From the Department of Pathology, The University of
Melbourne, Victoria 3010 and The Mental Health Research Institute,
Parkville, Victoria 3052, Australia, § Biota Structural
Biology Laboratory, St. Vincent's Institute of Medical Research, 9 Princes St., Fitzroy, Victoria 3065, Australia, ¶ Institut
für Biochemie-Chemie, Freie Universität Berlin, Thielallee
63, D-14195 Berlin, Germany,
School of Physics and Materials
Engineering, Monash University, Clayton, Victoria 3168, Australia,
** The Walter and Eliza Hall Institute of Medical Research,
Post Office Royal Melbourne Hospital, Victoria 3050, Australia, and
Center for Molecular Biology, The
University of Heidelberg, Im Neuenheimer Feld 282, 69120 Heidelberg, Germany
Received for publication, January 21, 2003, and in revised form, February 27, 2003
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ABSTRACT |
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A major source of free radical
production in the brain derives from copper. To prevent metal-mediated
oxidative stress, cells have evolved complex metal transport systems.
The Alzheimer's disease amyloid precursor protein (APP) is a major
regulator of neuronal copper homeostasis. APP knockout mice have
elevated copper levels in the cerebral cortex, whereas
APP-overexpressing transgenic mice have reduced brain copper levels.
Importantly, copper binding to APP can greatly reduce amyloid Alzheimer's disease
(AD)1 is characterized by
progressive neuronal dysfunction, reactive gliosis, and the formation
of amyloid plaques in the brain. The cause of the neuronal cell loss in
AD is unclear but may be related to increased oxidative stress from excessive free radical generation (1-4). A major source of free radical production in the brain is from the transition metals copper
and iron (3, 5). These metals are vital for life because of their high
redox activity and have been utilized in a number of enzymatic
pathways, including cellular respiration. However, if the redox
reactivity of copper and iron is not strictly regulated, this can
result in the generation of toxic reactive oxygen intermediates (2).
The potential for oxidative damage from reactive oxygen intermediates
in the aging brain is further enhanced by the high oxygen consumption
and relatively low antioxidant levels in brain tissue. To prevent
transition metal-mediated oxidative stress, cells have evolved
elaborate systems for copper storage and transport that deliver copper
and iron to metalloenzymes and proteins. A number of studies have
implicated cell surface metalloreductases in the reduction of Cu(II) to
Cu(I), which is the form of the metal ion that is delivered to the
cytoplasm of eukaryotic cells via copper transporters (6). To avoid
Cu(I) redox chemistry inside the cell, Cu(I) ions are escorted by
specific cytosolic metalloproteins such as the copper chaperones that
are involved in intracellular copper trafficking to Wilson's disease
copper ATPase and the copper/zinc superoxide dismutase (7). This
results in unbound copper being essentially absent in the intracellular environment (8). Therefore, cupro-proteins play an important role in
maintaining cellular copper metabolism (9).
Both APP and A The importance of copper to Alzheimer's disease is emphasized by the
neurotoxic interaction between the A Expression and Purification--
CuBD, encompassed by residues
124-189, was expressed using the methylotrophic yeast, Pichia
pastoris. The N-terminal border was determined based on
proteolysis of the whole extracellular domain (16). The C-terminal
border was selected to end before the start of the acidic region
(glutamate 191). APP-(124-189) was generated by PCR using the primers
GCT CGA GAA AA GAG AGG CTA GTG ATG CCC TTC TCG and GAA TTC TTA CAG TGG
GCA ACA CAC AAA CTC. The PCR product was cloned as a
XhoI-EcoRI fragment into the P. pastoris vector pIC9 (Invitrogen). The constructs were transformed
into P. pastoris strain GS115 as previously described (27).
