The Fourth Epidermal Growth Factor-like Domain of Thrombomodulin
Interacts with the Basic Exosite of Protein C*
Likui
Yang and
Alireza R.
Rezaie
From the Edward A. Doisy Department of Biochemistry and Molecular
Biology, St. Louis University School of Medicine,
St. Louis, Missouri 63104
Received for publication, November 19, 2002, and in revised form, January 10, 2003
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ABSTRACT |
Thrombomodulin (TM) functions as a cofactor to
enhance the rate of protein C activation by thrombin ~1000-fold. The
molecular mechanism by which TM improves the catalytic efficiency of
thrombin toward protein C is not known. Molecular modeling of the
protein C activation based on the crystal structure of thrombin in
complex with the epidermal growth factor-like domains 4, 5, and 6 of TM (TM456) predicts that the binding of TM56 to exosite 1 of thrombin positions TM4 so that a negatively charged region on this domain juxtaposes a positively charged region of protein C. It has been hypothesized that electrostatic interactions between these oppositely charged residues of TM4 and protein C facilitate a proper docking of
the substrate into the catalytic pocket of thrombin. To test this
hypothesis, we have constructed several mutants of TM456 and protein C
in which charges of the putative interacting residues on both TM4
(Asp/Glu) and protein C (Lys/Arg) have been reversed. Results of
TM-dependent protein C activation studies by such a compensatory mutagenesis approach support the molecular model that TM4
interacts with the basic exosite of protein C.
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INTRODUCTION |
Thrombin is the final serine protease of the coagulation cascade
that activates fibrinogen to form blood clots (1-3). Thrombin can
up-regulate its own production by activating platelets (4), factor XI
(5), and factors V and VIII (1). It can also down-regulate its own
production by forming a complex with thrombomodulin
(TM)1 on the surface of
endothelial cells (6). Upon interaction with TM, thrombin can no longer
clot fibrinogen but, instead, rapidly activates protein C to activated
protein C (6, 7). Activated protein C is a vitamin
K-dependent plasma serine protease that can shut down the
coagulation cascade by degrading factors Va and VIIIa by limited
proteolysis (8, 9). Structural and functional data have indicated that
TM binds to exosite 1 of thrombin to change the specificity of thrombin
from a coagulant to an anticoagulant enzyme (10-15). The occupancy of
exosite 1 by fibrinogen, the thrombin receptor on platelets and
cofactors V and VIII is required for the recognition and efficient
activation of these molecules by thrombin in the clotting cascade (3,
16, 17). Thus, the binding of TM to exosite 1 competitively blocks the
interaction of thrombin with these procoagulant proteins. It has been
demonstrated that the binding of epidermal growth factor (EGF)-like
domains 5 and 6 of TM to exosite 1 is sufficient to block all coagulant properties of thrombin; however, for catalyzing the activation of
protein C, the fourth EGF domain of TM (TM456) is also required for its
cofactor function (6, 10).
Calcium plays a key role in protein C activation by the thrombin-TM
complex. Using Gla-domainless forms of protein C, it has been
demonstrated that the Ca2+-binding site critical for
activation of protein C by the thrombin-TM complex is located in the
70-80-loop of the substrate (18, 19) (chymotrypsinogen numbering
system (20)). This is the same site in trypsin that is also known to
bind Ca2+ (21). Occupancy of this site by Ca2+
is a prerequisite for the ability of TM to catalyze the rapid activation protein C by thrombin (18, 19). Interestingly, the binding
of Ca2+ to this site of protein C is inhibitory for
activation by thrombin in the absence of TM (18). It has been
hypothesized that the binding of Ca2+ to this site of
protein C is associated with a conformational change in the activation
peptide of the substrate and that the Ca2+-stabilized
conformer of the activation peptide is complementary for the active
site pocket of thrombin in the presence of TM but noncomplementary in
the absence of the cofactor (6).
The exact molecular mechanism by which TM456 improves the catalytic
efficiency of thrombin toward protein C in the presence of
Ca2+ is not known. Previously, it has been hypothesized
that TM may function by inducing allosteric changes in the structure of
residues in the extended binding pocket of thrombin (6). Thus, it is believed that the TM-altered conformer of thrombin optimally recognizes the Ca2+-altered conformer of protein C (6). Recently, the
x-ray crystal structure of thrombin in complex with TM456 was resolved
(15). Surprisingly, structural data did not indicate any allosteric changes in the active site pocket of thrombin upon binding to the TM
fragment (15). Molecular modeling of protein C activation, based on the
structure of the complex, suggested that binding of the TM5 domain of
the cofactor to exosite 1 of thrombin positions the TM4 domain so that
a negatively charged region on this domain juxtaposes a positively
charged exosite of protein C (15). Several electrostatic interactions
between these oppositely charged regions are postulated to facilitate a
proper docking of protein C into the catalytic pocket of thrombin.
Based on this model, it has been hypothesized that "substrate
presentation" is the primary mechanism by which TM may function (15).
A previous study proposed a similar function for TM in protein C
activation by thrombin (22).
