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INTRODUCTION |
Prion diseases, which include Creutzfeldt-Jacob disease in
humans, mad cow disease in cattle, and scrapie in sheep, involve the
misfolding of the benign cellular prion protein
(PrPC)1 to the
infectious disease-causing scrapie isoform PrPSc (1-3).
The prion protein (PrPC) is a copper-binding cell surface
glycoprotein (4, 5). The role of copper in the normal function of PrP,
as well as in prion diseases, has been the subject of a number of
excellent reviews (6-8). The mature cellular form of PrP consists of
residues 23 to 231 and is tethered to the cell surface via a
glycosylphosphatidylinositol anchor at the C terminus. There are now a
number of NMR solution structures of copper-free mammalian PrPs
(9-12). A crystal structure of PrPC has also been
published; this structure is dimeric involving domain swapping of the
monomeric form (13).
PrPC contains a C-terminal domain that is largely
-helical. In the absence of Cu2+ the N-terminal half of
the protein, residues 23-124, is unstructured (11), with a large
degree of backbone flexibility (14). Residues 60-91 consist of an
octapeptide sequence, PHGGGWGQ, that is repeated four times. It is this
unstructured region that binds four Cu2+ ions in the
full-length protein and similarly in fragments from the octarepeat
region (5). This octarepeat region binds four Cu2+ ions
cooperatively with identical coordination geometry (5). There are now a
number of studies to suggest that copper binds to the octarepeat region
of PrPC with affinities ranging between fM (15)
and µM (16). In addition, a fifth Cu2+
binding site centered at residues His-96 and His-111 has also been
observed (15, 17).
The function of PrP is still the subject of debate. Elevated copper
levels promote endocytosis of PrP suggesting that PrP transports copper
into the cell (18, 19). An enzymatic role for Cu-PrP is also proposed
as it exhibits superoxide dismutase activity (20-22). It is also
suggested that PrP has a protective role binding Cu2+ in a
redox-inactive state (23-25). Mice deficient in cellular PrP show a
reduction in copper concentration in the brain relative to wild type
mice and a reduction in activity of copper/zinc superoxide dismutase (4), although this observation is contested (26).
Cu2+ has also been linked with prion diseases. For example,
the presence of copper can confer different strains of prion disease with different protease resistance properties (27, 28). Elimination of octarepeats slows disease progression (29). A mutant form of PrP
associated with familial prion disease contains nine additional octarepeats and fails to undergo copper-mediated endocytosis (19). Copper can enhance reversibility of scrapie inactivation (30). Metal
binding to prion protein is altered in human prion disease (31). Copper
can convert the cellular prion protein into a protease-resistant species (32). Metal imbalances are a feature of prion disease (33).
Copper-catalyzed redox damage of PrP has been implicated in prion
disease (34, 35).
There is now an impressive list of techniques that have been directed
at determining the structure and the copper binding properties of the
octarepeat region of PrPC. These include the following: CD
(5, 36, 37), electron paramagnetic resonance spectroscopy (EPR) (5, 38,
39), NMR spectroscopy (5, 15), mass spectrometry (40, 41), Raman
spectroscopy (42), infrared spectroscopy (43), voltammetry (37), and
fluorescence spectroscopy (36, 44). Despite this, the precise structure
of the copper-binding region is not fully established. Recently,
however, the crystal structure of Cu-HGGGW has been published (45). The
structure is in agreement with EPR data suggesting type II coordination
geometry involving three nitrogen ligands and oxygen ligand
coordination (5, 38). The structure is pentacoordinate with four
equatorial ligands in a square-planar arrangement with an additional
axial water molecule. Coordination includes an imidazole nitrogen
ligand, two deprotonated amides from the next two glycines with the
second glycine also contributing a carbonyl oxygen. The axial water
ligand is stabilized by hydrogen bonding to the tryptophan indole.
Despite numerous studies of copper binding to PrP there is still
intense disagreement in the literature as to the affinity of
Cu2+ for PrP and therefore its functional role (see Refs.
