From the Department of Neuropathology, Georg August
University of Göttingen, Robert-Koch-Strasse 40, 37075 Göttingen, Germany, the ¶ Department of Immunochemistry,
Max-Planck Institute for Experimental Medicine, Hermann-Rein-Strasse 3, 37075 Göttingen, Germany, the
Department of Biochemistry
II, Georg August University of Göttingen,
Heinrich-Düker-Weg 12, 37073 Göttingen, Germany, the
Laboratory of Molecular Medicine,
Children's Hospital, Boston, Massachusetts 02115, and the
§§ Institute of Molecular Virology,
GSF-Center for Environmental and Health Research, Technical
University of Munich, Trogerstrasse
4b, 81675 München, Germany
Received for publication, July 24, 2000, and in revised form, February 21, 2001
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ABSTRACT |
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The prion protein is known to be a copper-binding
protein, but affinity and stoichiometry data for the full-length
protein at a physiological pH of 7 were lacking. Furthermore, it was
unknown whether only the highly flexible N-terminal segment with its
octarepeat region is involved in copper binding or whether the
structured C-terminal domain is also involved. Therefore we
systematically investigated the stoichiometry and affinity of copper
binding to full-length prion protein PrP23-231 and
to different N- and C-terminal fragments using electrospray ionization
mass spectrometry and fluorescence spectroscopy. Our data indicate that
the unstructured N-terminal segment is the cooperative copper-binding domain of the prion protein. The prion protein binds up to five copper(II) ions with half-maximal binding at ~2 µM.
This argues strongly for a direct role of the prion protein in copper
metabolism, since it is almost saturated at about 5 µM,
and the exchangeable copper pool concentration in blood is about 8 µM.
Prion diseases are fatal neurodegenerative diseases thought to be
caused by conformational transition of the native and predominantly PrPC is nearly ubiquitously distributed to all tissues. The
highest levels are found in the brain (6). Native PrPC is
an asparagine-linked sialoglycoprotein that is attached to the surface
of the plasma membrane via a C-terminal
glycophosphatidylinositol anchor (7). The structured C-terminal
half of the full-length recombinant PrP23-231 (amino acids
23-231) from amino acids 126-231 is made up of two strands of
one small antiparallel Although many efforts have been made the physiological function of
PrPC has not yet been identified (11, 12). In 1995 it was
suggested that the octarepeat region ([PHGGGWGQ]4) from
amino acids 60-91 within the flexible N-terminal half of the prion
protein may play a role in binding copper (13). We have shown that
PrPC has indeed a function in synaptic copper binding (14).
Heavy metal binding studies indicated that PrPC seems to
specifically bind copper (15). Half-maximal binding of two copper ions
at 14 µM was reported for Syrian hamster
ShamPrP29-231 at pH 6.0 (15). In contrast,
binding of 4 and 5.6 copper ions to the octarepeat region peptide (16,
17) and humPrP23-98 (18) at pH 7.4 with
half-maximal binding at about 6 µM were found,
respectively. The copper binding to the octarepeat region was shown
to be strongly pH-dependent in the range from 5 to 7 (14)
with almost no binding at pH 5.
The lack of affinity and stoichiometry data for the full-length prion
protein at a physiological pH of 7, as well as the question of which
part of the prion protein is responsible for the copper binding,
prompted us to systematically investigate the copper binding to the
full-length prion protein as well as to different N- and C-terminal
fragments using fluorescence spectroscopy and electrospray ionization
mass spectrometry.
In this paper we present for the first time copper binding data on the
full-length prion protein under physiological conditions at pH 7. Our
data show that the prion protein binds up to five copper ions
and is almost saturated at 5 µM copper(II). Furthermore, our data suggest that the highly flexible N-terminal half is the cooperative copper-binding domain of the prion protein.
Materials and Reagents--
MOPS, dodecyltrimethylammonium
chloride, CuSO4·5H2O, and
N-ethylmorpholine were purchased from Fluka (Deisenhofen,
Germany). Chelite P was obtained from Serva (Heidelberg, Germany). Ion
exchange matrices EMD-COOH, EMD-TMAE, and thrombin were ordered
from Merck (Darmstadt, Germany). All other reagents used were of
analytical grade.
Synthesis and Purification of humPrP60-91 and
humPrP60-109--
The peptides
humPrP60-91 and humPrP60-109
were synthesized on an 9050-peptide synthesizer (Millipore) using amino acids protected with Fmoc (1-fluorenylmethoxycarbonyl) and activated with
benzotriazol-1-yl-oxy-tris(pyrrolidino)phosphonium-hexafluorophoshpate (PyBOP) (19). After cleavage from resin and removal of protecting groups, peptides were first purified by reversed-phase HPLC using a
1.9 × 30-cm2 Delta Pac C18 column (Millipore) with a
gradient from 0 to 50% acetonitrile including 0.1% trifluoroacetic
acid for 50 min. Both peptides were further purified on
an EMD-COOH column eluting with a gradient from 0.02 to 1 M
ammonium acetate, pH 5.0. Finally, peptides were purified by
reversed-phase HPLC on a Vydac C4 column (4 × 250 mm)
using a linear water acetonitrile gradient of 10-50% acetonitrile
including 0.1% trifluoroacetic acid.
Expression and Purification of Full-length
murPrP23-231 and N-terminal Fragments
humPrP23-98 and humPrP23-112--
The
N-terminal fragment humPrP23-98 was
expressed in E. coli according to Brown et al.
(18). After expression and thrombin cleavage of the
GST-humPrP23-98 fusion protein, the reaction
mixture was applied to an EMD-COOH column and
humPrP23-98 was eluted with a gradient from 0 M up to 1 M ammonium acetate pH 6.0.