To examine the validity of this molecular model by a mutagenesis
approach, we developed a bacterial periplasmic expression system for
TM456 and constructed several mutants of TM456 in which the negative
charges of five residues in TM4 (Glu346,
Asp349, Glu357, Glu382, and
Glu374) and two residues (Asp398 and
Asp400) in the interface between TM4 and TM5 were reversed
by substituting each residue with a Lys in separate constructs. In
addition, a double mutant of TM456 containing Glu346
Arg and Asp349
Lys substitutions was constructed and
expressed in the same system. Then the positive charges of basic
residues of the protein C exosite including Lys62,
Lys63, Arg74, Arg75, and
Lys78 were reversed by substituting these residues
individually or in a combination of two with Glu in a mammalian
expression/purification vector system. The protein C mutants were
prepared in Gla-domainless forms and expressed in HEK293 cells as
described (23). Kinetic characterization of these mutant molecules by
this compensatory mutagenesis approach supports the molecular model
that the TM4 domain of TM456 in the complex interacts with the basic
exosite of protein C. Moreover, analysis of results suggest that the
70-80-loop of protein C must be stabilized by the Ca2+ ion
before it can productively interact with TM4. This accounts for the
unique Ca2+ dependence of protein C activation by thrombin
in the presence of TM.
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EXPERIMENTAL PROCEDURES |
Mutagenesis and Expression--
The wild type and charge
reversal mutants of TM456 including Asp349 (D349K),
Glu346/Asp349 (E346R,D349K), Glu357
(E357K), Glu374 (E374K), Glu382 (E382K),
Arg385 (R385E), Asp398 (D398K), and
Asp400 (D400K) were prepared by PCR methods and expressed
in the periplasmic space of Escherichia coli using the
pIN-III-pelB expression/purification vector system as described
previously (24). Wild type and mutant protein C derivatives were
constructed in Gla-domainless forms (GD-PC) as described previously
(23). The charge reversal mutants of GD-PC including Lys62
(K62E), Lys63 (K63E), Lys62 and
Lys63 (K62E,K63E), Arg74 (R74E),
Arg75 (R75E), Lys78 (K78E), and
Arg74 and Arg75 (R74E,R75E) were prepared by
PCR methods and expressed in HEK293 cells using RSV-PL4
expression/purification system as described (23). Both mammalian and
bacterial expression vectors contain the sequence of a 12-residue
epitope for the Ca2+-dependent monoclonal
antibody, HPC4, for purification. Thus, both GD-PC and TM456
derivatives were purified by immunoaffinity chromatography using the
HPC4 antibody linked to Affi-gel 10 (Bio-Rad) as described (23). TM456
derivatives were further purified by anion exchange chromatography on a
Mono Q column developed with 30-ml linear gradient from 0.1 to 0.6 M NaCl and 0.02 M Tris-HCl, pH 7.5, as
described for the purification of the mammalian TM456 (23). Accuracies
of all mutant constructs were confirmed by DNA sequencing prior to
their expression.
Protein C Activation--
The initial rates of GD-PC activation
by thrombin were measured in 0.1 M NaCl, 0.02 M
Tris-HCl, pH 7.5 (TBS), containing 1 mg/ml bovine serum albumin (BSA),
0.1% polyethylene glycol (PEG) 8000, and 2.5 mM
Ca2+ in both the absence and the presence of TM456. In the
absence of TM456, the time course of activation of each GD-PC
derivative was studied by incubating 1 µM substrate with 50 nM thrombin at room temperature. At each time point (5-80
min), samples of reactions were transferred to TBS buffer containing
500 µg/ml human antithrombin to inhibit thrombin activity. At this
concentration of antithrombin, the activity of thrombin was rapidly
inhibited, whereas the amidolytic activity of active GD-PC remained
stable for more than 120 min. The amidolytic activities of active GD-PC
derivatives in the activation reactions were monitored by hydrolysis of
400 µM Spectrozyme PCa (American Diagnostica Inc.,
Greenwich, CT) in the TBS buffer containing 1 mg/ml BSA and 0.1% PEG
8000. The rate of hydrolysis was measured at 405 nm at room temperature
in a Vmax kinetic plate reader (Molecular Devices, Menlo Park, CA). The concentration of active protein C
derivatives in reaction mixtures was determined by reference to
standard curves that were prepared by total activation of GD-PC derivatives with excess thrombin at the time of experiments. This was
accomplished by total activation of 1 µM protein C with
10 nM thrombin in complex with 100 nM TM456 and
2.5 mM Ca2+ for 90 min at 37 °C. Under these
experimental conditions, the wild type zymogen was completely activated
in less than 30 min. The GD-PC mutants, which were resistant to
activation by the thrombin-TM456 complex in the presence of
Ca2+, were totally activated with thrombin alone in 1 mM EDTA as described (23). In this case, 1 µM
of each protein C derivative in TBS containing 1 mM EDTA
was activated with 50 nM thrombin for 4 h at 37 °C.
Time course analysis of the activation reactions indicated that all
GD-PC derivatives were totally activated under these conditions.
Characterization of the TM456 Derivatives--
The ability of
TM456 derivatives to bind thrombin
(Kd(app) values) as well as to function
as a cofactor to enhance the catalytic efficiency of thrombin in
activation of the GD-PC derivatives (kcat and
Km values) were evaluated. The Kd(app) values were determined from the
cofactor dependence of the initial rate of the activation of a
subsaturating concentration of GD-PC (1 µM) in the
presence of a fixed concentration of TM456 (~5 nM) and
increasing concentrations of thrombin (1-60 nM) as described (25). The Kd(app) values were
estimated from the saturable dependence of the GD-PC activation rates
on thrombin concentration using a hyperbolic equation as described
(25). The cofactor dependence of the initial rate of activation also allowed an accurate determination of the concentration of TM456 derivatives, since TM456 devoid from the chondroitin sulfate moiety binds to thrombin with a 1:1 stoichiometry (26). The time course of the
initial rate of protein C activation in the presence of TM456
derivatives was determined by incubating each GD-PC derivative (1 µM)
with thrombin (1-50 nM) in complex with saturating
concentrations of each TM456 derivative (>10 × Kd(app) values). Under these
experimental conditions, the concentrations of the thrombin-TM456 complexes were in proximity to the concentration of free thrombin. After inhibition of thrombin activity by antithrombin, the initial rate
of activation was measured from the rate of activated protein C
generation as described above. When a detailed kinetic analysis was
done to determine values for Km and
kcat of protein C activation, the initial rates
of activation were measured as a function of the GD-PC concentration
with 1 nM thrombin in complex with a saturating
concentration of TM456 as described above.