15 and 16). The specificity of PrP for Cu2+ ions is
disputed (see Refs. 44 and 46), and the number of Cu2+ ions
binding to PrP is in dispute. Copper binding to the octarepeats is
often described in terms of isolated single copper centers; this is
inconsistent with the cooperative nature of copper binding found in
multiple repeats. It is the aim of this paper to use direct
spectroscopic methods to resolve some of these inconsistencies.
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EXPERIMENTAL PROCEDURES |
Peptide Synthesis and Purification--
Peptides representing
various lengths of the repeat region of PrP were synthesized employing
solid phase Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry
using the University of London service at Imperial College. After
removing from the resin and deprotection the samples were purified
using reverse phase high pressure liquid chromatography and
characterized using mass spectrometry and 1H NMR. To mimic
the peptides within the full-length prion protein all peptides were
blocked at the N terminus with N-acetyl and with ethyl ester
at the C terminus. Peptides synthesized included those shown in
Table I.
pH and Buffers--
The pH was measured before and after each
spectrum was recorded. Unless otherwise stated all spectra were
recorded at pH 7.5. The effect of various buffers on Cu2+
binding to PrP (58-91) was investigated. We have found that Tris buffer will compete very successfully for Cu2+ ions. HEPES,
acetate, and N-ethylmorpholine buffer were found not to
interfere with the Cu2+ binding to the octarepeats as CD
signals were unaffected by the addition of the buffers at constant pH.
Typically, 50 mM N-ethylmorpholine buffer
was used for CD studies in the visible region whereas in the UV region
the pH was adjusted using small aliquots of 10 mM NaOH or
HCl. The effect of ionic strength on the UV-CD data was investigated
for a number of peptides with addition of NaCl; no effect was observed.
Titration of Metal Ions--
The peptide concentrations were
determined using extinction coefficients at 280 nm. The extinction
coefficient of the peptides were calculated for the octarepeat peptides
using 5690 M
1 cm
1 multiplied by
the number of Trp residues or 1280 M
1
cm
1 for Tyr residues (47). Typically, the freeze-dried
peptides contained 5 to 10% moisture by weight. The addition of metal
ions to the octarepeat peptides was performed using small aliquots from
stock aqueous solutions of CuCl2·2H2O and
MnCl2·4H2O.
CD--
CD spectra were recorded on an AVIV instrument at
25 °C. Typically a cell with a 0.1-cm path length was used for
spectra recorded between 185 and 260 nm with sampling points every 0.5 nm. A 1-cm cell path length was used for data between 260 and 800 nm
with a 2-nm sampling interval. A minimum of three scans were recorded, and baseline spectra were subtracted from each spectrum. It was not
necessary to apply smoothing methods to any of the data presented. Data
were processed using KaleidaGraph spread sheet/graph package. The
direct CD measurements (
, in millidegrees) were converted to molar
ellipticity, 
(M
1 cm
1)
using the relationship 
=
/33,000 × c × l = [
]/3,300, where [
] =
/cl, c is the concentration, and l
is the path length. The molar ellipticity [
] is in units deg
cm2 dmol
1; therefore, [
] = {millidegrees}/{(10) × (mol liter
1) × (cm)}.
Absorption Spectroscopy (UV-visible)--
UV-visible electronic
absorption spectra were obtained with a Hitachi U-3010 double beam
spectrophotometer, using a 1-cm path length.
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RESULTS AND DISCUSSION |
Stoichiometries: Each Histidine-containing Octarepeat Will Bind a
Single Cu2+ Ion--
To ascertain the minimal binding
motif of the Cu-PrP octarepeat complex, CD spectra of a series of
peptides representing various lengths of the PrP octarepeat region were
obtained. On the addition of increasing amounts of Cu2+
changes in the visible and UV region of the CD spectra were observed. Ellipticities, at specific wavelengths for
each peptide, have been plotted against
copper addition to assess the copper saturation point, as shown
in Fig. 1 and Table
II. The binding curves exhibit a sharp
transition at the saturation point indicating tight binding at peptide
concentrations of typically 0.05 mM (Kd < 50 µM). The Cu2+ stoichiometries for the
various peptides are summarized in Table II.

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Fig. 1.
