From the Division of Clinical Chemistry, Department
of Laboratory Medicine, Lund University, University Hospital
Malmö, Malmö S-205 02, Sweden and § INSERM U428,
University of Paris V, 4 Avenue de L'Observatoire,
75006 Paris, France
Received for publication, July 21, 2000, and in revised form, September 28, 2000
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
C4b-binding protein (C4BP) is a plasma
glycoprotein involved in regulation of the complement system. C4BP
consists of seven C4b-binding protein
(C4BP)1 is a regulator of the
classical pathway of complement that also plays a role in the
anticoagulant protein C pathway (1, 2). In the complement cascade, C4BP acts as a cofactor to factor I in the degradation of C4b (3). In
addition, C4BP inhibits the formation and accelerates the decay of the
classical C3 convertase pathway, i.e. the C4b2a complex (4).
C4BP is a large plasma glycoprotein of 570 kDa consisting, for the main
isoform, of seven The In plasma, ~70% of protein S is in complex with C4BP. Only free
protein S functions as an activated protein C cofactor (11). In
contrast, C4BP in complex with protein S can still exert its regulatory
functions on the complement system (14). C4BP regulates the plasma
availability of free protein S since the concentration of free protein
S represents the molar excess of protein S over C4BP (15). The
biological importance of the protein S-C4BP interaction is emphasized
by the fact that only the concentration of free protein S can clearly
be linked to thrombotic risk in patients suffering from protein S
deficiencies (16). The physiological purpose of the interaction between
C4BP and protein S is not yet fully understood. However, protein S,
being a vitamin K-dependent protein, has a very high
affinity for negatively charged phospholipids; and therefore, it could
localize C4BP to surfaces where such phospholipids are exposed (17,
18).
Our group has shown that the Fernández and Griffin (24) used synthetic peptides to probe the
protein S-C4BP interaction and suggested residues 31-45 on C4BP to be
important for protein S binding. This hypothesis was supported in a
subsequent report (25), where it was found that preincubation of
C4BP with monoclonal antibody 6F6 (directed against a region located
nearby residues 31-45) inhibited the C4BP and protein S interaction.
It was then concluded that the antibody interfered with the interaction
most likely because of steric hindrance (25).
We have previously shown that binding of protein S to C4BP varied only
to a small extent with the concentration of salt, in a manner implying
a significant contribution from hydrophobic interactions, with minor
roles played by electrostatic forces (26). These experiments were the
first to confirm the hypothesis that a hydrophobic cluster at the
surface of CCP1 may be the main binding site for protein S (27).
In this study, we have mutated potentially important amino acids
located in this hydrophobic cluster on C4BP CCP1. The mutations were
chosen based on the homology-based, computer-generated
three-dimensional structure of the C4BP Cloning Procedure
The prokaryotic expression vector used for expression of the
recombinant C4BP -chains and one unique
-chain, all constructed
of repeating complement control protein (CCP) modules. The
-chain,
made up of three CCPs, binds tightly to vitamin K-dependent
protein S, a cofactor to anticoagulant activated protein C. When bound
to C4BP, protein S loses its activated protein C cofactor function. In
this study, we have mutated potentially important amino acids located
at the surface of CCP1 of the
-chain to probe the protein S-C4BP
interaction. The substitutions were designed after analysis of a
homology-based three-dimensional structure of the
-chain and were
L27T/F45Q, I16S/V18S, V31T/I33N, I16S/V18S/V31T/I33N, L38S/V39S, and
K41E/K42E. The mutants were expressed in a prokaryotic system, purified
using an N-terminal His-tag, refolded using an oxido-shuffling system,
and tested in several assays for their ability to bind protein S. Our
data define Ile16, Val18,
Val31, and Ile33 as crucial for protein S
binding, with secondary effects from Leu38 and
Val39. In addition, Lys41 and Lys42
contribute slightly to the interaction. Our results further confirm that surface hydrophobicity analysis may be used to identify ligand recognition sites.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-chains and one
-chain, which are held together
by a central core. Both the
- and
-chains are composed of
multiple complement control protein (CCP) domains. A CCP domain is
~60 residues long and contains two disulfide bridges and a central
antiparallel
-sheet (5). CCP domains are present in numerous
proteins both within and outside the complement system (6). Some
CCP-containing molecules have been investigated by NMR or x-ray
crystallography, e.g. vaccinia virus complement control protein (7),
2-glycoprotein I (8), and CD46 (9). The knowledge of the three-dimensional structure of these domains enabled
us to construct a homology-based model of C4BP.
