues 1, 6, 13 and 14 are not visible even with no
copper added. As well as the selective effects described
above, there is a general copper-induced reduction of
the crosspeak intensities, which could be ascribed to
aggregation of the peptide. At high copper concentra-
tions, the crosspeak signal of Ala21 is also lost. This
confirms the presence of a second, weaker binding site
in the central part of the peptide. All the signals affec-
ted by the addition of copper reappeared, even if not
completely, upon EDTA addition (EDTA concentra-
tion exceeded metal concentration about 20-fold) (data
not shown).
Fluorescence spectroscopy
The peptide has a tyrosine residue in the N-terminal
part of the peptide, Tyr10, and the copper-binding site
is close to this site, as shown above. Addition of cop-
per quenches the tyrosine fluorescence signal [31,38].
Fluorescence quenching can be used to directly meas-
ure the dissociation constant of copper ions, and indi-
rectly to estimate the dissociation constant of zinc.

Full-length Ab has a tendency to adhere to the wall
of the quartz cuvette, and this slow process imparts a
time dependence to the fluorescence spectra. Treatment
with ethylenimine [42] increased the stability, but adhe-
sion was still detectable. Use of the full-length peptide
yielded an approximate affinity for copper, K
d

0.5 lm, but with large deviations of the data points
from the fitted line (data not shown). The shorter frag-
ment Ab(1–28) showed less tendency to adhere, and
the sample was stable for more than 3 days. The fluor-
escence binding studies were therefore performed using
the shorter fragment. The dissociation constant for
copper was determined as K
d
¼ 0.36 ± 0.1 lm for this
fragment at pH 7.2, assuming a 1 : 1 stoichiometry
(Table 1). This K
d
is in good agreement with the pre-
liminary results obtained using the full-length peptide
and the dissociation constant determined earlier [31],
but it is significantly higher than that earlier reported
for the full-length peptide at physiologic pH [43].
There is a fractional fluorescence signal left (approxi-
mately 50%) at the end of titration, suggesting that
the quenching copper ions are not in direct contact
with the fluorescent side chain of tyrosine, but close
enough to cause partial quenching. There are some

systematic deviations of the residuals from the fit,
probably arising from induced aggregation. Including
a term accounting for the induced aggregation in the
binding equation essentially removes all systematic res-
iduals (this modified model is described in supplement-
ary Doc. S1). The dissociation constant is unchanged
upon extending the model with this term.
Zinc has no fluorescence-quenching abilities, but its
binding affinity can be estimated by competitive titra-
tions with copper, provided that the binding site is the
same. Ab(1–28) was incubated with various amounts
of zinc. Copper was added to these mixtures, and pro-
duced signal quenching similar to what was observed
after the addition of copper alone (Fig. 5).
Table 1. Dissociation constants of copper and zinc for Ab. The dis-
sociation constants were calculated from tyrosine fluorescence.
pH K
d
(Cu
2+
)(lM) K
d
(Zn
2+
)(lM)
7.2 0.4 ± 0.1
a
1.1 ± 0.08
a
1.2 ± 0.03

b
6.5 1.2 ± 0.08
a
3.2 ± 0.1
a
7.2 2.5 ± 0.2
c
6.6 ± 0.2
c
a
Dissociation constant for Ab(1–28) calculated using fluorescence;
10 m
M sodium phosphate buffer, 20 °C.
b
Dissociation constant for
Ab(1–40) calculated using
1
H–
13
C HSQC crosspeak intensity;
10 m
M sodium phosphate buffer, 20 °C.
c
Dissociation constant for
Ab(1–28) calculated using fluorescence; 10 m
M Hepes buffer,
20 °C.
Zinc-binding site of the amyloid b -peptide J. Danielsson et al.
52 FEBS Journal 274 (2007) 46–59 ª 2006 The Authors Journal compilation ª 2006 FEBS
Addition of EDTA to release the copper from the