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RESULTS |
Expression and Purification of Recombinant Proteins--
Wild type
and mutant GD-PC derivatives were expressed in HEK293 cells and
purified to homogeneity on the HPC4 immunoaffinity column as described
(23). The SDS-PAGE analysis of zymogens indicated that protein C
derivatives were expressed as two closely migrating subforms that are
glycosylation variants observed previously with this protein (23, 27,
28) (data not shown). TM456 derivatives were expressed in the
periplasmic space of the XL1-B strain of E. coli using the
pIN-III-pelB expression/purification vector system as described (24).
The relative positions of the mutant residues in the fourth epidermal
growth factor domain of TM are schematically shown in Fig.
1A. TM456 expressed in this
system contains a 12-residue epitope for the
Ca2+-dependent monoclonal antibody HPC4 at its
N-terminal domain for an easy purification. The periplasmic extract was
prepared by a hypotonic shock and passed through the HPC4 column in the
presence of Ca2+ as described (24). The column was
extensively washed with TBS containing 1 M NaCl and 2.5 mM Ca2+ followed by elution with TBS containing
5 mM EDTA as described (24). The HPC4 eluates were further
purified by anion exchange chromatography on a Mono Q column as
described (23). SDS-PAGE analysis under nonreducing conditions
suggested that all TM456 derivatives had been purified to homogeneity,
migrating as single bands with expected apparent molecular masses of
~15 kDa (Fig. 1B). Wild type forms of both mammalian and
periplasmic TM456 exhibited comparable cofactor activities (within
80%) as determined by protein C activation assays described below.

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Fig. 1.
Schematic representation of the fourth
EGF-like domain of TM and the SDS-PAGE analysis of the charge reversal
mutant of this domain in the TM456 constructs expressed in the
periplasmic space of bacteria. The mutant residues in TM4 and in
the N-terminal end of TM5 are shown in gray
circles in the top panel. The
disulfide bonds are shown by solid lines. The
last disulfide bond (between cysteine residues 390 and 395) belongs to
TM5. The SDS-PAGE analysis of the TM456 mutants under nonreducing
conditions is shown in the bottom panel.
Lane 1, wild type TM456; lane
2, D349K; lane 3, E346R,D349K;
lane 4, E357K; lane 5,
E374K; lane 6, E382K; lane
7, R382E; lane 8, D398K;
lane 9, D400K; lane 10,
molecular mass standards in kilodaltons.
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Initial Rate of GD-PC Activation--
The initial rates of
thrombin-catalyzed wild type and mutant GD-PC activation were studied
in both the absence and presence of TM456. GD-PC is known to contain a
single Ca2+-binding site with an affinity of ~50
µM, which is located in the protease domain of protein C
in a loop consisting of residues Glu70-Glu80
(19, 23). It is known that the binding of Ca2+ to this loop
of protein C is inhibitory for activation by thrombin in the absence of
TM (6, 19). However, the metal ion is an obligatory cofactor for
activation by thrombin in the presence of the cofactor (6, 19). Since
several residues of the 70-80-loop were targeted for the mutagenesis
study, it was essential to evaluate the extent to which the interaction
of these mutants with Ca2+ has been altered. Thus, the
initial rates of GD-PC activation by thrombin were studied as a
function of varying Ca2+ concentrations. To simplify the
comparisons of the Ca2+ dependence of activations, the data
were normalized to maximal inhibition values as described (23). As
shown in Fig. 2, the Ca2+ ion
inhibited the activation of all GD-PC derivatives with the exceptions
of R75E and R74E,R75E with a similar half-maximal value of
30-40 µM (see legend of Fig. 2). However, these values
were elevated ~2-3-fold for the R75E and R74E,R75E mutants. Since
the half-maximal values for the activation of the R74E mutant were not
affected, the altered effect of Ca2+ in the thrombin
activation of the double mutant R74E,R75E is also probably due to the
R75E mutation.

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Fig. 2.
The inhibitory effect of Ca2+ on
the activation of GD-PC derivatives by thrombin. The initial rate
of protein C (1 µM) activation by thrombin (10 nM) was measured in the presence of increasing
concentrations of Ca2+ (0-2.5 mM) at room
temperature in TBS containing 1 mg/ml BSA and 0.1% PEG 8000. The
activation reactions were stopped by adding 500 µg/ml antithrombin,
and the rate of activation was measured from the concentration of
activated protein C generated by an amidolytic activity assay using
Spectrozyme PCa as described under "Experimental Procedures." Data
were normalized to maximal inhibition (100% at 2.5 mM
Ca2+) and plotted as a function of different concentrations
of Ca2+. Kd(app) values were
determined from the hyperbolic binding equation. The symbols
and half-maximal values for inhibition are as follows: wild type GD-PC
( , 36 µM), K62E ( , 32 µM), K63E ( ,
31 µM), K62E,K63E ( , 37 µM), R74E ( ,
27 µM), R75E ( , 71 µM), K78E ( , 40 µM), and R74E,R75E ( , 110 µM).