Direct Cu2+ binding curves for
peptides from the octarepeat region. A, HGGGW;
B, HGGG; C, HGG; D, PrP(2octa). Change
in  with Cu2+ was measured at 220, 197, 202, and 590 nm, respectively. The stoichiometries of binding are summarized in
Table II. Binding curves for PrP3octa and PrP(4octa) have been
published previously (5).
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Table II
Stoichiometries and cooperativities for various fragments of octarepeat
region of mammalian PrPs
Studies were at pH
7.5.
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The number of Cu2+ ions binding to PrP has been the subject
of disagreement. Stockel et al. (44) reported two
Cu2+ ions binding to the four octarepeat peptide whereas
others (5) have reported four Cu2+ ions to four octarepeats
but only one Cu2+ ion bound to two octarepeats.
Disagreements as to the stoichiometries of Cu2+ binding to
PrP are probably due to binding Cu2+ below physiological
pH. In this study we confirm that each histidine-containing octarepeat
will bind a single Cu2+ ion (see Table II). In particular,
the 15-mer, GGGWGQPHGGGWGQP, which represents the longest sequence from
the octarepeat region that contains one histidine residue, binds only a
single Cu2+ ion. The peptide HGGG represents the
shortest element that binds a single Cu2+ ion. Surprisingly
the crystal structure of Cu-HGGGW (45) suggests that that HGG might
represent the shortest peptide to bind a single Cu2+ ion,
as direct coordination comes from these three residues. Presumably the
shorter less bulky tripeptide facilitates a second HGG peptide to bind
a single Cu2+ ion, giving the 2:1 stoichiometry.
Affinity of Cu2+ for PrP(4octa): Gly Competes Strongly
for Cu2+ Ruling Out 1015 Affinity--
There
is much disagreement as to the affinity of the Cu2+ binding
to PrP in the octarepeat region. A recent study (15) using tryptophan
fluorescence quenching and glycine competition suggests that the
affinity is as much as eight orders of magnitude higher than the
µM dissociation constants reported previously (16). With
this in mind we have used CD spectroscopy to study the binding of
Cu2+ under the competitive effects of the free amino acid
glycine. Fig. 2 shows the CD spectrum of
Cu-PrP(4octa). Before the addition of glycine the CD spectrum gives
characteristic CD bands because of d-d electronic transitions at 580 and 680 nm. Addition of increasing amounts of glycine gradually
decreases the intensity of all the CD bands in the visible region. The
intensity of the band at 580 nm is plotted versus Gly
addition, as shown in Fig. 2B. After only 8 mol equivalents
of glycine relative to the PrP(4octa) peptide, there is almost no
visible CD signal. Two glycine residues bind to a single
Cu2+ ion; therefore 8 mol equivalents of glycine gives the
same 2:1 ratio with Cu2+ ions. Note that the d-d absorption
bands for the Cu(Gly)2 complex are CD silent. To confirm
that there is strong competition for Cu2+ from glycine,
even for the first equivalent of copper bound to PrP(4octa), spectra of
PrP(4octa) at 0.9, equivalent to Cu2+, were obtained, as
shown in Fig. 2C. Subsequent addition of glycine caused
almost complete loss of the CD signal, also shown in Fig. 2C.

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Fig. 2.
Competitive effects of glycine on
Cu2+ binding to PrP. A, CD spectrum
450-650 nm, 4 mol equivalent of Cu2+ relative to
PrP(4octa) with addition of 0, 1, 2, 4, 8, 12, and 16 mol equivalent of
glycine, pH 7.5, PrP(4octa) 0.031 mM. Symbols , ,
, X, and + correspond to 0, 1, 2, 4, and 8 equivalent of Gly, respectively. B, direct binding curve at
575 nm. C, CD spectrum 300-800 nm, PrP(4octa) 0.027 mM with 0.9 mol equivalent Cu2+ (shown as ),
followed by addition of 20 mol equivalent glycine (shown as
X).