peptide by competition recovered most but not all of
the tyrosine fluorescent signal. Approximately 30%
was still missing, suggesting that this fraction is aggre-
gated and ⁄ or precipitated. Assuming that zinc and
copper compete for the same binding site, the dissoci-
ation constant for zinc can be calculated using an
expression for the bound fraction as a function of the
dissociation constants of the two metal ions. The dis-
sociation constant for zinc was estimated to be
1.08 ± 0.08 lm, indicating only slightly lower affinity
than for copper at pH 7.2, close to physiologic condi-
tions (Table 1). This dissociation constant for zinc is in
excellent agreement with that obtained from NMR
data, which was measured on the full-length peptide
and is also included in Table 1. Thus, the affinities of
zinc for the shorter fragment A b(1–28) and for Ab(1–
40) are the same.
Under mildly acidic conditions (pH 6.5) and in
phosphate buffer, the dissociation of copper is slightly
increased (K
d
¼ 1.16 ± 0.08 lm), whereas the dissoci-
ation constant for zinc increases from 1.08 lm to
3.19 ± 0.08 lm (Table 1). The changes in affinity due
to changes in pH close to pH 7 are not large.
To investigate the effect of the buffer used in these
experiments, the fluorescence measurements were
repeated in 10 and 50 mm Hepes buffer at pH 7.2 and
with 0, 10 and 20 lm Zn
2+

. Under these conditions,
the dissociation constant for the Ab(1–28)–copper
interaction was determined to be 2.5 ± 0.2 lm, and that
for the Ab(1–28)–zinc interaction was 6.6 ± 0.1 lm,
similar to what was estimated in the experiments using
phosphate buffer.
CD spectroscopy
To monitor the structural changes of A b induced by
metal ion interactions, CD spectra were recorded for
Ab with increasing amounts of added copper and zinc
(supplementary Fig. S1). The interaction of Ab with
copper reduces the amount of PII helix present in
apo-Ab, as demonstrated by examining the difference
spectra. The N-terminal part of the Ab peptide has a
relatively high propensity to adopt a PII helix [8].
Thus, a reduction of PII helix secondary structure is
consistent with a copper interaction in the N-terminal
part of the peptide. At low copper concentrations, the
structural transition is between two states, and there-
fore the CD spectra show an isoelliptic point.
Zinc binding gives similar, but not identical, results
to those obtained with copper. The zinc interaction
mainly reduces the signal intensity but does not clearly
reduce the amount of PII helix. This may be due to
induced aggregation and subsequent precipitation. At
neutral pH, as in this present study, zinc has a higher
propensity to induce aggregation than copper [40].
This aggregation can mask the structural change of the
peptide.
1

0.8
0.6
0.4
Fluorescence intensity / I/I
0

3020100
0.02
0.01
0
-0.01
-0.02
Copper Concentration / [
µ
M]
Residuals
Fig. 5. The tyrosine fluorescence intensity of 10 lM Ab(1–28) in
25 m
M phosphate buffer (pH 7.2) at 298 K and 305 nm as a func-
tion of copper concentration. The three datasets correspond to 0
(s), 60 (n) and 90 (h) l
M zinc acetate added. The solid lines are
the fitted curves of the one-to-one binding equation. The plateau
values differ between the attenuating intensities. This could be due
to different peptide aggregation propensities at different total metal
ionic strengths. In the bottom panel, the residuals from the fit are
shown.
J. Danielsson et al. Zinc-binding site of the amyloid b -peptide
FEBS Journal 274 (2007) 46–59 ª 2006 The Authors Journal compilation ª 2006 FEBS 53
Discussion

Zinc is suggested to have a major effect on aggregation
of Ab [19–21,44], either increasing the aggregation at
high zinc concentrations or reducing the aggregation
at low concentrations [27,28]. Here we have studied
the zinc-binding site in soluble monomeric Ab. Both
zinc and copper induce specific NMR changes, affect-
ing the same residues in the peptide. This is supported
by the fluorescence data, which also show that zinc
and copper compete for the same high-affinity bind-
ing-site (Fig. 5). This in agreement with the findings
for the shorter fragments Ab(1–16) and Ab(1–28) [45].
However, both copper and zinc have a putative second
weaker binding site, as shown by NMR. This is in
agreement with the finding of two binding sites for
copper in earlier studies [31,33].
The details of the high-affinity binding site for zinc
were studied by NMR.
1
H–
13
C HSQC and
1
H–
15
N
HSQC experiments showed a selective zinc-binding site
with His6, His13 and His14 and the N-terminal Asp1 as
ligands (Fig. 1). Direct study of the
1
H–