-chains of C4BP, consisting of eight CCPs, bind complement
protein C4b (1). A key recognition site for C4b on C4BP has been
recently ascribed to a cluster of positively charged amino acids on the
interface of CCP1-CCP2 of the
-chain (10). The unique C4BP
-chain, essentially made up of three CCPs, binds protein S, an
anticoagulant molecule that acts mainly as cofactor to activated
protein C in the degradation of coagulation factors Va and VIIIa. C4BP
and protein S form a high affinity, noncovalent complex with a 1:1
molecular ratio, which is greatly enhanced by calcium (11). The
C-terminal sex hormone globulin binding-like region of protein S is
involved in the interaction with C4BP (12, 13). This domain in protein
S is expected to have calcium-binding site(s), whereas it has never
been shown or proposed that C4BP interacts with any metal ion (12,
13).
-chain of C4BP (19) contains the
protein S-binding site (20); more precisely, CCP1 is required for
binding to occur (21). It was recently suggested by van de Poel
et al. (22, 23) that CCP2 also contributes to the binding to
a small extent. van de Poel et al. used a different approach
to study the binding. In their investigation, chimeras were constructed
composed of individual CCP modules (or the different CCPs in
combination) fused to the N-terminus of a modified tissue plasminogen
activator. They found that CCP2 increased the affinity for protein S
~5-fold.
-chain and previous
experimental data. In addition, two lysines that could be responsible
for the slight electrostatic component seen in the interaction were
also mutated and studied. Wild-type CCP1 and CCP2 of the
-chain and
the mutants were expressed in a prokaryotic system, purified on a
nickel-Sepharose column, and refolded. All recombinant proteins were
then tested for their ability to bind protein S. We found that
substitution of four hydrophobic amino acids by polar residues in the
first CCP of the
-chain decreased the apparent affinity for protein S 100-fold. Our results not only provide insights into the nature of
the protein S-C4BP interaction, but also have implications for the
prediction of binding sites at the surface of other CCP modules and may
be valuable for the understanding of protein-protein recognition.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-chain was pET-26b(+) (Novagen). It carries an
N-terminal pelB signal sequence and a C-terminal 6-His tag. CCP1 and CCP2 of the C4BP
-chain were cloned by polymerase chain reaction from full-length cDNA of the
-chain into pET-26b(+) using the following primers: 5'-ATC CAT GGG ATC AGA TGC AGA GCA C-3'
and 5'-CGC TCG AGA CTT TTG CAG ATG GGA AA-3'. This construct was then
used as a template, and the mutations were introduced using the
QuickChange site-directed mutagenesis kit (Stratagene). The templates
and sense primers used for mutagenesis as well as the resulting mutants
are shown in Table I. The various
-chain constructs were then transformed into Escherichia
coli DH5
bacteria, and mutations were confirmed using an
automated DNA sequencer (PerkinElmer Life Sciences).
Templates and sense primers used for mutagenesis
Expression and Purification of Recombinant Proteins
cDNAs coding for the recombinant proteins were transformed
into and expressed in E. coli strain BL21(DE3). About 1 ml
of overnight culture of the transformed bacteria grown in Luria broth
containing 30 µg/ml kanamycin was used to inoculate 500 ml of the
same medium. The bacteria were grown by shaking at 37 °C until the
absorbance at 600 nm was ~0.7. Expression of protein was induced by
the addition of 1 mM
isopropyl-1-thio--D-galactopyranoside, and incubation was continued for 3 h. The culture was then centrifuged at
7000 × g for 25 min at 4 °C, and the bacterial
pellet was resuspended in 100 ml of cold phosphate-buffered saline.
After incubation for 15 min at room temperature, with lysozyme added to
a final concentration of 100 µg/ml, the bacteria were sonicated at 10 micron peak to peak and centrifuged in the same way. The pellet obtained was suspended in 6 M guanidine HCl, 20 mM Tris-HCl (pH 8.0), and 10 mM reduced
glutathione and sonicated and centrifuged as described above. The
supernatant was then applied to a nickel-nitrilotriacetic acid
Superflow column (2.6 × 12 cm, QIAGEN) equilibrated with the same
buffer. The column was washed with 50 mM Tris-HCl (pH 8.0),
150 mM NaCl, and 20 mM imidazole, and the
protein was eluted with 10 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 100 mM EDTA. Fractions containing
protein were chosen by measurement of the absorbance at 280 nm and
pooled. Tris-HCl (pH 8.3) and dithiothreitol were then added, both to a
final concentration of 100 mM. After incubation for 2 h at 4 °C, the sample was diluted in 50 mM Tris-HCl (pH 8.3), 3 mM cysteine, and 0.3 mM cystine so that
the absorbance at 280 nm was equal to 0.1, and folding of the protein
was accomplished by overnight dialysis at 4 °C against the same
buffer. Iodoacetamide was then added to the dialyzed sample to a final
concentration of 5 mM, and dialysis was continued overnight
at 4 °C against 50 mM Tris-HCl (pH 8.0) and 10%
glycerol. The dialyzed sample was then applied to a MonoQ column (2 ml,
Amersham Pharmacia Biotech) equilibrated with the same buffer; protein
was in the flow-through. The protein was concentrated using an Amicon
concentrator. Finally, the protein was dialyzed against 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 10%
glycerol and stored at
70 °C until further use. Exact
concentrations of recombinant protein were determined by analysis of
amino acid composition after hydrolysis in 6 M HCl for
24 h.