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Glycine is able to coordinate to Cu2+ via its amino and
carboxylate group. The affinity of Cu2+ for glycine at pH
7.4 is reported to be Ka ~1.2 * 108
(48). The binding curve shown in Fig. 2 shows a rapid loss of
Cu2+ bound to PrP(4octa) with glycine addition. At Cu:Gly
ratios of 2:1, little copper remains bound to PrP(4octa). We can
conclude that Gly competes strongly with Cu2+ indicating
that the affinity of Cu2+ for PrP(4octa) is less than that
of Cu2+ for glycine. The tight binding observed in the CD
experiments at 10 µM concentrations therefore puts the
Kd in the 10-µM to 10-nM range.
The affinity we observed is in sharp contrast the study that reported
fM dissociation constants for the four octarepeats. The
study describes a biphasic binding of Cu2+ to the
octarepeats, with a single Cu2+ ion binding with a
Kd of 8 fM followed by weaker
Cu2+ binding of 10 µM (15). There is,
however, no evidence for two modes of binding to the octarepeats. As we
can see from Fig. 2C, the shape of the CD bands for 0.9 mol
equivalents of Cu2+ is identical to that observed for
subsequent additions of Cu2+ to PrP(4octa) as seen in Fig.
3A and Ref. 5. The EPR spectra for 1 equivalent Cu2+ added to PrP (58-91) is also not
significantly different from EPR spectra of subsequent additions of
copper (5). The addition of Gly as shown in Fig. 2C should
not be sufficient to have any effect on the 8 fM site (if
one was present), but it is clear from Fig. 2C that Gly is a
very effective competitor. To reconcile the observations made by
Jackson et al. (15), we suggest that the copper-bound Gly
complex, (Gly)2Cu, interferes with apo-PrP(4octa) tryptophan fluorescence signal, causing it to partially quench.

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Fig. 3.
The CD spectrum, 300-800 nm, of a range of
PrP octarepeat fragments. A, four, three, and two
octarepeats are shown in black, and the single octarepeats
are shown in gray for PrP (70-84), PrP(1octa), and HGGGW.
The multiple repeats have had the molar ellipticities divided by four,
three, and two for four-, three-, and two-octarepeat peptides
respectively, reflecting the Cu2+ ion concentration added.
B, peptides for a PrP(1octa), HGGGW, HGGG, and HGG.
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A Single Repeat Is Found in a Different Environment than Multiple
Repeat Complexes--
From the stoichiometries alone (see Table II)
one might conclude that HGGG (or HGGGW) represents the minimal binding
motif for Cu2+. Indeed, the EPR spectra for HGGGW are
indistinguishable from larger multiple repeats (38). However, it is
clear from a comparison of the CD spectra in the visible region (Fig.
3) that for various peptides, including, HGGG, HGGGW,
GGGWGQPHGGGWGQP, PrP(2octa), PrP(3octa), and PrP(4octa),
there is a distinct difference between those peptides binding a single
Cu2+ ion and multiple repeat peptides.
Fig. 3A compares the spectra for PrP(2octa), PrP(3octa), and
PrP(4octa) peptides containing 2, 3, and 4 mol equivalent of Cu2+ added, respectively (i.e. the peptides are
fully loaded with copper). The CD spectra have been plotted using

(divided by 4, 3, and 2, reflecting the Cu2+ ion
concentration added) for a direct comparison. As observed previously
(5) for the four-octarepeat peptide, the visible region of the CD
spectrum shows two bands due to d-d electronic transitions. These are
centered at 625 nm (visible absorption E625 nm = 40 cm
1 M
1) with a positive
ellipticity band at 580 nm and negative band at 680 nm. In addition,
there is a positive band at 330 nm of comparable intensity. It is clear
that the multiple octarepeats give strikingly similar visible CD
spectra. In contrast, if we compare these multiple octarepeats with
octapeptides containing a single histidine residue, the single
octarepeats spectra show some significant differences (Fig.
3A). The position of the maximum shifts to longer wavelength
from 580 nm (±3 nm) in the multiple octarepeats to 610 nm (±3 nm) for
various versions of the single octapeptide. The crossover point from
positive to negative also shifts by ~40 nm to a longer wavelength.