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Next, the time course of the initial rates of GD-PC activation by
thrombin alone or by the thrombin-TM456 complex was studied at
saturating concentration of Ca2+ (2.5 mM). As
shown in Fig. 3A, thrombin
activated all GD-PC derivatives with comparable or improved initial
rates. The greatest improvement (~6-7-fold) was observed for the
thrombin activation of the R74E,R75E double mutant. A detailed kinetic
analysis was not possible, since protein C exhibits a
Km of greater than 60 µM for thrombin in the presence of Ca2+ (29). In contrast to activation by
thrombin alone, the TM-dependent activations of the GD-PC
derivatives were all impaired at varying degrees. Under conditions
described in the legend to Fig. 3, no TM456-dependent
activation by thrombin was detected for the R74E and R75E mutants of
GD-PC, and the rates were impaired ~2-fold for K63E and K78E and
~5-fold for the K62E and K62E,K63E mutants (Fig. 3B).
These results suggested that the basic residues of both 60- and
70-80-loops are critical for the thrombin activation of protein C in
the presence of TM. It should be noted that in addition to these
residues, it is known that basic residues of the 39-loop
(Lys37-Lys39) are also required for the
TM-dependent protein C activation by thrombin (30).
Consistent with these results, we found that the activation of a triple
GD-PC K37E,K38E,K39E mutant by thrombin alone was normal in both EDTA
and Ca2+, but it was severely impaired in the presence of
TM456 (data not shown).

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Fig. 3.
Initial rates of GD-PC activation by thrombin
in the absence and presence of TM456. A, the time
course of the initial rate of GD-PC (1 µM) activation by
thrombin (50 nM) was measured in 96-well plates at room
temperature in TBS containing 1 mg/ml BSA, 0.1% PEG 8000, and 2.5 mM Ca2+. At the indicated time points, aliquots
of the activation reactions were stopped by adding 500 µg/ml
antithrombin, and the rate of activation was measured from the
concentration of activated protein C generated as described under
"Experimental Procedures." , GD-PC; , K62E; , K63E; ,
K62E,K63E; , R74E; , R75E; , K78E; , R74E,R75E.
B, the same as above, except that the time course of
activation of GD-PC derivatives was measured by 1 nM
thrombin in complex with 250 nM TM456. , GD-PC; ,
K62E; , K63E; , K62E,K63E; , K78E. The solid
lines are drawn according to a linear equation.
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Initial Rates of Protein C Activation by Thrombin in Complex with
TM456 Derivatives--
The initial rates of wild type and mutant GD-PC
activation by thrombin in complex with different TM456 derivatives were
measured as described above. As shown in Fig.
4, relative to wild type TM456, the
cofactor activities of all TM456 mutants in catalyzing the thrombin
activation of wild type GD-PC were impaired. The impairments ranged
from a maximum of 90-95% for the E357K, D398K, and D400K mutants to a
minimum of 50-60% for the E374K and R385E mutants of TM456. These
results appear to indicate that charged residues under study in TM4
play a role in the cofactor function of TM in enhancing the thrombin
activation of protein C. This is consistent with the literature (25,
31).

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Fig. 4.
Comparison of the cofactor effect of TM456
mutants. The initial rate of GD-PC (1 µM) activation
by thrombin (1-10 nM) in complex with a saturating
concentration of each TM456 derivative (500 nM) was
measured at room temperature in TBS containing 1 mg/ml BSA, 0.1% PEG
8000, and 2.5 mM Ca2+. Following inhibition of
thrombin activity by antithrombin, the rate of activated protein C
generation was measured by an amidolytic activity assay as described
under "Experimental Procedures."
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The rationale for the preparation of the R385E mutant of TM456 was that
in the previous molecular model of protein C activation it was proposed
that the interaction of Arg385 of TM4 with a basic residue
of the 70-80-loop of protein C (particularly Glu80) might
be responsible for the unique Ca2+ dependence of activation
(15). Thus, the initial rate of wild type GD-PC activation by thrombin
in complex with saturating concentrations of TM456 derivatives were
studied as a function of varying concentrations of Ca2+.
The metal ion enhanced the rate of GD-PC activation by thrombin in
complex with wild type TM456 by an order of magnitude with Kd(app) of ~20 µM
at room temperature (data not shown). Interestingly, however, no
Ca2+ dependence of GD-PC activation was observed by
thrombin in complex with D349K, E357K, D398K, and D400K mutants of
TM456. Although a similar Kd(app) of
~20 µM for GD-PC activation by thrombin in complex with
other mutants of TM456 was observed, the extent of the cofactor effect
of Ca2+ was, nonetheless, reduced to ~2-fold with these
mutants. Similarly, no Ca2+-dependence of activation was
observed for the thrombin activation of the R74E and R75E mutants of
GD-PC in the presence of wild type TM456. Since the interaction of
thrombin with TM456 is not known to be
Ca2+-dependent (6, 19), it follows that the
cofactor defect of TM456 derivatives must primarily be caused by their
inability to interact with the Ca2+-stabilized conformer of
GD-PC in the complex.
Next, the cofactor functions of the TM456 mutants in enhancing the
thrombin activation of the compensatory GD-PC mutants were evaluated.
Similar to wild type TM456, none of the mutants exhibited any cofactor
activity toward thrombin activation of the 70-80-loop mutants of GD-PC
(data not shown). However, the cofactor functions of both the D349K and
E346R,D349K mutants in enhancing the activation of the compensatory
60-loop mutants of GD-PC were improved (Fig. 5, shown for the double mutant only).