Structural Analysis of Recombinant Proteins
Binding of Monoclonal Antibodies--
All recombinant proteins
were tested for their ability to bind to seven monoclonal antibodies
raised against the recombinant wild-type -chain using a standard
procedure. Microtiter plates were coated with 50 µl of purified
antibody at 10 µg/ml in 75 mM sodium carbonate (pH 9.6)
at 4 °C overnight. The plate was then washed three times with wash
buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl,
0.1% (w/v) Tween 20, and 2 mM CaCl2), quenched
in wash buffer with 3% fish gelatin for 2 h, and washed as
described above. Increasing amounts of recombinant
-chain (wild-type
and mutant) plus trace amounts of 125I-labeled recombinant
wild-type
-chain were added for 5 h at room temperature. The
plates were then washed five times, and bound radioactivity was
measured in a
-counter.
Gel Filtration-- All recombinant proteins were analyzed by gel filtration (Superose 12 HR 10/30, Amersham Pharmacia Biotech). Fifty µg of each protein was applied to the column, previously equilibrated with Tris-buffered saline (pH 8.0). The flow rate used was 0.5 ml/min. During each run, the absorbance of the eluate at 280 and 214 nm was constantly monitored.
Mass Spectrometry-- Mass spectrometry, carried out at the Protein Analysis Center of the Karolinska Institute (Stockholm, Sweden), was performed on all recombinant proteins using quadrupole time-of-flight (Q-TOF) mass spectrometry (Micromass) (28). The proteins were dialyzed against 2% HAc. Samples were subsequently analyzed by nanoelectrospray mass spectrometry in 1% acetic acid and 60% acetonitrile.
Circular Dichroism-- Recombinant proteins were dialyzed against 10 mM sodium phosphate (pH 7.4) before analysis. Approximately 50 µg of each protein was analyzed in the far-UV region (185-250 nm). The resolution was 1 nm; the speed was 10 nm/min; and the response was measured every 8 s. Sensitivity was 20 millidegrees.
Proteins--
Human C4BP and protein S were purified as
described before (29). The concentrations were determined by measuring
the absorbance at 280 nm. The extinction coefficients
(1 cm1%) used were 14.1 (C4BP) and 9.5 (protein S). Protein S and recombinant wild-type C4BP
-chain CCP1 and CCP2 were labeled with 125I using the
chloramine-T method.
Electrophoretic and Blotting Techniques-- Recombinant proteins were run on 15% SDS-polyacrylamide gel under reducing (~1 µg) and nonreducing (~0.5 µg) conditions for silver staining and under nonreducing conditions for radioligand blotting (~2 µg). For radioligand blotting, the proteins were transferred from the gel to a polyvinylidene difluoride membrane. The membrane was then incubated for 1 h at room temperature in a quenching solution composed of buffer A (50 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 0.5% (w/v) Tween 20) supplemented with 3% fish gelatin. The buffer was changed to buffer A with 2 mM CaCl2 and trace amounts of 125I-labeled protein S, and the membrane was incubated overnight at 4 °C. The membrane was then washed with buffer A, dried, and exposed in a cassette. Finally, the membrane was scanned using a PhosphorImager (Molecular Dynamics). In addition, the intensities of the bands detected were estimated by densitometry using ImageQuant software (Molecular Dynamics).
Binding Assays
Direct Binding--
Microtiter plates were coated with 50 µl
of protein (plasma-purified C4BP or recombinant wild-type or mutant
-chain) at 10 µg/ml in 75 mM sodium carbonate (pH 9.6)
at 4 °C overnight. The plate was then washed three times with wash
buffer and quenched in wash buffer with 3% fish gelatin for 2 h.
The plate was washed as described above, and protein S was added at
increasing concentrations (0-240 nM final concentration)
with trace amounts of 125I-labeled protein S at 4 °C
overnight. Finally, the plate was washed five times with wash buffer,
and bound radioactivity was determined in a
-counter.