13
C HSQC cross-
peaks of the aromatic amino acid side chains shows that
Tyr10 is not directly involved in the binding, but is
located close to the bound metal (Fig. 2). The quenching
effect of copper on the tyrosine fluorescence signal con-
firms this view. From the present data, a second binding
site for zinc can be proposed, which involves residues
23, 24, 26 and 28. For Cu
2+
, a similar central region is
involved, manifested by a loss of signal intensity of resi-
due 21. A more detailed study of the second binding site
of copper is not possible, due to the general paramag-
netic line-broadening exhibited by copper.
Different ligands for metal coordination by Ab were
suggested in earlier studies, but they were mainly per-
formed on truncated fragments of Ab with or without
acetylated N-terminals. All studies, however, showed
the histidines to be necessary ligands [40,46,47]. The
truncated fragments show varying binding modes with
respect to the fourth ligand. Acetylation of the N-ter-
minus does not inhibit zinc binding to the N-terminal
fragment Ab(1–16), but the fourth ligand is proposed
to be Glu11 in this variant [36]. In the same fragment
but without an acetylated N-terminus, Asp1 was sug-
gested to be the fourth ligand [35]. Recently Syme
et al. published a study on Ab(1–16) and Ab(1–28) in
which they also suggested the fourth ligand to be the
N-terminus, and indeed an N-terminal-blocked variant

of Ab(1–28) showed less effects when zinc was added
[45].
In a recent paper by Hou et al., the interaction of
copper and zinc with full-length Ab(1–40) was studied
using
1
H–
15
N HSQC. They suggested that, after
anchoring of the copper ion by the histidine side
chains, a less precise binding mode of metal prevails
for full-length Ab, compared to the shorter fragments.
They also observed a reduction on signal intensity that
they interpreted as being due either to deprotonation
of amides or line-broadening due to an intermediate
chemical exchange rate between the apo-form and
holo-form [48]. In the present study, we used the full-
length Ab(1–40) and combined the use of
1
H–
13
C
HSQC and
1
H–
15
N HSQC. Under these conditions,
zinc binding occurs with His6, His13, His14 and Asp1
as ligands. The binding seems to be specific and affects
mainly the ligands and the neighboring residues. The

detected residue-specific signal loss upon metal binding
arises from line-broadening due to chemical exchange
between conformations in an intermediate rate regime
with respect to the NMR timescale (Fig. 1).
The N-terminal Asp1 may bind zinc either with the
amine group or with the carboxylate groups on the
side chain. Our data give no direct evidence for which
of these is responsible for binding. However, the
1
H–
13
C HSQC findings shows that the C
a
crosspeak of
Asp1 is more affected by zinc binding (both intensity
and chemical shift) than are the C
b
crosspeaks (data
not shown). The reason could be that C
a
is closer than
C
b
to the binding site, suggesting zinc binding to the
amine group of Asp1, in agreement with the recent
findings of Mekmouche et al. [35]. However, our data
cannot rule out the possibility of zinc binding to the
Asp1 side chain, and this would be in agreement with
EPR studies that have reported copper coordination
by three nitrogens and one oxygen, 3N1O, suggesting

involvement of the carboxylate oxygen in divalent
metal binding [32,47].
The dissociation constant K
d
for copper and Ab has
been reported earlier to be approximately 1–5 lm
[29,38,48]. For zinc, a K
d
of 3–300 lm has been repor-
ted for full-length Ab(1–40) [38,49,50]. Our present
results (Table 1) show micromolar dissociation con-
stants for both copper and zinc, with a somewhat
higher affinity for copper, in good agreement with the
earlier reports. This holds for experiments in phos-
phate as well as in Hepes buffer. NMR measurements
were similar to fluorescence measurements made under
the same conditions. Use of the induced chemical shift
changes to estimate the dissociation constant yields
K
d
 2.6 lm (supplementary Fig. S2). This is close to
the values obtained with other techniques, and hence
provides further evidence for N-terminal involvement
in the metal binding of Ab. As previously mentioned,
these quantitative results may be somewhat biased, due
to metal–phosphate complex formation and peptide
aggregation, which in turn may depend on precise
Zinc-binding site of the amyloid b -peptide J. Danielsson et al.
54 FEBS Journal 274 (2007) 46–59 ª 2006 The Authors Journal compilation ª 2006 FEBS
experimental conditions such as choice of buffer and