The intensity of the CD bands at 330, 580, and 680 nm is very similar
for 4, 3, and 2 octarepeats (divided by 4, 3, and 2 respectively,
reflecting the number of copper ions bound); however, this is quite
different in the single repeat peptides, as shown in Fig. 3. The pH
dependence of the CD spectrum in the visible region was obtained to
confirm that the differences in the spectra were not because of slight differences in pH. The maximum positions of the CD bands and crossover points did not change by more than 2 nm between pH 7.4 and 7.9. The pH
dependence of binding has been described previously that shows complete
release of Cu2+ below pH 6 (5).
The visible absorption bands and accompanying CD bands are very
sensitive to ligand coordination geometry around the Cu2+
ion. For example, increasing the number of peptide nitrogens coordinating the Cu2+ ion for the complex with
acetyl-Gly-Gly-His results in a decrease in wavelength maximum from 765 nm for 1 nitrogen to 540 nm for 4 nitrogens (49). The hexadecant
rule has been used in an attempt to interpret CD bands in terms of
coordination geometry around the metal center (50). The sign of the CD
band indicates the position of ligands around the Cu2+
coordination plane. The single octarepeat has the same sign of CD bands
as the multiple repeats, and we can conclude that the difference in
ligand position is small, as it is not sufficient to move coordinating
ligands into neighboring hexadecant sectors.
EPR data indicate that a very similar coordinating geometry exists in
HGGGW, relative to multiple repeats, with the fundamental coordination
ligands unchanged (38). It appears that, in this instance, the visible
CD spectra are more sensitive to differences in the coordination
geometry than the EPR spectra. The CD spectra are sensitive to the
interactions between Cu2+ centers, and this is reflected in
the differences seen in the CD spectra between multiple and single
repeats. The strong cooperativity of Cu2+ binding in
multiple repeats (see Table II) also indicates that the four
Cu2+ ions are not isolated from each other.
Octarepeats Are Not Isolated from Each Other, but the Main Chain
Folds Together to Produce Cooperative Binding--
Addition of
Cu2+ to various octarepeat peptides causes a profound
structuring of the main chain from an unstructured conformation, as
indicated by the CD spectrum in the UV region. Comparisons of the CD
spectra in the secondary structure region (185-260 nm) have been made
(see Fig. 4) in an attempt to establish
whether the main chain conformation of the Cu2+-bound
octarepeat region behaves differently in a single octarepeat compared
with when present in multiple octarepeats. Fig. 4B shows the
resultant spectrum in the UV region when the CD spectrum for HGGGW
(multiplied by four) is subtracted from the four-octarepeat peptide
spectrum. EPR data suggest that HGGGW encompasses the residues directly
binding a single Cu2+ ion in multiple octarepeats (38, 45).
The difference between the spectra should therefore give an indication
of the difference in the backbone conformation for an isolated
octarepeat relative to multiple octarepeat peptides. Interestingly, the
resultant difference between the spectra is not characteristic of an
irregular/unstructured peptide. A resultant random coil spectrum would
be expected if the Cu2+ binding residues HGGGW were
isolated from each other in successive octarepeats. The resultant CD
spectrum shown in Fig. 4B gives a maximum at 205 nm and a
negative band at 225 nm. These bands indicate profound structuring of
the intervening (GQP) residues between successive HGGGW motifs.

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Fig. 4.
CD difference spectra of PrP octafragments in
the UV region. A, Cu4PrP (58-91) and
Cu(HGGGW). The intensity of Cu(HGGGW) has been multiplied by four for a
direct comparison. B, the resultant spectra of
{Cu4PrP (58-91)} {Cu(HGGGW) * 4}, plotted
as per mean residue, i.e. divided by 14, because {34
residues} {5 * 4 residues}. C, Cu-PrP (70-83)
and Cu(HGGGW). D, the resultant spectra of {PrP
(70-83)} {Cu(HGGGW)} plotted as per mean residue,
i.e. divided by 10 residues.