Competition Assay--
Microtiter plates were coated with 50 µl of plasma-purified C4BP at 10 µg/ml in 75 mM sodium
carbonate (pH 9.6) at 4 °C overnight. The plate was then washed
three times with wash buffer, quenched in wash buffer with 3% fish
gelatin for 2 h, and washed as described above. Increasing amounts
of plasma-purified C4BP or recombinant -chain (wild-type and mutant)
plus trace amounts of 125I-labeled protein S were added
overnight at 4 °C. The next day, the plates were washed five times
with the same buffer, and bound radioactivity was measured in a
-counter.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Expression and Purification of Recombinant Proteins--
To study
a possible binding site for protein S on the C4BP -chain, the
following mutations were introduced in the first CCP of the
-chain:
L27T/F45Q, I16S/V18S, V31T/I33N, I16S/V18S/V31T/I33N, K41E/K42E, and
L38S/V39S. The mutations were chosen after analysis of a homology-based
three-dimensional model of the
-chain (Fig. 1) and previous experimental data on the
protein S-C4BP interaction (21, 24-27). The residues selected for
mutagenesis were all solvent-exposed, and their replacement should be
structurally well tolerated, as removal of hydrophobic residues from
the surface generally tends to stabilize a protein. This results from
the fact that surrounding water molecules should be able to form
hydrogen bonds with the newly introduced polar amino acids.
Furthermore, substitution of amino acids expected to be positively
charged at physiological pH (Lys41 and Lys42)
with two negatively charged residues at the surface of CCP1 should not
alter the structure of the domain. Indeed, the two Lys side chains
point in opposite directions in the three-dimensional model, in part
due to charge-charge repulsion. This phenomenon is also expected for
the two resulting Glu residues.
|
All proteins (recombinant wild-type and mutant) were expressed in a
prokaryotic system and purified utilizing a C-terminal His-tag. Since
the expressed proteins were localized to inclusion bodies in the
BL21(DE3) bacteria, the almost pure but misfolded protein obtained was
then refolded using an oxido-shuffling system (30, 31) and subsequently
purified on a MonoQ column. All recombinant proteins were of similar
apparent molecular mass (14 kDa) as judged by nonreducing SDS-PAGE
(Fig. 2B). (A slight increase in apparent size was seen under reducing conditions for all recombinant proteins (Fig. 2A).) Approximately 3 mg of purified protein
was obtained from each liter of bacterial culture.
|
Characterization of Recombinant Proteins--
Introduction of
mutations did not affect the expression levels or electrophoretic
mobilities of corresponding proteins compared with the wild-type
protein. Furthermore, all constructs bound with similar apparent
affinity to seven monoclonal antibodies raised against the recombinant
wild-type protein. Results for two different antibodies are shown as an
example of the binding curves obtained (Fig.
3).
|
The exact masses of all recombinant proteins (except L38S/V39S) were analyzed by mass spectrometry. All masses were the precise expected match given the change in mass due to the changes of amino acids (Table II).
|
Upon gel filtration, all proteins eluted with a major single peak at a volume of ~15 ml, corresponding to a protein size of 13 kDa, as judged by a standard curve obtained from proteins with a known molecular mass, indicating that aggregates were not present (data not shown). The amount of protein eluted in the major peak, compared with the total amount of protein eluted, varied between 63 and 99%, the lowest being for the L27T/F45Q mutant. The major eluted peak for the mutant with four hydrophobic amino acids mutated, I16S/V18S/V31T/I33N, contained 84% of the total eluted protein.
Circular dichroism analysis of all recombinant proteins gave very
similar spectra, once again confirming that introductions of mutations
did not cause folding changes. The signal was not possible to judge
below 205 nm due to background noise. Results are presented as strength of the signal
relative to the lowest point (millidegrees/s) measured (Fig.
4).
|
Radioligand Blotting--
To assess the binding of protein S to
the various mutants, we used radioligand blotting, in which unreduced
wild-type and mutant proteins immobilized on a polyvinylidene
difluoride membrane were allowed to bind 125I-labeled
protein S (Fig. 5, one representative
experiment is shown). Mutant L27T/F45Q bound with similar or increased
strength compared with the recombinant wild-type -chain. I16S/V18S,
V31T/I33N, I16S/V18S/V31T/I33N, and L38S/V39S all lost the binding
ability for protein S as judged by the absence of bands on the blot
after analysis by the PhosphorImager. The K41E/K42E mutant displayed weaker binding than the recombinant wild-type
-chain. For better quantification of differences between mutants, the intensities of all
bands were estimated by measurement of density using ImageQuant software. The results are shown in Table
III; each value represents the mean ± S.D. of three different experiments. No binding was detectable when
proteins were reduced (data not shown), implying that the two
characteristic disulfide bonds present in the CCP modules were
appropriately formed and, as expected, are crucial for the domain
folding and thus for the interaction with protein S.
|
|
Direct Binding Assay--
To further confirm the results obtained
by radioligand blotting, a ligand binding assay was performed (Fig.