Uniformly 15N- and 13C,15N-labeled
proteins were prepared by a standard protocol (28) using
15NH4Cl, [13C]glucose, and
[13C]methanol as the isotope sources. The proteins were
purified to homogeneity using a QHyperD 1.6 × 13-cm column
(Biosepra) followed by a Superdex 75 HR 10/30 gel filtration
column (Amersham Biosciences). N-terminal amino acid sequencing and
mass spectrometry (matrix-assisted laser desorption/ionization-time of
flight) analysis confirmed that the N terminus was intact, and the mass
correlated with the predicted sequence. Protein concentration was
determined using a Bio-Rad protein assay. Inductively coupled plasma
mass spectrometry for metal analysis was performed using an Ultramass
700 (Varian, VIC, Australia).
NMR Spectroscopy--
NMR spectra were acquired at 30 °C on a
Bruker DRX-600 spectrometer equipped with triple-resonance
pulsed-field gradient probes. Sequential resonance assignments were
made using a series of triple-resonance spectra (29) acquired on either
uniformly 15N- or 13C,15N-labeled
CuBD using the methods described previously (30). Spectra were obtained
on samples that were typically 0.5 mM protein in 20 mM phosphate buffer (pH 6.9), 10%
2H2O, and 1 mM EDTA, which was
either removed or titrated out in the metal binding studies. An
essentially complete set of resonance assignments was determined from
spectra acquired using 15N and
13C,15N-labeled protein. The structures were
calculated with CNS (31) using protocols described elsewhere (30). The
final 21 structures (see Fig. 2) were selected on the basis of
their stereochemical energies. Structural statistics are presented in
Table I.
Electron Paramagnetic Resonance (EPR)
Spectroscopy--
Continuous-wave X-band EPR spectra were obtained
using a Bruker ECS106 spectrometer equipped with a temperature
controller and flow-through liquid nitrogen cryostat.
65Cu(II) was added to CuBD (residues 133-189), and spectra
were collected at 105 K from samples contained in 4-mm internal
diameter Suprasil quartz EPR tubes (Wilmad). Measurement of spectral
parameters was carried out using either the instrument software or,
off-line, Bruker WineprTM, and spectra were simulated with
the aid of Bruker SimFoniaTM. For the calculation of g
values the instrument was equipped with a Bruker EIP548B frequency
counter, and the magnetic field was calibrated using a sample of
Structure of CuBD--
We previously identified the second domain
of APP, residues 124-189 (CuBD) (Fig.
1), as the copper binding domain of APP
(10, 20). The CuBD was expressed recombinantly from P. pastoris. The copper binding and redox activity of CuBD in
isolation was very similar to that of the intact protein (20). Mass
spectrometry revealed that the purified protein was essentially free of
metal ions, indicating it was in the apo form. To characterize the
interaction of copper with APP at the molecular level, we have
determined the three-dimensional structure of the CuBD by triple
resonance multidimensional NMR spectroscopy. Sequential resonance
assignments were made using a series of triple-resonance spectra (29)
acquired at pH 6.9 on either uniformly 15N- or
13C,15N-labeled CuBD using methods described
previously (30). Resonance assignments were essentially complete. We
used an Ellman's test and the NMR data to show there are six
half-cystine residues in CuBD linked in three disulfide bonds. Klaus
et al. show that
Ci
The structures were calculated with CNS (31) using protocols described
elsewhere (30). The final 21 structures (Fig.
2) were selected on the basis of their
stereochemical energies. Structural statistics are presented in Table
I. The structures are well defined and
have good stereochemical properties, with all residues falling in the
allowed regions (33) of the Ramachandran plot. The structure consists
of an Metal Binding to CuBD--
To determine the metal binding site on
CuBD, Cu(Gly)
There was a general decrease in the signal-to-noise ratio of the
spectrum after Cu(II) addition, suggesting that higher order aggregates
formed. To further characterize the metal binding site, the diamagnetic
ions, Zn(II) and Ni(II), were titrated separately into CuBD solutions,
and similar changes in the NMR spectra were observed with either metal.
Decreases in the signal-to-noise consistent with metal-induced
aggregation were observed with all metals, the addition of Zn(II)
leading to a visible precipitate. Resonances corresponding to the
aromatic protons of Tyr-168 broadened and disappeared, as did
C
The NMR studies identified His-147, His-151, Tyr-168, and Met-170 as
probable ligands for copper in a tetrahedral coordination sphere (Fig.