temperature. Binding of zinc to Ab does not induce
any such well-defined structure of the peptide that can
be determined by NMR methods. This is in agreement
with previous reports [45,48]. However, the relaxation
data show that the N-terminus becomes more struc-
tured upon zinc binding. The results indicate that the
N-terminal region folds around the ion, similar to ear-
lier suggested structures induced by copper [31,33,40].
The CD data show that copper and zinc have only
minor and somewhat different effects on the spectra.
The reason may be that copper binding to the histi-
dines induces a charge transfer from the ligand imidaz-
ole to the metal (so called ligand to metal charge
transfer, LMCT), and thus changes the chiral proper-
ties of the peptide. The copper-induced changes may
therefore have this origin, and need not necessarily be
due to a change in secondary structure. Zinc does not
have this effect on the histidines, and the zinc-induced
changes in CD are very small. We conclude that CD
under the present conditions does not provide much
information on the potential metal-induced changes in
the secondary structure of Ab.
From the relaxation and diffusion data, we conclude
that no stable, soluble, metal-induced dimers ⁄ oligo-
mers are present. This is in contrast to what has been
reported for Ab(1–16) [26]. The full-length peptide dif-
fers from the shorter fragments also in this respect.
Our results give rise to a model of the induced struc-
ture of the peptide when bound to zinc (Fig. 6). The
binding involves the histidines and the N-terminus.

There is a turn at Glu3, bending the N-terminus
towards His6. We propose a second turn at Gly9, to
put His13 and His14 close to the metal ion. The model
is similar to the model of Ab(1–28) bound to copper,
proposed by Syme et al. [31]. We also propose that
zinc has a second, possibly cooperative, binding site
involving the middle segment Asp23, Val24, Asn26
and Lys28 with an induced turn at Gly25.
The N-terminal region of free Ab in aqueous solu-
tion has an extended conformation rich in PII helix
that is proposed to help to keep the peptide soluble
and protected from amorphous aggregation [8,51–53].
When the N-terminus binds zinc (or copper), it folds
around the metal, forming another relatively well-
defined structure. The previous reports on differential
metal-binding effects [20,27] on Ab aggregation at low
and high metal ion concentrations may now be under-
stood in the following terms. Metal ions at high
concentrations saturate the binding site(s) of Ab and
lower the electrostatic repulsion between the overall
negatively charged Abs (net charge nominally ) 3at
pH 7). This effect predominates at high metal ion con-
centrations, and explains the higher aggregation pro-
pensity under these conditions. The structure induction
brought about by the metal-induced fold of the N-ter-
minus counteracts aggregation. This effect, masked at
high metal ion concentrations, should dominate at low
ion concentrations, thereby explaining the decreased
aggregation propensity under these conditions.
Experimental procedures

The peptides, unlabeled Ab(1–40), as well as
15
N-labeled
and the
13
C–
15
-N-labeled Ab(1–40), were purchased from
rPeptide (Athens, GA, USA) and were used without further
purification. Ab(1–28) was purchased from Neosystems
(Strasbourg, France). All peptides were nonmodified in the
termini. Solvation of the peptide was performed using the
protocol suggested by Zagorski et al. [54]. This protocol
prescribes that the peptide is dissolved in a base, here
10 mm NaOH, at high concentration (up to 2 mgÆ mL
)1
),
and sonicated in a water ⁄ ice bath for 2 min. The stock
solution was diluted first with water, and then with buffer
to the desired concentration and pH. In the present study,
the NMR peptide concentration was 50–80 lm and the pH
was 7.0–7.3. The peptide was in 10 mm phosphate buffer,
and NMR samples contained 10% D
2
O. The peptide and
N
H
N
CH
2

N
H
N
CH
2
N
H
CH
2
N
N
H
N-terminus
C-terminus
His6
His13
His14
Fig. 6. A schematic representation of the structural model of Ab
binding zinc or copper. The structure was constructed using a com-
bination of signal intensity changes, relaxation data and induced
amide proton stability.
J. Danielsson et al. Zinc-binding site of the amyloid b -peptide
FEBS Journal 274 (2007) 46–59 ª 2006 The Authors Journal compilation ª 2006 FEBS 55
solvents was kept cold, < 8 °C throughout the sample pre-
paration. The pH was adjusted using NaH
2
PO
4
and
Na