The plasmid pEThuPrP23-112 was constructed for the
expression of humPrP23-112 following the
cloning of the human PRNP ORF from genomic DNA (20). The coding region
of the N-terminal segment of PrP (aa 23 to 112) was amplified via PCR
under standard conditions using primers huPrP23-112up
(5'-GGCCGGTCATGAAGAAGCGCCCGAAGCCT-3') and huPrP23-112do
(5'GCCGGGAATTCTTATCACATGTGCTTCATGTTGGT-3'). Primer huPrP23-112up
contains a BspHI cleavage site and a start codon. Primer
huPrP23-112do contains two stop codons and an EcoRI cleavage site. The construct was cloned into the NcoI and
EcoRI sites of the inducible expression vector pET-21d
(Novagen) and the sequence of the insert was verified by DNA sequence
analysis
The expression of the humPrP23-112 in E. coli was induced with IPTG. Harvested cells were lysed with 1%
Triton X-100 and centrifuged at 18,500 g for 15 min. The supernatant
was applied to an EMD-COOH column, washed with 10 mM
ammonium acetate pH 6.0. humPrP23-112 and
eluted with a linear gradient up to 1 M ammonium acetate pH
6.0 from the column.
Finally, both N-terminal PrP fragments were purified by reversed phase
HPLC on a Vydac C4 column (4 mm x 250 mm) using a linear water acetonitrile gradient of 10% to 50% acetonitrile including 0.1% trifluoroacetic acid.
The full-length murPrP23-231 was expressed and
purified according to Liemann et al. (21). Before
lyophilization, protein was dialyzed with Chelite P to remove traces of
bound copper. The C-terminal fragment
murPrP121-231 was expressed and purified as
described earlier (22).
Fluorescence Titration of PrP23-231 and Its
Fragments--
Fluorescence spectroscopy was carried out on an LS 50B
from PerkinElmer Life Sciences (Überlingen, Germany).
Measurements were performed in 20 mM MOPS, pH 7.2, 100 mM NaCl, and 1 mM DTAC. MOPS buffer was treated
with Chelite P to remove traces of heavy metal ions. To excite
selectively tryptophan fluorescence, a wavelength of 295 nm was chosen,
and the fluorescence signal was detected on the emission side at 355 nm. Excitation slit width varied from 3 to 5 nm, and emission slit
width was in the range of 10-15 nm. For reasons of sensitivity the
excitation wavelength for murPrP121-231 was set
to 285 nm. Copper sulfate was added carefully to prevent possible
precipitation (monitored by systematic excitation peak broadening in
the emission spectra). Each point in the fluorescence titration curves
represents the average of at least three measurements. Fluorescence
titrations were performed at 20 °C. Due to absorption of copper in
the UV range, fluorescence titration curves were corrected for inner
filter effect (23). All curves were corrected for background fluorescence.
ESI Mass Spectrometry--
Mass spectrometry of N-terminal PrP
fragments with and without copper were performed on a TSQ7000
(Finnigan) in the nano spray mode. The buffer was 1 mM
NEMO/formic acid at pH 7.4. The applied spray voltage was 0.8-1.0 kV.
Capillary temperature was set to 150 °C.
Peptide and Protein Quantification--
Peptide and protein
concentrations were determined in 6 M guanidinium chloride
according to Gill and von Hippel (24). Peptide quantification by
amino acid analysis for humPrP60-91 resulted in
identical values.
Electrospray Ionization Mass Spectrometry of Full-length
PrP23-231 and Its Fragments with Copper--
ESI mass
spectrometry was used to elucidate the stoichiometry of copper binding
to the different N-terminal fragments
humPrP60-91, humPrP60-109,
humPrP23-98, and
humPrP23-112 as well as for the C-terminal
fragment murPrP121-231 and for the full-length
prion protein murPrP23-231. The experimental conditions for metal interaction studies at pH 7.4 with respect to
suitable buffers and additives were intensively investigated. Finally,
we introduced N-ethylmorpholine at pH 7.4 as a new buffer system for metal interaction studies with ESI MS, because it did not
interfere with copper binding to peptides and protein compared with
ammonium salt-containing buffers. Furthermore, addition of solvents
like acetonitrile or methanol significantly affected the copper
population in the ESI mass spectra (data not shown) and were therefore
omitted. To avoid unspecific binding the copper concentration as well
as the peptide concentration were kept as low as possible.
For a negative control experiment we used hen egg lysozyme, since it is
similar to the prion protein with respect to its size and isoelectric
point. Besides the potential unspecific copper binding sites at the
N-terminal amino group as well as C-terminal and side chain carboxyl
groups, it has one histidine at amino acid position 15 just after the
first helix, which is exposed to the solvent. As clearly shown in the
spectra of lysozyme with (Fig. 1,
B and C) and without copper (Fig. 1A),
even at a 10-fold excess of free copper(II) no significant binding
occurred. For the first and second copper adduct mass differences of
61.7 and 61.3 average mass units were obtained, respectively. The low
unspecific copper binding to lysozyme as a negative control
demonstrated that NEMO is an excellent new buffer system for copper
interaction experiments with proteins at a physiological pH of 7.
ESI mass spectrometry of the N-terminal fragments (Fig.
2) resulted in the expected average
masses for the apopeptides of humPrP60-91,
humPrP60-109, and
humPrP23-98 (Table I). In the spectrum of apo
humPrP60-109, a by-product (Fig. 2C,
marked with triangles) with a corresponding mass of 4934.3 amu is observed. The mass difference of 127.6 suggests a missing lysine
or glutamine residue in the by-product. The mass calculated from the
spectrum of humPrP23-112 (Fig. 2G)
was 1 amu higher than the theoretically expected 9354.2 amu. This is
probably due to the hydrolysis of Asn108 to
Asp108 (25). The same holds for the by-product (marked with
triangles) for which a mass of 8958.6 amu was obtained. The
mass difference indicated that the three C-terminal amino acids KHM
after Met109 have been cleaved off from
humPrP23-112.