Relative to activation of wild type GD-PC, the activation of K62E,
K63E, and K62E,K63E by the thrombin-TM456 complex were impaired
2-5-fold (Fig. 5). Interestingly, however, the defect in the
activation of these mutants by thrombin was restored if the E346R,D349K
mutant of TM456 was used as the cofactor in the activation reaction. As
shown in Fig. 5, the initial rate of activation of K62E,K63E by
thrombin in complex with the double mutant of TM456 was essentially a
mirror image of the GD-PC activation rate by thrombin in complex with
the wild type TM456. Thus, 100% of the cofactor activity of the
Glu346 and Asp349 charge reversal mutants of TM
could be restored if compensatory mutations were made in residues of
the 60-loop of protein C. These results clearly suggest that TM4
provides a site for interaction with protein C in the activation
complex.

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Fig. 5.
Restoration of the defective cofactor effect
of the TM456 mutant by a compensatory GD-PC mutant. The initial
rate of GD-PC (1 µM) activation by thrombin (1-10
nM) in complex with a saturating concentration of each
TM456 derivative (500 nM) was measured at room temperature
in TBS containing 1 mg/ml BSA, 0.1% PEG 8000, and 2.5 mM
Ca2+. Following inhibition of the thrombin activity by
antithrombin, the rate of activated protein C generation was measured
by an amidolytic activity assay as described under "Experimental
Procedures." The solid black bars
are derived from activation studies in the presence TM456, and
blank bars are derived from the same data in the
presence of TM456 E346R,D349K.
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Evaluation of Kd(app) Values--
The cofactor
dependence of the initial rate of protein C activation was also used to
evaluate the affinities of the TM456 derivatives for interaction with
thrombin in the ternary complex. With the wild type GD-PC as a
substrate, a Kd(app) value of 5.7 nM for the interaction of mammalian TM456 with thrombin was
observed, which is consistent with previously reported values in the
literature (31). The bacterial TM456 exhibited a similar Kd(app) of 4.2 nM,
suggesting that the periplasmic protein has been properly folded (Table
I). The
Kd(app) values for the TM456 mutants
ranged from 5 to 15 nM (Table I). The TM456 E346R,D349K
mutant exhibited ~2.5-fold impairment in binding to the activation
complex. Interestingly, the Kd(app)
values for the E346R,D349K mutant resembled the value for the wild type TM456 if the K62E,K63E mutant of GD-PC was used as a substrate in the
activation reaction. Thus, in contrast to a
Kd(app) value of 10.1 nM for
the thrombin-TM456 E346R,D349K interaction as shown in Table I, this
value was decreased to 4.5 nM if the K62E,K63E mutant of
GD-PC was used as the substrate in the reaction. These results further
support the conclusion that the TM4 domain of the cofactor in the
activation complex provides a binding site for the 60-loop of protein C
in the ternary complex. The results further suggest that the
interaction of protein C with TM4 in the ternary complex contributes to
Kd(app) of the thrombin-TM456 interaction. The Kd(app) values with the
two mutants D398K and D400K, which are located outside TM4 in the
2-
3 loop of TM5 (15) were elevated more than all other mutants.
To determine whether the loss of cofactor function with these mutants
is due to their impaired binding to thrombin or a specific effect
related to the protein C activation complex, the cofactor dependence of the amidolytic activity of thrombin toward Spectrozyme TH was studied
at saturating concentrations of TM456 (500 nM) for all mutants. It is known that the occupancy of exosite 1 by hirugen, thrombin receptor on platelets, and TM456 is associated with
~60% enhancement in the amidolytic activity of thrombin toward this substrate (32). As shown in Table I, the cofactor functions of all
mutants were normal in this assay, since they enhanced the catalytic
efficiency of thrombin toward the chromogenic substrate to a similar
extent (70-90%). This is in contrast to protein C activation, in
which the cofactor functions of these mutants were dramatically
impaired under similar assay conditions (500 nM TM456). Thus, the loss of cofactor function of these mutants is not likely to
be due to their impaired interaction with thrombin, but rather their
impaired interaction with protein C in the activation complex. These
results are also consistent with the essential role of these residues
in the cofactor function of TM as previously shown by others (25,
31).
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Table I
Apparent Kd and kinetic constants for thrombin activation of
wild type GD-PC in the presence of TM456 derivatives
The Kd(app) values were determined from the
cofactor-dependence of the initial rate of activation of 1 µM GD-PC in the presence of a fixed concentration of
TM456 ( 5 nM) and increasing concentrations of thrombin
(1-60 nM) as described under "Experimental
Procedures." Km(app) and
Vmax values were determined from the concentration
dependence of the initial rate of wild type (WT) GD-PC activation by
thrombin (1 nM) in complex with a saturating concentration
of each TM456 derivative (500 nM) according to the
Michaelis-Menten equation. The percentage increase in SpTH hydrolysis
by thrombin (2 nM) in complex with each TM456 derivative
(500 nM) was determined in TBS containing 0.1 mg/ml BSA,
0.1% PEG 8000, and 2.5 mM Ca2+. ND, not
determined.
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Km and kcat Analysis--
To study the
effect of cofactor mutagenesis on the kinetics of GD-PC activation, the
initial rate of protein C activation by thrombin in complex with a
saturating concentration of each TM456 derivative (500 nM)
was determined as a function of increasing concentrations of wild type
GD-PC. Analysis of data by the Michaelis-Menten equation suggested that
all TM456 derivatives enhanced the rate of GD-PC activation with
similar kcat values (Table I). However, the
Km(app) values with all mutants were
impaired. These results appear to suggest that the primary defect in
the protein C activation reactions with these mutants is due to
impaired Km(app) values. Interestingly,
the impairment in the Km(app) of
GD-PC activation by thrombin in complex with the E346R,D349K mutant of
TM456 was totally restored if the compensatory K62E,K63E mutant of
GD-PC was utilized as the substrate in the activation reaction (Fig.