2
HPO
4
and measured using an Orion PerpHecT pH-
meter (San Diego, CA, USA). The peptide concentrations
of the samples were determined by weight. The metal ions
were purchased from Merck (Darmstadt, Germany) and
Sigma-Aldrich (Steinheim, Germany) as chloride and acet-
ate. The metal salt purity was higher than 98.2%.
Titrations
The metal titrations were performed as metal ion additions
to the peptide sample. For each titration, a freshly prepared
sample was used. Small amounts of highly concentrated
zinc or copper solution, as chloride or acetate, were added
to the peptide. The small changes in volume were taken
into account in the binding constant calculations.
CD spectroscopy
CD spectra were measured using a Jasco (Easton, MO,
USA) J-720 spectropolarimeter equipped with a PTC-343
temperature controller. A 1 mm path-length quartz cell was
used, and the spectral range was 190–250 nm. The resolu-
tion was 0.2 nm, and the bandwidth was 2 nm. The back-
ground was corrected for in all spectra. The CD signal, in
mdeg, was converted to molar ellipticity.
Fluorescence
Fluorescence spectra were collected using a Jobin Yvon
Horiba Fluorolog 3 (Longjumeau, France) spectrometer. A
4 mm quartz cuvette was used. The excitation wavelength
was 276 nm, and the measured emission was between 290
and 350 nm. The excitation and emission slits were 4 nm.

NMR
NMR experiments were performed on Bruker (Karlsruhe,
Germany) Avance 500, 800 and 900 MHz spectrometers, all
equipped with cryogenically cooled probeheads. A Varian
(Palo Alto, CA, USA) 800 MHz spectrometer was also
used. All NMR experiments were performed at 286 K and
pH 7.0–7.3, unless stated otherwise. The
1
H–
15
N HSQC
and
1
H–
13
C HSQC experiments were performed using
2048 · 128 increments and 32 scans, and with the carrier
placed on the water resonance frequency.
15
N relaxation
experiments were performed using the same parameters as
above. The
15
N backbone longitudinal relaxation rates, R
1
,
were measured as previously described [55], using delays in
the pulse sequence of 20, 40, 80, 160, 320, 640 and
1280 ms, and for transverse relaxation rates, R
2

, the delays
were 16, 32, 64, 128, 240 and 500 ms. Peak intensities were
fitted to a single exponential decay.
Diffusion experiments were performed using a pulsed-
field gradient sequence including an eddy current delay
and longitudinal storage with mono-phase square-shaped
gradient pulses of 32 strengths. The gradient pulse was
4 ms and the diffusion length 100 ms. Nonlinear gradient
profiles were accounted for using the modified Stejskal–
Tanner equation method developed by Damberg et al.
[56]. Amide proton exchange was estimated by studying
the temperature stability of the
15
N HSQC peak intensity.
The peak intensities at 281 K (S
8
) and 289 K (S
16
) with
and without zinc were compared. The intensity ratio
S
16
⁄ S
8
is a coarse measure of the temperature stability of
the amide proton, inversely proportional to the exchange
propensity of the amide proton. Comparing this ratio for
the apo-form and holo-form of the peptide gives an esti-
mate of the amide proton stability change upon metal
interaction.

Acknowledgements
This study was supported by a grant from the Swedish
Research Council and by the European Commission,
Contracts LSHG-CT-2004-51 and QLK3-CT-2002-
01989.
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Supplementary material
The following supplementary material is available
online:
Doc. S1. Extended binding model. A theoretical model
for metal binding using tyrosine fluorescence quench-

ing data, including a correction for metal-induced pep-
tide aggregation.
Fig. S1. CD spectra of 15 lm Ab(1-40) in 25 mm
phosphate buffer (pH 7.2) at 298 K with increasing
amounts of metal ion. (A) CD spectra of Ab with 0,
3, 6, 12, 18 and 24 lm copper acetate. The arrow
indicates the apo-form of Ab. (B) Ab(1-40) with 0, 3,
6, 9, 15 and 21 lm zinc acetate. With no metal, the
random coil conformation is predominant. (C, D) The
respective difference spectra with increasing amounts
of metal added to Ab. (C) The difference spectrum
shows the features of a PII helix, indicating that bind-
ing of copper induces a loss of PII helix. The arrows
indicate the change in the difference spectra with
increased metal concentration.
Fig. S2.
1
H NMR chemical shift of Asp1 as a func-
tion of zinc concentration. The fitted line is a first-
order one-to-one binding model, and the fitting yielded
K
d
¼ 2.6 lm. The Ab concentration was 50 lm,in10
mM sodium phosphate buffer at pH 7.2 and 286 K.
Zinc was added as chloride, and the three data points
correspond to 0, 20 and 50 lm Zn
2+
.
This material is available as part of the online article
from

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than missing material) should be directed to the corre-
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J. Danielsson et al. Zinc-binding site of the amyloid b -peptide
FEBS Journal 274 (2007) 46–59 ª 2006 The Authors Journal compilation ª 2006 FEBS 59