Adding a 5-10-fold excess of copper(II) to the apopeptides resulted in
mass spectra (Fig. 2, B, D, F, and
H) with several copper adduct peaks. On addition of 100 µM copper sulfate to 20 µM
humPrP60-91, new m/z peaks appeared
(Fig. 2B) corresponding to up to four bound copper(II) per
peptide molecule. The two small peaks between the two following
m/z copper peaks are always due to unspecific sodium and
potassium adducts commonly observed in mass spectrometry of peptides
and proteins at neutral pH. Adding 100 µM copper(II) to
10 µM humPrP60-109
resulted in the appearance of additional m/z peaks due to
the complex formation of up to five bound copper per
humPrP60-109 (Fig. 2D). Note that
the peaks apparently corresponding to zero (marked by
diamonds) and one bound copper for
humPrP60-109 are also the main peaks for the
by-product with two and three bound copper, because their masses differ
only by 4 amu and thus appeared as poorly resolved double peaks.
Addition of 70 µM copper(II) to 10 µM
humPrP23-98 (Fig. 2F) led to the
appearance of m/z peaks for up to six bound copper
ions. It should be noted that adding a 10-fold excess (data not
shown) above all significantly raised the intensity of the m/z peaks for six bound copper ions, suggesting a
less specific copper binding site. In contrast, only adduct peaks for
up to five bound copper were observed on addition of 100 µM copper sulfate to 10 µM of the 14 amino
acids longer humPrP23-112 (Fig. 2H).
The significant shift of m/z peaks to lower charge values z and thus higher m/z values on addition of
copper suggests conformational changes associated with copper binding,
resulting in a more folded or structured conformation compared with the apopeptides.
The structured C-terminal domain of the prion protein
murPrP121-231 was analyzed by ESI mass
spectrometry (Fig. 2I) and yielded a mass of 13335.5, which
corresponds to the expected average molecular weight of 13334.8. On
addition of up to 100 µM copper to 13 µM
protein, only one bound copper was observed (Fig. 2J). This
could be seen easier from the deconvoluted spectra (Fig.
3A). This site appears to be
of lower affinity, because at 20 µM copper(II), which is
the 1.5-fold excess in copper, only a peak of little intensity is
observed. At 40 µM copper (a 3-fold excess of copper) the
apoprotein peak almost disappeared, and the molecular weight peak for
one bound copper became prominent. This remained almost unchanged,
increasing the copper concentration up to 100 µM and
indicating that there is probably one copper-binding site with lower
affinity in murPrP121-231. In contrast to the
N-terminal fragments, there is no shift of the m/z charge envelope to lower values. Thus, presumably no conformational change occurs on binding of the copper ion.
From the mass spectrum of the apoprion protein
murPrP23-231 (Fig. 2K) an
average mass of 23110.6 with a S.D. of 3.1 is obtained, which is about
6 amu higher than the expected molecular mass of 23104.4. The reason
for this difference was unclear. On addition of up to 100 µM copper, several not very well resolved copper adduct
peaks (Fig. 2L) appeared. Due to the broad half-peak width
and the high charges, the copper adduct peaks could be observed better
from the deconvoluted spectra (Fig. 3B). The deconvoluted spectra showed molecular weight peaks for up to five bound copper on
addition of 20-100 µM copper. In contrast to the
preceding spectra (20, 40, 60, and 80 µM) there are only
minor changes in the spectra going from 80 to 100 µM
copper, suggesting a saturation of murPrP23-231
with copper.
The average molecular masses for all used peptides and proteins are
summarized in Table I. From this table the average mass differences
between the copper sites almost corresponds to 61.5 amu, which is
typically observed in copper(II) binding ESI mass spectrometry
experiments. Because the average mass of copper is 63.5 amu, the
difference of 2 amu is explained as displacement of two hydrogens on
binding of each copper ion (M + n·Cu
Comparing the results of humPrP60-109,
humPrP23-98, and
humPrP23-112 only to
humPrP23-98, an additional sixth copper
ion appears to be bound. For
humPrP23-112, which is almost identical to
humPrP23-98 but C-terminally extended, only
five bound copper were observed. Also
humPrP60-109, which shares an almost identical
C-terminal extension to the octarepeat region, binds only five copper
ions. Therefore the binding of a sixth copper ion to
humPrP23-98 appears to be an unspecific copper-binding site. Thus up to five copper ions appear to be bound specifically to humPrP60-109,
humPrP23-98, and humPrP23-112, whereas four copper ions
are bound to the octarepeat region, suggesting an additional
copper-binding site within the N-terminal segment. Because the
additional copper-binding site occurs already in
humPrP60-109, which is compared with humPrP60-91 C-terminally extended, this
additional binding site has to be located C-terminally of the
octarepeat region.
Fluorescence Titration of PrPC and Its N-terminal
Fragments with Copper--
Fluorescence measurement and quantification
of the copper binding to PrPC and its fragments were
difficult with respect to some items. First, to prevent unspecific
binding of copper to PrPC and its fragments, 100 mM NaCl was included. Second, to prevent unspecific
aggregation and adsorption to surfaces 1 mM DTAC was added
at a concentration clearly below critical micelle concentration (26).
Third, for the quantification of affinities the peptide or
PrPC concentrations were chosen to be significantly lower
than the dissociation constants, to assume that the free copper
concentration is approximately equal to the added copper concentration.
Fourth, in contrast to, e.g. calcium, the fluorescence of
peptides and proteins is not only quenched by conformational changes
induced by copper(II) binding but also by diffusion-controlled
collision of copper(II) with tryptophan and tyrosine side chains
(27-29). Thus fluorescence titration curves have to be recorded at
copper concentrations far beyond the saturation point to get
quantitative data from the fluorescence titration curves.