6). Thus, in contrast to
Km(app) and kcat values of 43.1 ± 3.0 µM and 15.1 ± 0.8 mol/min/mol for the GD-PC activation by the thrombin-TM456 E346R,D349D
complex, the corresponding values were 8.0 ± 0.3 µM
and 15.7 ± 0.3 mol/min/mol if the K62E,K63E mutant of GD-PC was
used as the substrate in the activation reaction (Fig. 6). These
results suggest that the interaction of basic residues of protein C
with acidic residues of TM4 primarily decreases the
Km of the substrate activation by thrombin in the presence of Ca2+. Noting that the Km
constant in an enzymatic reaction partly represents the affinity of the
enzyme-cofactor complex for binding the substrate, these results
further suggest that the two acidic residues of TM4 are part of a
sequence of TM4 that interact with the basic residues of the 60-loop in
protein C. It should be noted that the highest available concentration
of GD-PC in these reactions was only 20 µM; thus, no
complete saturation could be observed for all reactions. Hence, kinetic
constants for such reactions are estimates that were derived from the
best fit of data to the Michaelis-Menten equation.

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|
Fig. 6.
Kinetic analysis of GD-PC activation by
thrombin in complex with TM456. A, the initial rate of
wild type (WT) GD-PC ( ) or GD-PC K62E,K63E ( )
activation were determined as a function of GD-PC concentration by 1 nM thrombin in complex with 500 nM wild type
TM456 in TBS containing 1 mg/ml BSA, 0.1% PEG 8000 and 2.5 mM Ca2+ as described under "Experimental
Procedures." B, the same as A, except that 500 nM of TM456 E346R,D349K mutant replaced the wild type TM456
in the activation reaction. The kinetic constants,
Km and kcat were determined
from nonlinear regression fits to the Michaelis-Menten equation
(solid lines).
|
|
 |
DISCUSSION |
Previous molecular modeling of protein C activation, based on the
structure of the thrombin-TM456 complex, predicted that the binding of
TM56 domains of the cofactor to exosite 1 of thrombin may orient TM4 so
that a negatively charged region in this domain could contact a basic
exosite of protein C in the ternary activation complex (15). Based on
this model, it was postulated that electrostatic interactions between
the oppositely charged residues of these two regions could facilitate a
proper docking of protein C into the catalytic pocket of thrombin (15).
In this study, we tested the validity of this molecular model by a
compensatory "gain of function" mutagenesis approach. Thus, we
reversed the charges of the acidic residues of TM4 in TM456 constructs
and expressed the mutant cofactor fragments in the periplasmic space of
bacteria. We then prepared compensatory mutants of protein C in
Gla-domainless forms by reversing the charges of certain basic residues
of the putative exosite that is believed to interact with TM4. Based on
results of previous TM456 (25, 31) and protein C (33, 34) mutants, we
reasoned that such mutants would exhibit impaired cofactor or substrate
activities if they were individually evaluated in appropriate
functional assays. However, if the molecular model is correct, it was
expected that the defective cofactor effect of certain TM456 mutants in
catalyzing the thrombin activation of certain GD-PC mutants would
fully, or at least partially, be restored. Indeed, the results
suggested that the basic residues of the 60-loop of protein C interact
with Glu346 and Asp349 of TM4 in the ternary
activation complex. This was evidenced by the restoration of full
cofactor activity of the double mutant of TM456 in catalyzing the
thrombin activation of the K62E,K63E mutant of GD-PC.
In addition to basic residues of the 60-loop, the molecular model of
the protein C activation predicts that the basic residues of the
Ca2+-binding 70-80-loop (particularly Arg74
and Arg75) also interact with the acidic TM4 domain of the
cofactor. The results of this and other previous mutagenesis studies
(33, 34) clearly suggest that these basic residues are essential for
the TM-dependent activation of protein C by thrombin.
However, by the compensatory mutagenesis approach of this study, we
could not provide any information as to possible sites of the
interaction of the 70-80-loop residues of protein C with TM4. The
reason for the lack of success in mapping these interaction sites by
this approach is not known. One possibility is that the guanidinium groups of Arg74 and Arg75 are involved in
bifurcated salt bridges with two acidic residues in TM4, and thus the
choice of Lys in the compensatory TM456 mutants may have flawed the
strategy. The second possibility is that, with the exception of
Glu346 and Asp349, other residues of TM4
mutated in this study are located on
-sheet structures and that the
mutagenesis of these residues may cause subtle conformational changes
in TM4, thereby hindering the proper alignment of these residues with
basic residues of the Ca2+-loop. Such adverse
conformational effects in Glu346 and Asp349
residues of TM4 would be minimal, since these residues are more flexible and lie in the C-terminal loop and are also solvent-accessible (14). Finally, the other possibility is that Ca2+ may have
a complex electrostatic effect on the conformations of charged residues
of the 70-80-loop in the protein C mutants. For instance, in addition
to three basic residues, the 70-80-loop of protein C also has four
acidic residues, two of which (Glu70 and Glu80)
are involved in ligating the Ca2+ ion. It is likely that,
in the absence of Ca2+, the acidic and basic residues of
this loop are in random and disordered conformations and that the
binding of the metal ion to the 70-80-loop leads to the
internalization of acidic residues to ligate Ca2+ and
exposure of basic residues to facilitate their interaction with TM4 in
the thrombin-TM complex. Thus, reversing the charges of
Arg74 and Arg75 could interfere with such
electrostatic steering of this loop by the Ca2+ ion. In
agreement with this hypothesis, previous spectroscopic studies have
indicated that binding of Ca2+ to the 70-80-loop of GD-PC
is associated with a conformational change in the loop that is detected
by quenching of the intrinsic fluorescence of two Trp76 and
Trp79 residues in this loop (residues 231 and 234 in the
protein C numbering) (35).