6A). Increasing amounts of
unlabeled and 125I-labeled protein S were added to
immobilized plasma-purified C4BP or the recombinant -chain
(wild-type or mutant). After washing, bound radioactive protein S was
measured using a
-counter. The recombinant wild-type
-chain
construct bound protein S with similar affinity as plasma-purified
C4BP. This result also supports the structural integrity of the
recombinant molecule and further confirms the fact that this expression
system combined with the proper refolding technique leads to the
production of a protein sharing the same characteristics as
plasma-purified C4BP, but with the absence of glycosylation. It is
known, however, that the carbohydrate side chains are not important for
protein S binding, as a truncated recombinant wild-type
-chain
composed of three CCP modules was able to bind to protein S in a
similar fashion as plasma-purified C4BP (20), which is also confirmed
in the present study. As in the radioligand blotting, the L27T/F45Q
mutation seemed to increase the binding of C4BP to protein S. For the
remaining mutants, I16S/V18S, V31T/I33N, I16S/V18S/V31T/I33N, and
L38S/V39S, the binding was essentially lost. K41E/K42E again displayed
weaker binding, never reaching more than ~70% of binding of
125I-labeled protein S, compared with the wild-type
-chain.
|
Competition Assay--
In the competition assay, increasing
amounts of plasma-purified C4BP or recombinant -chain (wild-type or
mutant) were allowed to compete with immobilized C4BP for binding of
fluid-phase 125I-labeled protein S. After washing, bound
radioactive protein S was measured using a
-counter. The wild-type
-chain bound protein S equally well compared with plasma-purified
C4BP (Fig. 6). In this assay, mutant K41E/K42E bound in a similar
fashion compared with the wild-type
-chain (Fig. 6C).
L38S/V39S displayed ~10-fold less apparent affinity (Fig.
6C), whereas I16S/V18S, V31T/I33N, and I16S/V18S/V31T/I33N
had an ~100-fold lower apparent affinity (Fig. 6B).
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In this study, we show that a key binding surface for protein S is
centered on Ile16, Val18, Val31,
and Ile33 on CCP1 of the -chain of C4BP. This cluster of
solvent-exposed hydrophobic residues on the first CCP of the
-chain,
together with two lysines, became apparent during analysis of a
predicted three-dimensional model structure for C4BP (26, 27)
based upon the NMR structure reported by Norman et al. (5).
Since relatively large patches of solvent-exposed hydrophobic residues tend to destabilize the native state of a protein, possibly by shifting
the folding equilibrium toward denaturation (32), it could have been
argued that this region of C4BP was not appropriately modeled and was a
computational artifact. However, we have also predicted the structure
of this first CCP using other experimental templates (e.g.
2-glycoprotein I, CD46, and vaccinia virus complement control protein) and found that this exposed hydrophobic cluster is
essentially present in all models (data not shown). Because of this
observation and the data showing that the protein S-C4BP interaction is
not significantly altered by the presence of increasing concentrations
of NaCl, it was most likely that the solvent-exposed cluster indeed
plays a significant role in protein S binding. The mutations introduced
were chosen based on the analysis of the homology-based
three-dimensional structure of the
-chain of C4BP (Fig. 1).
Substitution of solvent-exposed residues by more polar ones should be
favorable to the protein stability and/or folding, and we do not expect
that structural problems could be induced because of the mutations. For
example, the Ile16, Val18, Val31,
Ile33, and Leu38 cluster has a surface area of
~300 Å2. With a hydrophobic solvation free energy of
~20 cal/mol/Å2, the energetic cost of exposing a patch
of 300 Å2 is high and ~6 kcal/mol.
The substitutions used were I16S/V18S, V31T/I33N, I16S/V18S/V31T/I33N,
L38S/V39S, L27T/F45Q, and K41E/K42E. Our results strongly suggest that
the protein S-binding site is indeed composed of a hydrophobic patch
containing Ile16, Val18, Val31,
Ile33, Leu38, and Val39, but that
Leu27 and Phe45 are not involved in the
binding. The weak contribution of electrostatic forces, observed
earlier (26), could be due in part to the presence of Lys41
and Lys42. Although these amino acids affect the binding,
they do not, by themselves, seem to be crucial for the interaction. It
is clear that the change of Ile16/Val18 or
Val31/Ile33 is sufficient to abrogate the
protein S-C4BP interaction. The residues involved in protein S binding
are shown in Fig. 7. These amino acids
form a cluster located in the direct vicinity of the second CCP (Figs.