The fluorescence titration curves with copper(II) for the
N-terminal fragments humPrP60-91,
humPrP60-109,
humPrP23-98, and
humPrP23-112; the structured C-terminal
fragment murPrP121-231; as well as for the
full-length recombinant murPrP23-231 are shown
in Fig. 4, A-F. All curves,
except that for murPrP121-231, show more
or less clearly visible sigmoidal changes in fluorescence intensity
upon addition of copper, suggesting a basic cooperative binding
mechanism. It appears that the peptides/protein cannot be saturated
with copper at high concentrations. This is due to collisional
quenching, which can be shown by plots according to the Stern-Volmer
equation (28),
From these Stern-Volmer plots, the Stern-Volmer constants as well as
the fraction of conformational fluorescence change caused by binding of
copper fconf are calculated by linear regression analysis at high copper concentrations. For this purpose Equation 1 has
to be modified, because it is only valid where exclusively collisional
quenching occurs. In the presence of conformational fluorescence
changes true values for KSV can be calculated,
introducing a correction factor according to Equation 2.
From the fluorescence titration curves (Fig. 4, A-E) it is
apparent that there is a significant increase in affinity for
copper(II) going from humPrP60-91 (Fig.
4A) to humPrP60-109 (Fig. 4B). Again a small increase in affinity can be observed
going further to humPrP23-98 (Fig.
4C), whereas the affinities of
humPrP23-98 and
humPrP23-112 (Fig. 4D), as well as
for the full-length recombinant murPrP23-231
(Fig. 4E) seem to be identical.
To get quantitative copper binding data the titration curves were
fitted by nonlinear regression analysis. ESI mass spectrometry has
confirmed that four copper ions bind to the octarepeat region (humPrP60-91). The mass spectrometry data
suggest that one additional copper binding site exists for
humPrP60-109, humPrP23-98,
humPrP23-112, and
murPrP23-231. This site was assumed to be
independent, because particularly the titration curves for
humPrP23-98,
humPrP23-112, and
murPrP23-231 could only be fitted with a
mathematical term for an independent binding site. Assuming almost
equal fluorescence changes per cooperative binding site, data of
titration curves were fitted according to Equation 3,
The quantitative data (Table II) show that C- and N-terminal
extensions in the sequence of the octarepeat region
humPrP60-91 have a significant influence on
both dissociation constant Kcoop and Hill
coefficient n. The dissociation constants for the
cooperative binding of four copper ions to the octarepeat
region decrease significantly from 5.5 to 2.5 µM for
humPrP60-91 and finally only slightly to 2.2 µM for humPrP23-98,
humPrP23-112, and
murPrP23-231. Also Hill coefficients
significantly increase from 2.4 to values ranging from 3.6 to 4.2 for
humPrP60-109, humPrP23-98,
humPrP23-112, and
murPrP23-231, suggesting almost perfect
cooperativity for the four cooperative copper-binding sites in the
full-length prion protein. Similar changes in the dissociation constant
are observed for the independent binding site. Here the values for
Kind significantly decreases from 8.8 µM to values between 1.4 and 2.4 µM for
humPrP23-98, humPrP23-112, and
murPrP23-231. For the one putative copper-binding site in murPrP121-231, a
dissociation constant of about 8 µM was estimated
considering that no precise mathematical fitting was obtained (see Fig.
4F).
Here we present for the first time affinity and stoichiometry data
on the copper binding to humPrP60-109,
murPrP121-231, and the full-length prion
protein murPrP23-231 under physiological conditions as well as for humPrP23-112
representing a natural cleavage product of the prion protein (30, 31).
Our results clearly demonstrate that the full-length prion protein
binds copper within the physiological concentration range with binding
of five copper ions per molecule. Furthermore, we proved that
the positive cooperativity of copper binding is not restricted to
N-terminal fragments but is a property of the full-length protein. The
systematical comparison of the data for full-length PrP with C- and
N-terminal fragments indicates that the N-terminal segment represents
the cooperative copper-binding domain of the prion protein.
Mass spectrometry has evolved as a method in the assessment and
elucidation of copper and heavy metal binding stoichiometry of proteins
and peptides (32, 33). In contrast to Whittal et al. (34) we
used ESI MS only for the copper binding stoichiometry studies for
several reasons. First, our fluorescence affinity data indicated the
copper binding sites of the prion protein and its fragments to be
almost saturated with copper under the chosen concentrations in ESI MS.
Therefore, it is expected to obtain mass spectra where predominantly
the four or five copper adduct peaks are observed. This is not the case
as readily visible from the spectra (see Fig. 2). It might be explained
by the high flexibility of the N-terminal copper binding domain and
thus its higher sensitivity to mechanical stress during the spraying
procedure than more classical copper binding domains in which the
copper binding sites are surrounded by several stabilizing secondary
structure elements (35). Thus the ESI mass spectra appear not to
reflect quantitatively the situation in solution but rather
qualitatively for the prion protein and its peptides. Second, further
investigations showed that ESI spectra are quite sensitive to changes
of buffer and additives. The effect of ammonium salts on the ESI mass
spectra of humPrP60-91 compared with NEMO as
buffer is shown in Fig. 5. In contrast to the ammonium buffer system the third and fourth copper-binding sites
are significantly populated using the N-ethylmorpholine instead. Additionally, the use of Tris with a functional primary amine
prevented copper binding to humPrP58-91 in CD
spectroscopy experiments (17). Summarized, ESI mass spectrometry of
copper binding to prion protein and peptides appears to be quite
sensitive to the chosen buffer and additive conditions. Therefore we
have introduced N-ethylmorpholine as new buffer system in
ESI mass spectrometry for metal interaction studies at a physiological pH of 7.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helical prion protein
(PrPC)1 to the
significantly more
-sheet-containing pathogenic isoform (PrPSc) (1). This conformational transition apparently
induces the formation of PrPSc aggregates (2), which are,
in contrast to PrPC, highly protease-resistant (3). During
infection, PrPSc appears to serve as a template for the
conversion of the native prion protein, because host PrPC
expression is required for infection with prions (4). There is evidence
that copper plays a role in the formation of PrPSc (5).
-sheet and three
-helices (8). The
N-terminal half from amino acids 23-125 showed no structure in NMR
analysis and is therefore postulated to be highly flexible (9, 10).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
ESI mass spectra of hen egg lysozyme with and
without copper(II) as negative control. To 10 µM
lysozyme (A) at pH 7.4, 50 µM (B)
and 100 µM (C) copper sulfate were added,
respectively.