4). To confirm this experimentally, we
used EPR spectroscopy to determine the coordination sphere about the
metal. However, to avoid the physiologically irrelevant second copper
site, a protein construct was prepared where the unstructured
N-terminal residues were deleted such that the domain consisted of
residues 133-189. NMR studies on unlabeled protein (data not shown)
indicated that the removal of the unstructured N-terminal residues had
no effect on the fold of the domain. The X-band EPR spectrum of 0.3 mM 65Cu(II) bound to 0.5 mM
CuBD-(133-189) in 20 mM pH 6.9 phosphate buffer (Fig.
5) gave a signal due to a single bound
Cu(II), indicating a specific high affinity metal binding site, and
there was no signal due to free Cu(II). The observed g values (gxx
2.055, gyy 2.091, gzz 2.253) were typical of distorted square planar
complexes with a similar ligation to that suggested by the NMR data
(34). The decrease in the observed A APP has a copper binding domain located in the N-terminal
cysteine-rich region that can strongly coordinate Cu(II) and reduce it
to Cu(I) (Fig. 1). It has been demonstrated that this domain can
modulate copper homeostasis and production of A His-147 and His-151 were shown previously to be necessary for copper
binding (11). The orientation of these residues in the
three-dimensional structure indicates that, with very small side-chain
movements, a tetrahedral metal binding site suitable for coordinating
Cu(I) is formed (Fig. 4). Such a site is reminiscent of the blue copper
proteins that bind copper with a tetrahedral arrangement of ligands
consisting of two histidines, a methionine, and a cysteine residue
(36). The binding site in CuBD appears novel; a search of the PDB
failed to identify a copper site with the same ligands. The closest
example was peptidylglycine monoxygenase (PDB code 1PHM), which
contains a redox active Cu(II) binding site consisting of two histidine
residues, a methionine residue, and a water molecule in a tetrahedral
coordination about the metal (37). Beyond this, there was no sequence
or structural similarities between the two proteins.
The coordination of Cu(II) to the tetrahedrally arranged His-147,
His-151, Tyr-168, and Met-170 (Fig. 4) can explain the redox chemistry
associated with Cu binding to APP. In general, four coordinate Cu(II)
ions favor a square planar coordination sphere about the metal, whereas
Cu(I) generally prefers a tetrahedral arrangement (38). The EPR data
(Fig. 5) suggest that Cu(II) bound to APP CuBD is distorted away from
the square plane toward a tetrahedral structure. Histidine residues are
common ligands for Cu(I) sites, and thioether ligands are known to
stabilize Cu(I) in model compounds (38). Oxygen ligands are more common in Cu(II) complexes, and an oxygen ligand in stellacyanin is thought to
be a major factor in this protein having the lowest reduction potential
of all blue proteins (39). Hence, the tyrosine ligand in APP may
facilitate binding of Cu(II), and this is subsequently followed by
redox reactions. Because the copper binding site of CuBD appears to be
a relatively rigid tetrahedral site, Cu(I) binding would be preferred,
and the geometry would facilitate the reduction of Cu(II), which in the
absence of any exogenous reductants, results in Met-170 oxidization
(Fig. 3c). The oxidation of Met-170 in vivo is
unlikely because this would alter the characteristics of the binding
site, making it less likely to stably bind Cu(I); the presence of
exogenous reductants such as ascorbate and thiols would also render
metal reduction via Met-170 redundant.
Cu(I) sites are normally sequestered inside proteins because exposure
could lead to the generation of reactive oxygen species via Fenton
chemistry. Indeed such chemistry is observed when copper binds to this
domain (21). The APP copper binding site described here is unusual in
that it is surface-exposed but similar to copper chaperone proteins
that also possess surface Cu(I) sites (40). It is thought that the
surface location ensures that the metal can be sequestered on binding
of the chaperone to its target. Because exposed Cu(I) sites are prone
to Fenton chemistry it would seem imperative that copper binding to APP
would result in a rapid response. One possible scenario is as follows
(Fig. 6). 1) Membrane-bound APP acts as a
copper sensor/scavenger (17, 18). Cu(II) binding to APP leads to Cu(II)
reduction since the CuBD binding site is optimized for Cu(I) binding.