1 and 6). van de Poel et al. (22, 23) showed that CCP2 seems
to have a weak contribution to the binding between protein S and C4BP.
It is possible that the large sex hormone globulin binding-like domain
of protein S not only binds to CCP1, but also interacts with CCP2 of
the -chain. Another explanation for the influence of CCP2 on the
binding of protein S could be that the second CCP sterically
facilitates the binding of protein S to CCP1. However, our data are in
agreement with our previous results stating that CCP1 contains
the key binding site for protein S (21), as the substitution of four
residues dramatically alters the interaction, fully consistent with the
structural observations.
|
The -chain is glycosylated, and two consensus sequences for
N-linked glycosylation are present on the first CCP module
(33). The difference between the expected molecular mass of the
-chain based on its amino acid composition (26.4 kDa) and the
apparent molecular mass as judged by SDS-PAGE (45 kDa) implies that
most or all of the glycosylation sites are occupied (33). This is further emphasized by the fact that, after digestion with
endoglycosidase F, an enzyme that removes N-linked
carbohydrates, the apparent molecular mass on SDS-PAGE of the
-chain
is 29 kDa (33). It is known that oligosaccharide moieties can
contribute to protein-protein interactions (34). For instance, the
N-linked glycan attached to the second CCP of CD46 is
essential for virus binding (35). However, it has been shown that
sugars are not important for the binding of protein S to C4BP (20).
Therefore, assuming one or both Asn residues (at positions 47 and 54)
to be glycosylated, the glycan should be located outside the key
binding surface for protein S. To study the spatial arrangement of
these sugars relative to the defined protein S-binding site, glycan
core structures were modeled as shown in Figs. 1 and 7. The glycan
molecule was taken from the x-ray structure of CD46 (9) and grafted
onto C4BP Asn47 and Asn54. Because we show that
the key binding residues for protein S are Ile16,
Val18, Val31, and Ile33 and since
the glycans are away from this region, the predicted structure and
experimental data are again in full agreement.
Fernández and Griffin (24) used synthetic peptides to probe the protein S-C4BP interaction and suggested that residues 31-45 are important for protein S binding. Our results in part confirm their observations since we show that Leu38 and Val39 have great influence on the protein S-C4BP interaction. However, we show that also Ile16 and Val18 are crucial for the binding of protein S to C4BP. Furthermore, changing the hydrophobic residues Leu27 and Phe45 to polar residues did not lessen the binding to protein S. Rather, the substitution seemed to slightly enhance the binding, suggesting that these residues directly or indirectly repulse protein S.
The role of exposed hydrophobic residues in protein-protein interaction is not unique for the CCPs of C4BP. Our data are consistent with the analysis of other macromolecular interactions, as in many systems, a solvent-exposed hydrophobic cluster has been observed (36-38). For instance, it has been proposed upon analysis of the CD46 x-ray structure that the CD46-measles virus hemagglutinin interaction is dependent on a critical set of hydrophobic residues at the protein interfaces and that this reaction resembles the CD4-HIV gp120 interaction (9). Recent investigations have highlighted the importance of solvent-exposed hydrophobic patches (ranging from 200 to 1200 Å2) (39, 40). Many different types of binding surfaces are expected to be found in nature to allow proper folding of the molecules and to render high, medium, or low affinity as well as specificity. Thus, it is obvious that some protein interfaces are very rich in charged residues, whereas others display solvent-exposed hydrophobic clusters. The importance of hydrophobic contacts for protein interactions is appealing, as such interactions appear to glue two molecules together. However, these interactions may not be very specific and so should be complemented by hydrophilic interactions. This situation is expected in the protein S-C4BP interaction, as it seems that the key hydrophobic binding surface is supplemented by hydrophilic interactions. Long-range electrostatic interaction may not be the only force that can affect association. Important contributions to the binding free energy involve also desolvation (i.e. the removal of solvent from nonpolar and polar atoms). Indeed, when a large apolar surface is exposed to solvent, other long-range attractive forces are expected to contribute significantly to protein assemblies (41). There seem to be many reasons to maintain large or small hydrophobic clusters within a binding site area of transient or very stable molecular complexes, despite the overall energetic cost of such a structural feature.
Investigation of the protein S-C4BP interaction is of importance for numerous reasons. First, protein S deficiency is a risk factor for thrombosis, and a better understanding of the protein S-C4BP interaction could be valuable for better diagnosis and treatment of coagulation disorders. Second, CCP modules are present in numerous proteins involved in various important biological processes. Thus, analysis of a specific CCP domain could provide information that may be a general consensus for CCP modules. Third, a better understanding of protein-protein interaction guides the design of approaches aimed at the prediction of hot spots at the surface of a molecule. This is of importance because many research projects involve characterization of binding sites, which then helps the understanding of molecular mechanisms and thus the generation of new therapeutic compounds or diagnostic tools.