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Fig. 2.
ESI mass spectra of PrPC
and its fragments with and without copper(II) at pH 7.4. A, C, E, G, I,
and K show the mass spectra of the apopeptides/proteins of
humPrP60-91,
humPrP60-109,
humPrP23-98,
humPrP23-112,
murPrP121-231, and
murPrP23-231. B, D,
F, H, J, and L represent
the mass spectra of the corresponding holopeptides of 20 µM humPrP60-91, 10 µM humPrP60-109, 10 µM humPrP23-98, 10 µM humPrP23-112, 13 µM murPrP121-231, and 10 µM murPrP23-231 with 100 µM copper(II) and 70 µM copper(II) for
humPrP23-98, respectively. If present,
open circles indicate m/z peaks for the
apopeptides. The triangles in C and G
mark the m/z peaks of by-products (see
"Results"). The diamonds in D mark
the m/z peaks for two copper ions bound to the by-product
and not for the apopeptide of
humPrP60-109.
Molecular masses for the copper binding of the prion protein and its
fragments calculated from ESI mass spectra
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Fig. 3.
Deconvoluted ESI mass spectra of
murPrP121-231 (A) and
murPrP23-231 (B) at
different copper concentrations (see the labeling of curves). The
concentrations of murPrP121-231 and
murPrP23-231 were 13 and 10 µM,
respectively. Peaks labeled with Cu, K, and
Na represent the corresponding adducts for copper, sodium,
and potassium.
2·n·H+).
where F is the fluorescence at the individual
concentration of the quencher [Q] (which is copper in this
case), Fo is the initial fluorescence without
quencher, and KSV is the quenching constant also
called the Stern-Volmer constant. Above the saturation point of copper
binding to the peptides or protein, the changes of
Fo/F should be linear in Stern-Volmer
plots if this is only due to collisional quenching. Indeed, with the
exception of murPrP121-231 (Fig. 4F,
inset) the linearity in the Stern-Volmer plots of
PrPC and its N-terminal fragments (Fig. 4, A
(Eq. 1)
E,
insets) showed that the fluorescence changes above ~30-50
µM are the result of collisional quenching. The
nonlinearity of the Stern-Volmer plot for
murPrP121-231 might be explained by the
different excitation wavelength of 285 nm instead of 295 nm
used for reasons of sensitivity. The fluorescence of
murPrP121-231, which has 11 tyrosines and
only 1 tryptophan, probably reflects not only the Trp fluorescence but
also the fluorescence of tyrosines, which are expected to have
different dynamic copper-quenching properties.
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Fig. 4.
Fluorescence titration curves of the prion
protein and its fragments with copper(II). A shows the
fluorescence data of 170 nM
humPrP60-91 (octarepeat), B of 145 nM humPrP60-106, C of
125 nM humPrP23-98, D of
120 nM humPrP23-112, E
of 85 nM murPrP23-231, and
F of 125 nM
murPrP121-231. Lines represent the
results of nonlinear regression analyses according to Equation 3. The
insets represent Stern-Volmer plots of fluorescence data
according to Equation 1. The result of linear regression analysis at
high copper concentrations is indicated by a straight
line.
where fconf is the fraction of
conformational fluorescence changes. Thus to obtain the true value for
KSV the slopes from the Stern-Volmer plots have
to be divided by the value of the ordinate 1/(1
(Eq. 2)
fconf). The Stern-Volmer plots yield values for fconf and KSV (Table
II). The Stern-Volmer constant is used
later to fit fluorescence data by nonlinear regression to obtain
copper binding data. The values for KSV
represent a measure for the accessibility of tryptophan residues. For
humPrP60-109,
humPrP23-98, and
humPrP23-112, KSV
decreases from almost 4000 M
1 to
about 2500 M
1, suggesting that
the tryptophans become more buried in the holopeptides with
longer fragment size. The reason for the at least 5-10-fold higher
value for murPrP23-231 is not known but has to
be an effect of the C-terminal domain. Although the Stern-Volmer plot
of murPrP121-231 (Fig. 4F,
inset) could not be fitted very well with Equation 2, a
similar Stern-Volmer constant of 22,000 M
1 could be estimated considering
only data from 30 to 80 µM copper.
Parameter for the dynamic and conformational fluorescence quenching of
the prion protein and its N-terminal fragments by copper(II)
where Frel is the relative fluorescence;
find and Kind are the
fraction of fluorescence change and the dissociation constant for the
independent binding site, respectively; fcoop,
Kcoop, and n are the fractional
fluorescence change, the dissociation constant, and the Hill
coefficient for the four cooperative binding sites, respectively;
KSV is the Stern-Volmer constant; and
[Cu2+] is the free copper concentration. Because the
peptide/protein concentration was chosen to be significantly lower than
the dissociation constants, the added or total copper concentration was
assumed to be equal to the free copper concentration. For the fitting procedure the Stern-Volmer constants obtained from Stern-Volmer plots
were inserted for KSV in Equation 3. Equation 3
was applied to all fluorescence titration curves except for
humPrP60-91, because only cooperative binding
sites were assumed, and thus the term for the independent site was
omitted. The same held for murPrP121-231, but
n was set to 1, because mass spectrometry suggested binding
of only one copper ion, and thus no cooperative binding can occur.
(Eq. 3)
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 5.
Relative population of the individual copper
binding sites for the ESI mass spectra of
humPrP60-91 for different buffers.
The spectra were recorded from either NEMO or an ammonium-containing
buffer at pH 7.4 at a final concentration of 1 mM. 100 µM copper sulfate was added to 18 µM
peptide.