2) The Cu(I) binding signals APP processing or proteolytic breakdown
via the non-amyloidogenic route (21, 22). The signal transduction
pathway could be triggered by conformational changes or oligomerization
caused by the reduction. This is supported by experimental evidence
showing that copper binding causes such changes (10); our metal
binding experiments were accompanied by varying degrees of protein
aggregation, and APP oligomerization plays a major role in APP
processing (41). 3) The release of the APP ectodomain from the
membrane (21, 22) would allow this secreted form to transport the metal
to a nearby copper transporter/receptor or for excretion from the body via the liver. This hypothesis would explain the need for a
surface location of the Cu(I) ion and provide a molecular basis for the
observed role of APP in copper homeostasis (17, 18) and copper
modulation of APP processing (22, 23).
production in vitro. To understand this interaction
at the molecular level we solved the structure of the APP copper
binding domain (CuBD) and found that it contains a novel copper binding
site that favors Cu(I) coordination. The surface location of this site,
structural homology of CuBD to copper chaperones, and the role of APP
in neuronal copper homeostasis are consistent with the CuBD acting as a
neuronal metallotransporter.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, a proteolytic breakdown product of APP, play a
central role in Alzheimer's disease and can strongly bind Cu(II)
(Kd APP
10 nM) and reduce it to
Cu(I) in vitro (10-15). The APP copper binding domain
(CuBD) is located in the N-terminal cysteine-rich region next to the
growth factor-like domain (10, 16) (Fig. 1). APP is a member of a
multigene family, and the CuBD sequence is similar among the different
APP family paralogs and orthologs, suggesting an overall conservation
in its function or activity. In vivo studies show that APP
expression is a key modulator of neuronal copper homeostasis since APP
knockout mice have increased copper levels in the brain (17).
Conversely, APP overexpressing transgenic mice have significantly
reduced copper levels in transgenic mouse brain (18). The APP CuBD can also modulate Cu(I)-mediated neurotoxicity (19) and, depending on the
ortholog, can either promote or inhibit copper neurotoxicity (20). The
interaction between the APP-Cu(I) species with hydrogen peroxide can
result in Cu(I) oxidation to Cu(II) and APP fragmentation (21). Of
importance to Alzheimer disease pathology is the finding that
increasing the copper concentration modulates APP processing, resulting
in greatly reduced A
production and increased levels of the
cell-bound and secreted forms of APP (22, 23). Mutagenesis of histidine
residues within CuBD inhibits the effects of copper on APP expression
and proteolysis (23).
peptide and copper. The A
peptide binds copper with a high affinity and reduces Cu(II) to Cu(I),
resulting in the catalytic generation of hydrogen peroxide (H2O2) and A
aggregation (24). A
and
copper can interact to form an oligomeric complex that binds copper at
a copper/zinc superoxide dismutase-like binding site (25). The
importance of copper in AD pathology has been demonstrated by the
ability of a chelator (clioquinol) to disaggregate amyloid both
in vitro and in a transgenic mouse model in vivo
(26). It is plausible that copper binding to CuBD and A
are linked
phenomenon. The modulation of copper levels by the APP CuBD would
influence A
-Cu interactions, resulting in increased
H2O2 from A
-Cu or an overall increase in
neuronal reactive oxygen intermediate production. Changes in copper
levels will also affect APP processing into A
, thus controlling the
production of neurotoxic A
. Therefore, defining the interaction of
copper with APP has important consequences for A
production and AD
pathogenesis and subsequent therapeutic intervention. To understand the
interaction of copper with the APP CuBD at the molecular level, we have
determined the three-dimensional structure of the CuBD (APP residues
124-189) by NMR spectroscopy. The structure has led to the
identification of a novel copper binding site. The CuBD has structural
homology to copper chaperones, thus suggesting the APP CuBD functions
as a neuronal metallotransporter and/or metallochaperone.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
,
'-diphenyl-
-picrylhydrazyl.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
H-Cj
H, and
Ci
H-Cj
H nuclear
Overhauser effects are predictive of disulfide pairings between
half-cystines i and j (32). This method allowed
the unambiguous assignment of pairings to 133/187, 144/174, and
158/186.