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For a control comparison, the difference between
GGGWGQPHGGGWGQP (the longest possible peptide containing a
single Cu2+ ion) and HGGGW is made as shown in Fig. 4,
C and D. In this case, the resultant spectrum is
similar to that found for unstructured/random coil peptides with a
single negative CD band at 200 nm and with a 
per mean residue of
1.5 M
1 cm
1. In this case, the
residues on either side of HGGGW do not form a regular secondary
structure. This is direct evidence to indicate that the
Cu2+ bound to HGGGW is not an isolated entity but interacts
with neighboring Cu2+ centers to cause the intervening
residues to fold up in an ordered conformation.
The endocytosis of PrP is triggered by the presence of Cu2+
(18, 19). The folding together of the four copper centers, as demonstrated by our CD measurements, is critical to endocytosis. Recent
work shows that mutations of just two histidine residues in the
octarepeats is sufficient to restrict endocytosis (19). The cooperative
fold of all four copper centers is essential to the recognition process
that triggers endocytosis.
Cu2+ Binds to Avian PrP Repeat Region but Forms a
Different Complex than That of Mammalian PrP--
The octarepeats are
the most highly conserved region of mammalian prion proteins primary
sequence. In chicken and other avian species, a hexameric repeat
(PHNPGY)7 is conserved (51). There has been disagreement as
to whether avian PrP also binds Cu2+. Studies have
suggested that synthetic peptides of the chicken hexarepeat will bind
copper with µM affinity (36), others (52) have indicated
no Cu2+ binding to full-length chicken PrP, and others (20,
53) have observed binding of Cu2+ to full-length chicken
PrP.
There is little spectroscopic evidence of Cu2+ binding to
avian PrP. With this in mind a peptide representing the longest avian sequence containing just two histidine residues has been synthesized, a
17-mer, NPGYPHNPGYPHNPGYP. Fig.
5 shows CD spectra in the visible and UV
region on the addition of increasing amounts of Cu2+ to
this avian PrP two-hexarepeat peptide. It is clear from changes in the
spectra with copper addition that Cu2+ does bind to avian
PrP.

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Fig. 5.
The CD spectrum of avian-PrP(2hexa) with the
addition of increasing amounts of Cu2+ at pH 7.5, 185-260
nm (A) and 300-800 nm (B). The
inset is a direct binding curve showing change in 
monitored at 330 nm with Cu2+ addition. The binding curve
indicates one Cu2+ ion binds tightly per avian-PrP(2hexa)
rather than two Cu2+ ions seen for mammalian PrPs.
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The mode of binding is very different from that of the mammalian
octarepeat sequence. The visible CD bands are quite different from
those observed for the mammalian repeats. Most strikingly, there is a
negative band observed at 330 nm rather than the positive band seen in
the mammalian octarepeats. The binding curve for a CD band at 330 nm
(Fig. 5B) indicates the stoichiometry is also very different
from mammalian PrP with only one Cu2+ ion binding to the
two-hexarepeat peptide. The d-d bands have the same sense as the
mammalian spectra but with a positive band at 550 nm, a crossover at
625 nm, and a negative band at 700 nm. The intensities of the bands are
much weaker by an order of magnitude relative to mammalian PrP. Changes
in the UV region of CD spectrum with Cu2+ addition (Fig.
5A) are also very different from the mammalian PrP(octas)
(see Fig. 4A for comparison). The copper-free form of avian
PrP hexarepeat peptide is not characteristic of random coil. This is
presumably because of the high proportion of proline in the sequence.
Addition of excess copper causes a decrease in the negative CD band at
210 nm.
The crystal structure of Cu-HGGGW (45) indicates copper coordination by
an amide nitrogen from the second glycine after the histidine residue.
In the case of PHNPGY the second residue following the histidine
residue is proline, which is not available for coordination to the
copper in the avian hexarepeat. For this reason, a very different
complex must form. The stoichiometry for the two-hexarepeat peptide
strongly suggests that both histidine residues coordinate to a single
copper ion. The preservation of copper binding in the repeat region in
avian, as well as mammalian PrP (although with different coordination
geometry), supports a functional role for this domain.