Our finding that the octarepeat region (humPrP60-91) binds four copper ions confirmed earlier mass spectrometry (13, 34) and circular dichroism spectroscopy data (17). The mass spectra of humPrP60-109, humPrP23-98, and humPrP23-112 revealed one additional copper-binding site. This is in good agreement with the binding of 5.6 copper(II) ions to humPrP23-98 from our earlier equilibrium dialysis experiments (18). From these experiments, as well as from the mass spectrum of humPrP23-98 (Fig. 2C), a sixth binding site, which was also observed earlier (34), might be assumed. But because only five copper ions were observed for humPrP60-109 and humPrP23-112, this site has to be unspecific. The involvement of the N-terminal amino group of humPrP23-98 for the unspecific site was proved by N-terminal acetylation (34).
Mass spectrometry data showed binding of up to five copper ions to the full-length protein, confirming one additional copper-binding site compared with the four of the octarepeat region. The data for the structured C-terminal domain murPrP121-231 containing three histidine residues revealed one possible copper-binding site with an estimated dissociation constant of 8 µM. This affinity is probably too low to be of physiological significance. For copper dissociation constants in the micromolar range, at least two side chains are essential from which one has to be a histidine or cysteine residue (36). Looking for this site in the three-dimensional NMR structure of the C-terminal fragment, we found a site made up of His140, Asp147, and Met138. These residues are on the surface and close enough to each other (distance: ~5 Å).
In contrast to the putative C-terminal copper(II)-binding site for the fifth binding site in humPrP23-98, humPrP23-112, and full-length prion protein, we obtained a dissociation constant of about 2 µM. Since the additional fifth copper is already bound by humPrP60-109, it has to occur C-terminal of the octarepeat region. This binding site might be located at His96, because it is known that besides the cysteine residue, the histidyl side chain is the major copper-binding site in peptides and proteins (36). In structured proteins at least two ligands, e.g. two histidine side chains, are necessary for copper binding in the micromolar concentration range (37). In flexible peptides without secondary structure only one histidine is essential for the copper binding. In such peptides the peptide backbone can adopt conformations in which peptide bond nitrogen is involved as a ligand in copper binding after deprotonation (38). Histidine hinders deprotonation of peptide bonds in the C-terminal position but promotes the deprotonation of N-terminally located peptide bonds as for N-acetyl-Gly-Gly-Gly-His (36). In this tetrapeptide the abstraction of the three adjacent peptide-bond hydrogens, together with the imidazole nitrogen, results in a 4N coordinated copper complex. The sequence at His96 ... -Gly-Gly-Gly-Thr-His- ... is strikingly similar to N-acetyl-Gly-Gly-Gly-His, but also His112 might be involved in copper binding. Further investigations are required to clarify the role of His96, His112, or His140 in the copper binding of prion protein.
The sigmoidal form of fluorescence titration curves for the prion protein and its N-terminal fragments clearly indicated an underlying positive cooperative binding mechanism. This agreed well with CD spectroscopy data for humPrP58-91, which is almost identical to our octarepeat fragment humPrP60-91 (17). In contrast, Whittal et al. (34) have obtained individual dissociation constants for humPrP58-91 clearly indicating a negative cooperativity. This is probably explained by the limitations of ESI mass spectrometry in quantitative analysis and the buffer conditions as discussed above. Our results demonstrate that the positive cooperative copper binding is not restricted to the octarepeat fragment but is a general feature of the full-length prion protein.
The fluorescence data for humPrP60-91 yielded a cooperative dissociation constant of 5.5 µM, which is almost identical to the value of 6 µM for humPrP58-91 obtained by CD spectroscopy (17). Compared with equilibrium dialysis experiments for humPrP23-98 with a dissociation constant of 5.9 µM (18), we have calculated significantly lower dissociation constants of 2.2 and 1.4 µM for the cooperative and the assumed independent site, respectively. This might be explained by the different experimental conditions. Our affinity data for humPrP60-91 and humPrP23-98 are difficult to compare with those obtained by quantitative ESI MS (34) for the already mentioned reasons and because we have obtained one cooperative constant instead of four individual constants from which only two were calculable in the case of humPrP23-98 (34).
The similarity of the fluorescence titration curves of humPrP23-98 and humPrP23-112 with the full-length prion protein murPrP23-231 as well as the obtained data (Table II) indicated that the N-terminal segment is a copper-binding domain. The putative low affinity copper-binding site in the C-terminal domain (murPrP121-231) apparently had no influence on the copper-binding mode and affinity of the N-terminal segment. Although the total N-terminal segment up to amino acid 126 is highly flexible, C- and N-terminal extensions of the octarepeat region (humPrP60-91) have a significant influence on the affinity and cooperativity of the independent and cooperative binding sites, respectively. They clearly increase if the octarepeat region is C-terminally extended to humPrP60-109 (Table II). The affinity of the assumed independent site becomes significantly higher on N-terminal sequence extension from humPrP60-109 to humPrP23-98 or humPrP23-112. Thus the fluorescence data of the four cooperative sites as well as for the assumed independent site indicated that there are additional interactions stabilizing the holocopper-binding domain.
Fluorescence titration curve data show that the full-length murPrP23-231 is almost saturated at about 5 µM copper(II). This is within the physiological concentration range of the total copper concentration in blood plasma of 18.6 µM (39). But this concentration does not represent the physiologically available copper pool, because about 10.2 µM copper (65%) is bound to ceruloplasmin in a nonexchangeable way. In contrast about 2.8 µM is bound to serum albumin, 1.9 µM to transcuprein, and 3.6 µM is bound to low molecular weight components like amino acids in an exchangeable way. Considering only the copper pool of 3.6 µM bound by low molecular weight components and the copper affinities of the prion protein, there is strong argument for a direct role of the prion protein in copper metabolism. Additionally, copper concentrations ranging from 0.5 to 2.5 µM are found in the cerebrospinal fluid, whereas 15 µM is present in the synaptic cleft (40). In contrast to blood plasma the conditions at the synaptic cleft and in the cerebrospinal fluid are probably different in that no ceruloplasmin, serum albumin, or transcuprein is present.