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Fig. 1.
Schematic representation of APP showing
important regions of the molecule. The N-terminal growth factor
domain is followed by CuBD (together these constitute the cysteine-rich
region of the molecule), an acidic region, Kunitz-type protease
inhibitor (KPI), and OX2 domains that occur in some APP
isoforms, a series of domains attached to carbohydrate, a transmembrane
(TM) region, and a cytoplasmic (cyto) tail. The
location of the A region, a major component of Alzheimer's disease
plaques, is shown. The sequence of CuBD is shown with the copper
binding ligands in underlined type.
-helix (residues 147-159) packed against a triple-stranded
-sheet (residues 133-139, 162-167, and 181-188) (Fig. 2). A
disulfide bond between Cys-133 and Cys-187 links strands
1 and
3
and another between Cys-158 and Cys-186 links the
-helix to strand
3. The Cys-144-Cys-174 disulfide bond connects two loops at the
other end of the molecule. There are very few buried hydrophobic
residues in the vicinity of this disulfide bond, and therefore, this
bond is probably very important in stabilizing the structure in a
region that does not have any secondary structure. In addition to the
three disulfide bonds, there is a small hydrophobic core to assist in
stabilizing the structure consisting of a small segment of residues
(Leu-136, Trp-150, Val-153, Ala-154, Leu-165, Met-170, Val-182, and
Val-185) from each of the secondary structure elements. With the
exception of the unstructured residues near the N terminus,
{1H}-15N heteronuclear nuclear
Overhauser effect data (data not shown) indicated that the molecule was
rigid along the entire backbone. The surface of CuBD is highly charged
with several areas of high negative (Glu-156, Glu-160, and Glu-183,
Asp-167 and Asp-131), and positive (Lys-132, Lys-134, Lys-161, His-147,
His-151, and Lys-155) potential.
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Fig. 2.
APP CuBD structure. a,
stereoview of the backbone (N, C , C) traces of the
structure of CuBD as determined by NMR spectroscopy showing the 21 lowest energy structures. The structures are well ordered with only the
three N-terminal residues unstructured (angular order parameter <0.9),
and they have good stereochemical properties with more than 98% of
backbone angles falling in the allowed regions of the Ramachandran
plot. b, ribbon diagram of the structure closest to the
mean.
Structural statistics for the 21 energy-minimized structures of CuBD
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Fig. 3.
Effects of metal addition. a,
1H,15N HSQC spectra of 15N-labeled
CuBD (residues 124-189) before (left) and after
(right) the addition of one equivalent of Cu(II). Peaks
still visible after the addition of Cu(II) are from protons distant
from the binding sites. This construct had signals broadened due to
Cu(II) binding to His-147, His-151, Tyr-168, and Met-170; in addition
resonances were also broadened by the physiologically irrelevant second
site at the N terminus. b, the top spectrum is a 600-MHz
1H spectrum of unlabeled protein. Peaks due to the
C H3 of Met-170, the H
2 of
His-151, and the H
1 and H
1 of Tyr-168 are labeled.
The bottom spectrum is after the addition of Zn(II).
c, region of the 1H,13C HSQC spectra
of 15N,13C-labeled protein showing Met
S-methyl 1H/13C cross-peaks. The
top spectrum shows the S-methyl resonances of
Met-141 on the left and Met-170 on the right. The
middle spectrum shows that the effect of adding 1 eq of
Cu(II). The bottom spectrum was measured after the protein
was incubated with Cu(II) for 48 h. The signal-to-noise of the
spectrum decreased (due to aggregation), which necessitated the use of
lower contour levels to observe the modified Met-170 signal. The
Met-141 peak appears to get larger because of the lower levels, but in
fact the peak width at half-peak height remains constant.