Proline and Glycine Are Also Critical Residues in the
Copper-Octarepeat Complex--
To investigate which residues are
essential in the Cu-HGGGW complex, in addition to histidine and
tryptophan, whose side chains are involved in coordination, two
alternative sequences were synthesized. In one peptide the glycine
residues were replaced with alanine to produce the peptide HAAAW. In
the second peptide, the proline residue preceding the histidine is
replaced with alanine to produce the peptide GQAHGGGW. Both
changes in the sequence were found to have a dramatic effect on
Cu2+ binding.
Fig. 6A shows the visible CD
spectrum of HAAAW with Cu2+ addition. Cu2+
still binds to the histidine residues but with a very different coordination geometry. The spectra in both the visible and UV regions
(see Fig. 6B) are very different from HGGGW, which is also
shown in Fig. 6A for comparison. Indeed, the
inset in Fig. 6A shows that the stoichiometry is
Cu(HAAAW)2 rather than 1:1 observed for HGGGW. It is clear
that glycine residues are required for main chain coordination. The
additional
and
space available with glycine residues is
necessary for coordination, in the manner observed for the octarepeats
of PrP.

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Fig. 6.
A and B, mutated PrP peptides
HAAAW and GQAHGGGW, respectively. The CD spectrum of HAAAW in the
visible and UV regions is shown, and Cu-HGGGW is also shown for direct
comparison. C and D, GQAHGGGW visible and UV
regions. The insets are direct binding curves for HAAAW and
GQAHGGGW showing change in  monitored at 650 and 550 nm,
respectively, with Cu2+ addition.
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The presence of proline is also necessary to form the complex observed
in the octarepeats, although it is not directly involved in
coordination. Fig. 6, C and D, shows the CD
spectrum of GQAHGGGW with copper addition. Again both the visible and
UV regions of the CD spectrum are profoundly different when compared
with the octarepeat spectrum. Cu2+ ions tend to coordinate
main chain amides to the N terminus of His residues when available
(49). The presence of proline proceeding the His residue in the PrP
octarepeats forces an alternative coordination geometry.
These data show that the sequence of amino acids required
to form the complex observed in the octarepeats is quite specific. Not
only are the side chains of His and Trp required but so are the
intervening Gly residues and also the proceeding proline. The sequence
of amino acids in four successive repeats is also necessary for
Cu2+ to bind to PrP with the required geometry for
recognition associated with Cu2+-induced endocytosis of
PrPC.
Mn2+ Does Not Bind to the Octarepeat Region of
PrP--
It has been reported that Mn2+ can bind to PrP
and can substitute for Cu2+ in the octarepeat region (46).
Mn2+ appears to alter PrPC to a
protease-resistant conformation that forms fibrils. Furthermore, PrPC expression influences uptake of Mn2+ into
cells. It is speculated that Mn2+could have a role in the
formation of the scrapie isoform of the PrP generated in sporadic prion
diseases. The possibility that imbalances in environmental cations may
induce conditions favoring the formation of protease-resistant PrP in
sporadic Creutzfeldt-Jacob disease is controversial (54, 55) but has
gained interest in the British popular press. Evidence for
Mn2+ binding to PrP is based on equilibrium dialysis
studies. As yet there have been no spectroscopic studies of
Mn2+ binding to PrP. Fig.
7A shows a CD spectrum in the
UV region of the PrP(4octa) peptide before and after the addition of
Mn2+. The apopeptide gives a negative CD band at 195 nm,

1.5 M
1 cm
1 per mean
residue, which is characteristic of an unstructured peptide. Addition
of 8 mol equivalent of MnCl2, pH 7.5, has no effect on the
CD spectrum. In contrast, subsequent addition of Cu2+ shows
a substantial change in the CD spectrum. The changes in the CD spectrum
in the presence of Cu2+ indicate marked structuring of the
peptide with Cu2+ binding. Fig. 7B shows a CD
spectrum for PrP(4octa) in the visible region in the presence of 4 mol
equivalent of Cu2+. The CD spectra gives characteristic CD
bands due to d-d electronic transitions at 580 and 680 nm. Addition of
as much as 400 mol equivalent of Mn2+ has no effect on the
spectrum.

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Fig. 7.