In conclusion, we have shown a large hydrophobic patch on the -chain
of C4BP to be crucial for binding of protein S to C4BP. In addition,
Lys41 and Lys42 could contribute to the modest
electrostatic component playing a role in this interaction. These data
are the first reported that clearly pinpoint the specific amino acids
on CCP1 responsible for the interaction between protein S and C4BP. Our
data are also in agreement with what has been observed for other
CCP-containing molecules as well as in several macromolecular
assemblies. Furthermore, our investigation emphasizes the rational of
using computer-based molecular modeling to predict the
three-dimensional structure of a protein and its potential in the
design of experiments aimed at a better understanding of the
relationships between structure and function.
![]() |
ACKNOWLEDGEMENTS |
---|
We gratefully acknowledge Jan Johansson (Department of Medical Biochemistry and Biophysics, Karolinska Institute) for help with mass spectrometry, which was carried out at the Protein Analysis Center of the Karolinska Institute. We are also grateful to Sara Linse (Physical Chemistry 2, Lund Institute of Technology) for assistance with circular dichroism analysis.
![]() |
FOOTNOTES |
---|
* This work was supported by grants from the Swedish Medical Council, the Network for Inflammation Research funded by the Swedish Foundation for Strategic Research, the Tore Nilson's Trust, the Greta and Johan Kock's Trust, the Österlunds Trust, the Craaford Trust, and the Louis Jeantet Foundation of Medicine and by the University Hospital Malmö and the Fondation pour la Recherche Médicale.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.: 46-40-331501; Fax: 46-40-337044; E-mail: Bjorn.Dahlback@klkemi.mas.lu.se.
Published, JBC Papers in Press, October 24, 2000, DOI 10.1074/jbc.M006541200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: C4BP, C4b-binding protein; CCP, complement control protein; PAGE, polyacrylamide gel electrophoresis; HIV, human immunodeficiency virus.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. |
Scharfstein, J.,
Ferreira, A.,
Gigli, I.,
and Nussenzweig, V.
(1978)
J. Exp. Med.
148,
207-222 |
2. | Dahlbäck, B. (1995) Thromb. Res. 77, 1-43[CrossRef][Medline] [Order article via Infotrieve] |
3. | Fujita, T., Gigli, I., and Nussenzweig, V. (1978) J. Exp. Med. 148, 1044-1051[Abstract] |
4. |
Fujita, T.,
and Nussenzweig, V.
(1979)
J. Exp. Med.
150,
267-276 |
5. | Norman, D. G., Barlow, P. N., Baron, M., Day, A. J., Sim, R. B., and Campbell, I. D. (1991) J. Mol. Biol. 219, 717-725[Medline] [Order article via Infotrieve] |
6. | Bork, P., Downing, A. K., Kieffer, B., and Campbell, I. D. (1996) Q. Rev. Biophys. 29, 119-167[Medline] [Order article via Infotrieve] |
7. | Wiles, A. P., Shaw, G., Bright, J., Perczel, A., Campbell, I. D., and Barlow, P. N. (1997) J. Mol. Biol. 272, 253-265[CrossRef][Medline] [Order article via Infotrieve] |
8. |
Bouma, B.,
de Groot, P. G.,
van den Elsen, J. M.,
Ravelli, R. B.,
Schouten, A.,
Simmelink, M. J.,
Derksen, R. H.,
Kroon, J.,
and Gros, P.
(1999)
EMBO J.
18,
5166-5174 |
9. |
Casasnovas, J. M.,
Larvie, M.,
and Stehle, T.
(1999)
EMBO J.
18,
2911-2922 |
10. |
Blom, A. M.,
Webb, J.,
Villoutreix, B. O.,
and Dahlbäck, B.
(1999)
J. Biol. Chem.
274,
19237-19245 |
11. |
Dahlbäck, B.
(1986)
J. Biol. Chem.
261,
12022-12027 |
12. | He, X., Shen, L., Malmborg, A. C., Smith, K. J., Dahlback, B., and Linse, S. (1997) Biochemistry 36, 3745-3754[CrossRef][Medline] [Order article via Infotrieve] |
13. |
Evenäs, P.,
García de Frutos, P.,
Linse, S.,
and Dahlbäck, B.
(1999)
Eur. J. Biochem.
266,
935-942 |
14. |
Schwalbe, R. A.,
Dahlbäck, B.,
and Nelsestuen, G. L.