H3 resonances of Met-170 (Fig.
3b). The backbone amide resonances from His-147, His-151 and
Tyr-168 in the two-dimensional 1H,15N HSQC
spectrum broadened beyond detection on the addition of excess metal.
The broadening of resonances upon the addition of zinc or nickel is
evidence for chemical exchange between the metal-bound and apo forms of
the protein at intermediate exchange kinetics, which implies a
Kd in the micromolar range. The lack of change in
resonances distant from the immediate metal binding sites indicated
there was no significant structural alteration upon metal binding. To
demonstrate a role for Met-170 in the binding and reduction of Cu(II),
changes in the two-dimensional 1H,13C HSQC
spectrum of CuBD upon the addition of Cu(II) were monitored with the
1H and 13C chemical shifts of the
C
H3 and C
of methionine being
diagnostic. Initially, the S-methyl resonance of Met-170
broadened beyond detection, whereas the Met-141 resonance, the only
other methionine in CuBD, was unaffected, indicating the close
proximity of Met-170 to the Cu(II). After incubating the sample at
30 °C for 48 h a new resonance was observed with 1H
and 13C chemical shifts typical of
C
H3 and C
from methionine
(Fig. 3c), indicating the presence of a modified Met-170.
The changes in the 13C and 1H chemical shift
are consistent with the sulfur of Met-170 being oxidized as the Cu(II)
is reduced to Cu(I).
value (Axx 15, Ayy 25, Azz 137) is consistent with a severe distortion away from a
square planar geometry toward a tetrahedral arrangement of the ligands coordinated to copper. Thus, the EPR data support a type 2 copper site
with a N2SO coordination sphere (35) about Cu(II) that is intermediate
between a square plane (preferred by Cu(II)) and a tetrahedral
configuration (preferred by Cu(I)). We would expect that the
coordination about the reduced form of copper would be the tetrahedral
structure depicted in Fig. 4c.
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Fig. 4.
The metal binding site. a and
b, orthogonal views of the putative metal binding site
consisting of residues His-147, His-151, Tyr-168, and Met-170.
c, a model of Cu(I) coordinated in a tetrahedral
configuration to His-147, His-151, Tyr-168, and Met-170.
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Fig. 5.
X-band EPR spectra of 0.3 mM
65Cu(II) bound to 0.5 mM CuBD
in 20 mM pH 6.9 phosphate buffer.
Instrument settings were: frequency, 9.6253 GHz; modulation frequency,
100 KHz; modulation amplitude, 1.015 G; microwave power, 1 milliwatt;
sweep time, 83.886 s; time constant, 10.240 mc. Spectra were averaged
over 36 sweeps.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, a peptide that
plays a central role in the progression of Alzheimer's disease. Here
we report the structure of this domain and identify the residues (His-147, His-151, Tyr-168, Met-170) involved in coordinating copper
and the possible mechanism for copper reduction. The nature and
orientation of these residues constitute a novel copper binding site.
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Fig. 6.
A model outlining the mechanism and
biological consequences of the CuBD-copper interaction. APP is a
transmembrane molecule that can be cleaved via the amyloidogenic
pathway by - and
-secretase cleavage to release A
.
Alternatively, cleavage can occur via the non-amyloidogenic pathway by
- and
-secretase cleavage to release P3 (truncated A
) (49). In
the depicted model the key steps are as follows. 1) APP will bind
copper via the CuBD (hatched box) in response to copper
levels in the extracellular milieu and/or inside the cell. 2) The CuBD,
as shown by its structure, will favor the reduction the reduction of
Cu(II) to Cu(I) (11) and subsequent APP dimerization (41). Whether
copper participates directly in the dimerization is not known. We would
predict other proteins involved in APP processing (secretases or
co-factors) could also bind to the APP·Cu(I) complex. 3) The
APP·Cu(I) complex promotes the processing of APP via the
non-amyloidogenic pathway (22), resulting in the secretion of
-secretase-cleaved APP·Cu(I) and the P3 peptide. This results in a
decrease in A
levels and an increase in
-secretase-cleaved APP
(22). The secreted APP·Cu could also be complexed with other
molecules/co-factors (4). The secreted APP·Cu(I) can then act as a
copper transporter to transport the Cu(I) away from the tissue for
excretion via the liver. This is consistent with the in vivo
APP knockout mouse data showing increased copper levels in APP knockout
liver and brain (17). Alternatively, the APP could act as a copper
chaperone and transfer the copper to an as yet unidentified
cupro-protein.