Ca2+ plays an intriguing role in protein C activation by
thrombin. Whereas the metal ion is required for protein C activation by
thrombin in the presence of TM, it inhibits the activation by thrombin
alone (6). Based on the molecular model of protein C activation by the
thrombin-TM456 complex, it has been proposed that the unique
Ca2+ dependence of protein C activation by thrombin may be
the result of direct contacts between TM4 side chains and protein C
residues that coordinate Ca2+ (particularly between
Arg385 of TM4 and Glu80 of protein C) (15). The
observation that the Ca2+ dependence of protein C
activation by thrombin in complex with several mutants of TM456 (D349K,
E357K, D398K, and D400K) was completely eliminated supports this
hypothesis. However, the results do not agree with the proposal that
interactions between Arg385 of TM and Glu80 of
protein C account for this phenomenon, since protein C activation with
the R385E mutant of TM456 remained
Ca2+-dependent. Moreover, we previously
demonstrated that the substitution of Glu80 of protein C
with Lys results in a protein C mutant whose activation by the
thrombin-TM456 complex is independent of Ca2+ (19). Thus,
the unique Ca2+ dependence of protein C activation is
mediated by direct contacts between acidic residues of TM4 and basic
residues of protein C (particularly Arg74 and
Arg75). This is supported by the observation that the
activation of both R74E and R75E mutants of GD-PC was severely impaired
and that Ca2+ did not stimulate the reaction.
It is known that TM promotes the catalytic efficiency of thrombin
toward activation of protein C in the presence of Ca2+ by 3 orders of magnitude (6). Such an improvement in the rate of protein C
activation by thrombin in the presence of TM is accounted for both by a
1-order of magnitude decrease in Km and a 2-order of
magnitude increase in Vmax. The mechanism by
which TM improves both kinetic constants of protein C activation by thrombin in the presence of Ca2+ is not known. Results of
this mutagenesis study suggest that the phospholipid-independent
improvement in the Km of activation is primarily
mediated by an interaction between the basic exosite of protein C and
acidic residues of TM4. This is derived from the observation that the
Km of activation was impaired with all mutants of
TM456. Based on results presented, we believe that the Ca2+
stabilization of the 70-80-loop of protein C culminates in the exposure of basic residues required for interaction with TM4 and that
such an interaction leads to a 1-order of magnitude improvement in
Km. However, the result of this study does not
provide insight into how TM improves the kcat of
protein C activation by thrombin. The observation that the
kcat values for the GD-PC activation by thrombin
in complex with all mutants of TM456 were comparable, with only
Km values being impaired suggests that the
interaction of basic residues of protein C exosite with the acidic
residues of TM4 may not contribute to the kcat
of the activation reaction. Thus, the open question is how does TM
improve the kcat of protein C activation by
thrombin? Previously, it was hypothesized that TM456 may induce
conformational changes in the active site pocket of thrombin that leads
to preferential improvement in the catalytic efficiency of thrombin
toward protein C activation (6). Although numerous kinetics (36),
mutagenesis (37), and spectroscopic (38) studies have supported this
hypothesis, no significant structural rearrangement in the active site
pocket of thrombin has been observed in the x-ray crystal structure of the thrombin-TM456 complex (15). Molecular modeling based on structural
data has indicated that the interaction of protein C with TM4, and thus
"substrate presentation" by TM456 may be the primary mechanism by
which the cofactor functions (15). However, the results presented in
this study suggest that the energy of such an interaction could only be
utilized to improve the Km but not the
kcat of the reaction. It should also be noted
that our approach is suitable for only probing the ionic electrostatic
interaction between protein C and TM4 but not other possible
noncovalent interactions between the two proteins. In the case of
thrombin, hydrophobic interactions between exosite 1 and TM5 make a
significant contribution to the energy of binding (15). Thus, it is
possible that similar hydrophobic interactions exist between protein C
and TM4 that is associated with an improvement in the
kcat of protein C activation by the thrombin-TM
complex. It is also possible that the lack of a structural change in
the active site pocket of thrombin stems from using an active site inhibited thrombin for preparing crystals for the complex. In any case,
further studies are needed to understand exactly how TM improves the
kcat of protein C activation by thrombin in the presence of Ca2+.
In summary, our results clearly show that TM4 in the thrombin-TM456
complex provides a binding site for interaction with the basic exosite
of protein C. The interaction of the basic exosite of protein C with
acidic residues of TM4 improves the Km of activation
in the presence of Ca2+, and this interaction also accounts
for the unique Ca2+ dependence of protein C activation by
the thrombin-TM complex.
 |
ACKNOWLEDGEMENT |
We thank Audrey Rezaie for proofreading of the manuscript.
 |
FOOTNOTES |
*
This work was supported by NHLBI, National Institutes of
Health, Grants HL 62565 and HL 68571 (to A. R. R.).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: Dept. of Biochemistry
and Molecular Biology, St. Louis University School of Medicine, 1402 S. Grand Blvd., St. Louis, MO 63104. Tel.: 314-577-8130; Fax:
314-577-8156; E-mail: rezaiear@slu.edu.