The cooperative copper-binding mode of the prion protein within the physiological concentration range suggests two possible functions. First, similar to the cooperative oxygen binding of hemoglobin, it might play a role in copper transport. Second, the prion protein might be a copper sensor similar to the intracellular calcium-binding protein calmodulin, which also binds calcium in a cooperative way.
In mammals, relatively little is currently known about the precise components involved in copper transport and the mechanism by which copper is transported across the plasma membrane into cells (41). Cellular copper uptake by the prion protein might be similar to transferrin, which is mediated by endocytosis. Both transferrin and prion protein release their bound iron and copper at an acidic pH of 5.0 (15). A possible transport function for PrPC is supported by the stimulation of endocytosis with copper (42), the pH-dependent copper binding (17), and the lower copper content of brain in PrPC-deficient mice (18). This was recently further supported by our results showing that PrPC is apparently located in presynaptic membranes and that the loss of PrPC in Prnp0/0 mice strongly affected the copper content of synaptosomes (14). This suggests that PrPC is involved in the synaptic copper homeostasis.
The in vivo binding mode of copper to the prion protein is not yet known. Even if PrPC does not bind copper under normal conditions, it might serve as an extracellular copper sensor. It is known that cancer and infections increase plasma copper concentrations in blood (39). Thus it might be that PrPC binds copper and regulates cells on increasing copper concentration.
Copper binding to PrP appears to play a role in the prion disease as well (43). Copper apparently stabilized prions, because it enhances the regeneration of partially denatured PrPSc (5). Our finding of a low affinity copper-binding site in the C-terminal domain of PrPC might be interesting for the conformational change to PrPSc.
Further investigations are necessary to explore the molecular nature of
the unique, cooperative, copper binding motif in the octarepeat region
as well as to elucidate the role of copper binding for the prion
protein in vivo.
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ACKNOWLEDGEMENT |
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We thank Prof. Hübner for critically reading the manuscript, Dr. Nietmann for the use of HPLC, and K. Kroll for the use of fluorescence spectrophotometer. Furthermore, we thank Tanja Wucherpfennig for assistance in expression of humPrP23-98 and Meike Barche for excellent technical assistance.
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FOOTNOTES |
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* This work was supported by the European Union Grant BMH4-CT98-6051, by the BMBF of Germany, and by a grant from the Boehringer Ingelheim Fonds (to S. H.).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.
§ To whom correspondence should be addressed: Tel.: 49-551-39-2700; Fax: 49-551-39-8472; E-mail: mkramer@med.uni-goettingen.de.
** Present address: Ludwig Maximilian University, Dept. of Neuropathology, Marchionini-Str. 17, 81377 München, Germany.
Published, JBC Papers in Press, February 27, 2001, DOI 10.1074/jbc.M006554200
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ABBREVIATIONS |
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The abbreviations used are: PrP, prion protein; ESI, electrospray ionization; MOPS, 4-morpholinepropanesulfonic acid; DTAC, dodecyltrimethylammonium chloride; NEMO, N-ethylmorpholine; HPLC, high performance liquid chromatography; amu, average mass unit; MS, mass spectrometry.
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REFERENCES |
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1. | Pan, K.-M., Baldwin, M., Nguyen, J., Gasset, M., Serban, A., Groth, D., Mehlhorn, I., Ziwei, H., Fletterick, R. J., Cohen, F. E., and Prusiner, S. B. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 10962-10966[Abstract] |
2. | Post, K., Pitschke, M., Schäfer, O., Wille, H., Appel, T. R., Kirsch, D., Mehlhorn, I., Serban, H., Prusiner, S. B., and Riesner, D. (1998) Biol. Chem. 379, 1307-1317[Medline] [Order article via Infotrieve] |
3. | McKinley, M. P., Bolton, D. C., and Prusiner, S. B. (1983) Cell 35, 57-62[Medline] [Order article via Infotrieve] |
4. | Büeler, H., Fischer, M., Lang, Y., Bluethmann, H., Lipp, H.-P., DeArmond, S. J., Prusiner, S. B., Auet, M., and Weissmann, C. (1992) Nature 356, 577-582[CrossRef][Medline] [Order article via Infotrieve] |
5. |
McKenzie, D.,
Bartz, J.,
Mirwald, J.,
Olander, D.,
Marsh, R.,
and Aiken, J.
(1998)
J. Biol. Chem.
273,
25545-25547 |
6. | Bendheim, P. E., Brown, H. R., Rudelli, R. D., Scala, L. J., Goller, N. L., Wen, G. Y., Kascsak, R. J., Cashman, N. R., and Bolton, D. C. (1992) Neurology 42, 149-156[Abstract] |
7. | Stahl, N., Borchelt, D. R., and Prusiner, S. B. (1990) Biochemistry 29, 5405-5412[Medline] [Order article via Infotrieve] |
8. | Riek, R., Hornemann, S., Wider, G., Billeter, M., Glockshuber, R., and Wüthrich, K. (1996) Nature 382, 180-182[CrossRef][Medline] [Order article via Infotrieve] |
9. | Riek, R., Hornemann, S., Wider, G., Glockshuber, R., and Wüthrich, K. (1997) FEBS Lett. 413, 282-288[CrossRef][Medline] [Order article via Infotrieve] |
10. |
Donne, D. G.,
Viles, J. H.,
Groth, D.,
Mehlhorn, I.,
James, T. L.,
Cohen, F. E.,
Prusiner, S. B.,
Wright, P. E.,
and Dyson, H. J.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
13452-13457 |
11. | Büeler, H., Aguzzi, A., Sailer, A., Greiner, R.-A., Autenried, P., Aguet, M., and Weissmann, C. (1993) Cell 73, 1339-1347[Medline] [Order article via Infotrieve] |
12. |
Lledo, P. M.,
Tremblay, P.,
DeArmond, S. J.,
Prusiner, S. B.,
and Nicoll, R. A.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
2403-3407 |
13. | Hornshaw, M. P., McDermott, J. R., and Candy, J. M. (1995) Biochem. Biophys. Res. Commun. 207, 621-629[CrossRef][Medline] [Order article via Infotrieve] |
14. |
Herms, J.,
Tings, T.,
Gall, S.,
Madlung, A.,
Giese, A.,
Siebert, H.,
Schürrmann, P.,
Windl, O.,
Brose, N.,
and Kretzschmar, H.