(1990)
J. Biol. Chem.
265,
21749-21757 |
15. | Griffin, J. H., Gruber, A., and Fernández, J. A. (1992) Blood 79, 3203-3211[Abstract] |
16. |
Zöller, B.,
Garcia de Frutos, P.,
and Dahlbäck, B.
(1995)
Blood
85,
3524-3531 |
17. | Furmaniak-Kazmierczak, E., Hu, C. Y., and Esmon, C. T. (1993) Blood 81, 405-411[Abstract] |
18. |
Schwalbe, R.,
Dahlback, B.,
Hillarp, A.,
and Nelsestuen, G.
(1990)
J. Biol. Chem.
265,
16074-16081 |
19. |
Hillarp, A.,
and Dahlbäck, B.
(1988)
J. Biol. Chem.
263,
12759-12764 |
20. |
Härdig, Y.,
Rezaie, A.,
and Dahlbäck, B.
(1993)
J. Biol. Chem.
268,
3033-3036 |
21. |
Härdig, Y.,
and Dahlbäck, B.
(1996)
J. Biol. Chem.
271,
20861-20867 |
22. |
van de Poel, R. H. L.,
Meijers, J. C. M.,
and Bouma, B. N.
(1999)
J. Biol. Chem.
274,
15144-15150 |
23. | van de Poel, R. H. L., Meijers, J. C. M., Dahlbäck, B., and Bouma, B. N. (1999) Blood Cells Mol. Dis. 25, 279-286[CrossRef][Medline] [Order article via Infotrieve] |
24. |
Fernández, J. A.,
and Griffin, J. H.
(1994)
J. Biol. Chem.
269,
2535-2540 |
25. | Fernández, J. A., Villoutreix, B. O., Hackeng, T. M., Griffin, J. H., and Bouma, B. N. (1994) Biochemistry 33, 11073-11078[Medline] [Order article via Infotrieve] |
26. | Blom, A. M., Covell, D. G., Wallqvist, A., Dahlbäck, B., and Villoutreix, B. O. (1998) Biochim. Biophys. Acta 1388, 181-189[Medline] [Order article via Infotrieve] |
27. | Villoutreix, B. O., Fernández, J. A., Teleman, O., and Griffin, J. H. (1995) Protein Eng. 8, 1253-1258[Abstract] |
28. | Jonsson, A. P., Carlquist, M., Husman, B., Ljunggren, J., Jörnvall, H., Bergman, T., and Griffiths, W. J. (1999) Rapid Commun. Mass Spectrom. 13, 1782-1791[CrossRef][Medline] [Order article via Infotrieve] |
29. | Dahlbäck, B. (1983) Biochem. J. 209, 847-856[Medline] [Order article via Infotrieve] |
30. | Jaenicke, R., and Rudolph, R. (1989) in Protein Structure: A Practical Approach (Creighton, T. E., ed) , pp. 191-223, IRL Press Ltd., Oxford |
31. | Stenberg, Y., Muranyi, A., Steen, C., Thulin, E., Drakenberg, T., and Stenflo, J. (1999) J. Mol. Biol. 293, 653-665[CrossRef][Medline] [Order article via Infotrieve] |
32. | Cordes, M. H., and Sauer, R. T. (1999) Protein Sci. 8, 318-325[Abstract] |
33. | Hillarp, A., and Dahlbäck, B. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 1183-1187[Abstract] |
34. | Quasba, P. K. (2000) Carbohydr. Polym. 41, 293-309[CrossRef] |
35. | Maisner, A., Alvarez, J., Liszewski, M. K., Atkinson, D. J., Atkinson, J. P., and Herrler, G. (1996) J. Virol. 70, 4973-4977[Abstract] |
36. |
DeLano, W. L.,
Ultsch, M. H.,
de Vos, A. M.,
and Wells, J. A.
(2000)
Science
287,
1279-1283 |
37. | Freund, C., Dotsch, V., Nishizawa, K., Reinherz, E. L., and Wagner, G. (1999) Nat. Struct. Biol. 6, 656-660[CrossRef][Medline] [Order article via Infotrieve] |
38. | Clackson, T., and Wells, J. A. (1995) Science 267, 383-386[Medline] [Order article via Infotrieve] |
39. | Lijnzaad, P., Berendsen, H. J. C., and Argos, P. (1996) Proteins Struct. Funct. Genet. 25, 389-397[CrossRef][Medline] [Order article via Infotrieve] |
40. |
Young, L.,
Jernigan, R. L.,
and Covell, D. G.
(1994)
Protein Sci.
3,
717-729 |
41. | Lum, K., Chandler, D., and Weeks, J. D. (1999) J. Phys. Chem. B 103, 4570-4577[CrossRef] |