Interestingly a search of the Protein Data Bank for similar folds (42)
yielded 51 structures with the same -helix packed over a triple
strand
-sheet topology. Three of these proteins are involved in
copper chaperone activity including the Menkes copper-transporting
ATPase fragment (PDB code 1AW0), metallochaperone Atx1 (PDB code 2U2F),
and SOD1 copper chaperone (PDB code 1QUP). These all have a different
metal coordination sphere compared with CuBD using two thiol residues
in a CXXC motif to bind Cu(I). However, these proteins are
intracellular, whereas APP is an extracellular protein with the
cysteine residues involved in disulfide bonds and, therefore, are
available for metal coordination. Although most copper chaperones
identified to date have been shown to utilize the high affinity of the
sulfhydryl group of cysteine residues to coordinate Cu(I), it has been
reported that CopB copper ATPase, a transmembrane protein from
Enterococcus hirae that is responsible for exporting excess
copper, has histidine-rich metal binding motifs (43). The
metallochaperone Atx1 displays a number of Lys residues on its surface,
and it is thought that these residues play a critical in Atx1
recognizing its partner (Ccc2, a copper transporting P-type ATPase)
(44). Intriguingly, CuBD also has conserved Lys residues at 155 and 158 that lie in a similar location on the structure as does Lys-24 and -28 for Atx1.
The observations that APP knockout mice show specific elevations in brain and liver copper levels (17), whereas APP overexpression in mice results in significantly reduced copper levels (18) highlights the important role that APP plays in modulating neuronal copper levels. The structure presented here defines how copper interacts with the extracellular region of APP at the atomic level. Modulation of neuronal copper is important because a large body of work has emerged that suggests copper has a significant role to play in a range of neurodegenerative disease (3) including AD, Creutzfeldt-Jakob disease (45), Parkinson's disease (46), and amyotrophic lateral sclerosis (47).
As a possible treatment for Alzheimer's disease it would
be highly desirable to develop a drug with specific high affinity binding to APP that would interfere with amyloidogenic APP processing in vivo. The interaction of copper with the CuBD effects APP
processing such that A production is significantly reduced (22).
This suggests that agonists of copper interaction with APP would have therapeutic potential. The design of such agonists is greatly assisted
by the structural information presented here. In addition, the recently
reported success in a small-scale phase II clinical trial of the metal
chelator clioquinol in reducing A
levels of treated patients
illustrates the potential benefits of targeting copper interactions
with APP/A
(48).
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ACKNOWLEDGEMENTS |
---|
We thank Irene Volitakis and Robert Cherny for inductively coupled plasma mass spectrometry analysis and Frosa Katsis for N-terminal sequencing.
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FOOTNOTES |
---|
* This work was supported by the National Health and Medical Research Council.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The atomic coordinates and the structure factors (code 1OWT) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
§§ To whom correspondence may be addressed: E-mail: mwp@rubens.its.unimelb.edu.au.
¶¶ To whom correspondence may be addressed: E-mail: r.cappai@unimelb.edu.au.
Published, JBC Papers in Press, February 28, 2003, DOI 10.1074/jbc.M300629200
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ABBREVIATIONS |
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The abbreviations used are: AD, Alzheimer's disease; APP, amyloid precursor protein; CuBD, copper binding domain; EPR, electron paramagnetic resonance; PDB, Protein Data Bank; HSQC, heteronuclear single quantum correlation.
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REFERENCES |
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