CD spectra showing Mn2+ ion binding to
PrP at pH 7.5, UV region (A), PrP(4octa), 0.04 mM. Addition of 8 mol equivalent Mn2+ and
subsequent addition of Cu2+ 4 equivalent are shown.
B, PrP(4octa), 0.04 mM in the presence of 4 equivalent Cu2+ and subsequent addition of 4, 40, and 400 mol equivalents of Mn2+.
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The complete lack of change in the CD spectrum on the addition of
Mn2+ suggests that even at high levels of Mn2+
and peptide (0.4 and 0.05 mM, respectively),
Mn2+ does not bind to the octarepeat region of PrP. The CD
spectrum indicates that upon subsequent addition of Cu2+,
the copper binds to PrP(4octa) in an identical manner as PrP(4octa) with no Mn2+ present. Clearly Mn2+ does not
inhibit Cu2+ binding to PrP(4octa). In the reverse
experiment, shown in Fig. 7B, the metal complex is
pre-formed with Cu2+. In this situation Mn2+ is
unable to displace Cu2+ from PrP(4octa) even when a
100-fold excess of Mn2+ is added.
It is clear that Mn2+ does not bind to the octarepeat
region under the conditions used in this study (pH 7.5 and no buffer
present). However, equilibrium dialysis studies have indicated that
Mn2+ can compete with Cu2+ ions bound to PrP
and displace them (46). It may be that Mn2+ binds elsewhere
to PrP other than the octarepeat region, but it is clear from our
studies that Mn2+ will not directly displace
Cu2+ bound to the octarepeat region.
L-His Competes for Copper Binding to PrP but Does Not
Form a Ternary Complex--
The effects of L-histidine
competition on Cu2+ binding to PrP(4octa) were also
investigated. As with the addition of glycine, L-His will
strongly compete for Cu2+ ions. The
Cu4-PrP(4octa) signal at 580 and 320 nm is completely lost
when between 4 and 5 mol equivalent of L-His is added
(Cu-His 1:1 ratio). This is consistent with L-His having a
higher affinity for Cu2+ than PrP. We considered the
possibility of a ternary complex of L-His, PrP, and
Cu2+, which is thought to occur for the square-planar
complex of Cu2+ bound to serum albumin. However there is no
evidence of a ternary complex with PrP. In Fig.
8A we see a simple diminution
of the Cu4-PrP signals with L-His addition and
the appearance of new CD signals identical to that observed for Cu-His
complex only. Fig. 8B shows the CD spectra of Cu-His complex
for comparison.

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Fig. 8.
Competitive effects of
L-histidine on Cu2+ binding to PrP(4octa).
A, CD spectrum (260-800 nm) for Cu4-PrP(4octa)
showing the effect of the addition of L-His and 0, 1, 2, 3, 5, 6, 7, 8, 10, and 12 mol equivalent of histidine relative to
Cu2+ concentration. For clarity, Cu4-PrP(4octa)
and Cu4-PrP(4octa) with 5 equivalent L-His is
shown with dark shading and symbols and ,
respectively. B, CD spectrum (260-800 nm) of Cu-His
complex, 0.5 mM Cu2+ with increasing mol
equivalent of L-histidine at 0.0, 0.5, 1, 1.5, 2, and 3 mol equivalents. 0.0, 1, and 2 mol equivalents are shown with
dark shading.
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CONCLUSIONS |
The affinity of Cu2+ for PrPC has
important implications for its function. The extracellular levels of
Cu2+ can vary between nM and µM,
reaching as high as 15 µM in the neocortex (56).
Cu2+ affinity for PrP is in the same range.
PrPC is therefore able to bind extracellular copper. The
affinity of PrPC for Cu2+ is in a range in
which it could act as a sensor with binding triggered during increased
levels of extracellular Cu2+. The copper binds
cooperatively causing the unstructured N terminus to fold up in a
specific manner; this then triggers increased endocytosis of
Cu-PrPC. The metal ion binding appears to be specific for
copper, and Mn2+ binding to the octarepeat region is not
observed. It is clear that the sequence of amino acids required for
cooperative binding and endocytosis is highly specific. The four copper
centers are not isolated, and the intervening residues between copper
centers become structured in the presence of copper.