(1999)
J. Neurosci.
19,
8866-8875 |
15. | Stöckel, J., Safar, J., Wallace, A. C., Cohen, F. E., and Prusiner, S. B. (1998) Biochemistry 37, 7185-7193[CrossRef][Medline] [Order article via Infotrieve] |
16. | Hornshaw, M. P., McDermott, J. R., Candy, J. M., and Lakey, J. H. (1995) Biochem. Biophys. Res. Commun. 214, 993-999[CrossRef][Medline] [Order article via Infotrieve] |
17. |
Viles, J. H.,
Cohen, F. E.,
Prusiner, S. B.,
Goodin, D. B.,
Wright, P. E.,
and Dyson, H. J.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
2042-2047 |
18. | Brown, D. R., Qin, K., Herms, J. W., Madlung, A., Manson, J., Strome, R., Fraser, P. E., Kruck, T., von Bohlen, A., Schulz-Schaeffer, W., Giese, A., Westaway, D., and Kretzschmar, H. (1997) Nature 390, 684-687[CrossRef][Medline] [Order article via Infotrieve] |
19. | Coste, J., Le-Nguyen, D., and Castro, B. (1990) Tetrahedron Lett. 31, 205-208[CrossRef] |
20. | Windl, O., Dempster, M., Estibeiro, J. P., Lathe, R., de Silva, R., Esmonde, T., Will, R., Springbett, A., Campbell, T. A., Sidle, K. C. L., Palmer, M. S., and Collinge, J. (1996) Hum. Genet. 98, 259-264[CrossRef][Medline] [Order article via Infotrieve] |
21. | Liemann, S., and Glockshuber, R. (1999) Biochemistry 38, 3258-3267[CrossRef][Medline] [Order article via Infotrieve] |
22. | Hornemann, S., and Glockshuber, R. (1996) J. Mol. Biol. 262, 614-619 |
23. | Birdsall, B., King, R. W., Wheeler, M. R., Lewis, C. A., Goode, S. R., Dunlap, R. B., and Roberts, G. C. K. (1983) Anal. Biochem. 132, 353-361[Medline] [Order article via Infotrieve] |
24. | Gill, S. C., and von Hippel, P. H. (1989) Anal. Biochem. 182, 319-326[Medline] [Order article via Infotrieve] |
25. | Sandmeier, E., Hunziker, P., Kunz, B., Sack, R., and Christen, P. (1999) Biochem. Biophys. Res. Commun. 261, 578-583[CrossRef][Medline] [Order article via Infotrieve] |
26. | Hjelmeland, L. M. (1986) Methods Enzymol. 124, 135-164[Medline] [Order article via Infotrieve] |
27. | Syvertsen, C., Melö, T. B., and Ljones, T. (1987) Biochim. Biophys. Acta 914, 6-18[Medline] [Order article via Infotrieve] |
28. | Chen, R. F. (1976) in Biochemical Fluorescence: Concepts (Chen, R. F. , and Edelhoch, H., eds) , pp. 573-606, Marcel Dekker, Inc., New York |
29. | Eftink, M. R., and Ghiron, C. A. (1981) Anal. Biochem. 114, 199-227[Medline] [Order article via Infotrieve] |
30. | Caughey, B., Raymond, G. J., Ernst, D., and Race, R. E. (1991) J. Virol. 65, 6597-6603[Medline] [Order article via Infotrieve] |
31. |
Chen, S. G.,
Teplow, D. B.,
Parchi, P.,
Teller, J. K.,
Gambetti, P.,
and Autilio-Gambetti, L.
(1995)
J. Biol. Chem.
270,
19173-19180 |
32. | Loo, J. A. (1997) Mass Spectrom. Rev. 16, 1-23[CrossRef][Medline] [Order article via Infotrieve] |
33. | Mann, M., and Wilm, M. (1995) Trends Biochem. Sci. 20, 219-224[CrossRef][Medline] [Order article via Infotrieve] |
34. | Whittal, R. M., Ball, H. L., Cohen, F. E., Burlingame, A. L., Prusiner, S. B., and Baldwin, M. A. (2000) Protein Sci. 9, 332-343[Abstract] |
35. | Adman, E. T. (1991) Adv. Protein Chem. 42, 145-197[Medline] [Order article via Infotrieve] |
36. | Sovago, I. (1990) in Biocoordination Chemistry/Coordination Equilibria in Biologically Active Systems (Burger, K., ed) , pp. 135-184, Ellis Horwood Limited, New York |
37. | Regan, L. (1995) Trends Biochem. Sci. 20, 280-285[CrossRef][Medline] [Order article via Infotrieve] |
38. | Regan, L. (1993) Annu. Rev. Biophys. Biomol. Struct. 22, 257-281[CrossRef][Medline] [Order article via Infotrieve] |
39. | Linder, M. C., and Hazegh-Azam, M. (1996) Am. J. Clin. Nutr. 63, 797S-811S[Abstract] |
40. |
Hartter, D. E.,
and Barnea, A.
(1988)
J. Biol. Chem.
263,
799-805 |
41. | Vulpe, C. D., and Packman, S. (1995) Annu. Rev. Nutr. 15, 293-322[CrossRef][Medline] [Order article via Infotrieve] |
42. |
Paul, P. C.,
and Harris, D. A.
(1998)
J. Biol. Chem.
273,
33107-33110 |
43. | Wadsworth, J. D., Hill, A. F., Joiner, S., Jackson, G. S., Clarke, A. R., and Collinge, J. (1999) Nat. Cell Biol. 1, 55-59[CrossRef][Medline] [Order article via Infotrieve] |