Published, JBC Papers in Press, January 14, 2003, DOI 10.1074/jbc.M211797200
 |
ABBREVIATIONS |
The abbreviations used are:
TM, thrombomodulin;
EGF, epidermal growth factor;
TM456, TM fragment containing the
EGF-like domains 4, 5, and 6;
GD-PC, Gla-domainless protein C
from which the N-terminal amino-terminal residues 1-45 have been
removed by the recombinant DNA methods;
PEG, polyethylene glycol;
BSA, bovine serum albumin.
 |
REFERENCES |
1.
|
Mann, K. G.,
Jenny, R. J.,
and Krishnaswamy, S.
(1988)
Annu. Rev. Biochem.
57,
915-956[CrossRef][Medline]
[Order article via Infotrieve]
|
2.
|
Furie, B.,
and Furie, B. C.
(1988)
Cell
53,
505-518[Medline]
[Order article via Infotrieve]
|
3.
|
Fenton, J. W., II
(1995)
Thromb. Haemostasis
74,
493-498[Medline]
[Order article via Infotrieve]
|
4.
|
Bevers, E. M.,
Comfurius, P.,
and Zwaal, R. F. A.
(1991)
Blood Rev.
5,
146-154[Medline]
[Order article via Infotrieve]
|
5.
|
Gailani, D.,
and Broze, G. J., Jr.
(1991)
Science
253,
909-912[Medline]
[Order article via Infotrieve]
|
6.
|
Esmon, C. T.
(1993)
Thromb. Haemostasis
70,
1-5[Medline]
[Order article via Infotrieve]
|
7.
|
Dang, Q. D.,
Vindigni, A.,
and Di Cera, E.
(1995)
Proc. Natl. Acad. Sci. U. S. A.
92,
5977-5981[Abstract/Free Full Text]
|
8.
|
Walker, F. J.,
Sexton, P. W.,
and Esmon, C. T.
(1979)
Biochim. Biophys. Acta
571,
333-342[Medline]
[Order article via Infotrieve]
|
9.
|
Fay, P. J.,
Smudzin, T. M.,
and Walker, F. J.
(1991)
J. Biol. Chem.
266,
20139-20145[Abstract/Free Full Text]
|
10.
|
Ye, J.,
Liu, L.,
Esmon, C. T.,
and Johnson, A. E.
(1992)
J. Biol. Chem.
267,
11023-11028[Abstract/Free Full Text]
|
11.
|
Tsiang, M.,
Lentz, S.,
and Sadler, J. E.
(1992)
J. Biol. Chem.
267,
6164-6170[Abstract/Free Full Text]
|
12.
|
Suzuki, K.,
Hayashi, T.,
Nishioka, J.,
Kosaka, Y.,
Zushi, M.,
Honda, G.,
and Yamamoto, S.
(1989)
J. Biol. Chem.
264,
4872-4876[Abstract/Free Full Text]
|
13.
|
Mathews, I. I.,
Padmanabhan, K. P.,
and Tulinsky, A.
(1994)
Biochemistry
33,
13547-13552[Medline]
[Order article via Infotrieve]
|
14.
|
Meininger, D. P.,
Hunter, M. J.,
and Komives, E. A.
(1995)
Protein Sci.
4,
1683-1695[Abstract/Free Full Text]
|
15.
|
Fuentes-Prior, P.,
Iwanaga, Y.,
Huber, R.,
Pagila, R.,
Rumennik, G.,
Seto, M.,
Morser, J.,
Light, D. R.,
and Bode, W.
(2000)
Nature
404,
518-525[CrossRef][Medline]
[Order article via Infotrieve]
|
16.
|
Vu, T. H.,
Hung, D. T.,
Wheaton, V. I.,
and Coughlin, S. R.
(1991)
Cell
64,
1057-1068[Medline]
[Order article via Infotrieve]
|
17.
|
Esmon, C. T.,
and Lollar, P.
(1996)
J. Biol. Chem.
271,
13882-13887[Abstract/Free Full Text]
|
18.
|
Rezaie, A. R.,
Esmon, N. L.,
and Esmon, C. T.
(1992)
J. Biol. Chem.
267,
11701-11704[Abstract/Free Full Text]
|
19.
|
Rezaie, A. R.,
Mather, T.,
Sussman, F.,
and Esmon, C. T.
(1994)
J. Biol. Chem.
269,
3151-3154[Abstract/Free Full Text]
|
20.
|
Bode, W.,
Mayr, I.,
Baumann, U.,
Huber, R.,
Stone, S. R.,
and Hofsteenge, J.
(1989)
EMBO J.
8,
3467-3475[Abstract]
|
21.
|
Bode, W.,
and Schwager, P.
(1975)
J. Mol. Biol.
98,
693-717[Medline]
[Order article via Infotrieve]
|
22.
|
Vindigni, A.,
White, C. E.,
Komives, E. A.,
and Di Cera, E.
(1997)
Biochemistry
36,
6674-6681[CrossRef][Medline]
[Order article via Infotrieve]
|
23.
|
Rezaie, A. R.,
and Esmon, C. T.
(1992)
J. Biol. Chem.
267,
26104-26109[Abstract/Free Full Text]
|
24.
|
Rezaie, A. R.,
Fiore, M. M.,
Neuenschwander, P. F.,
Esmon, C. T.,
and Morrissey, J. H.
(1992)
Protein Expression Purif.
3,
453-460[Medline]
[Order article via Infotrieve]
|
25.
|
Nagashima, M.,
Lundh, E.,
Leonard, J. C.,
Morser, J.,
and Parkinson, J. F.
(1993)
J. Biol. Chem.
268,
2888-2892[Abstract/Free Full Text]
|
26.
|
Ye, J.,
Esmon, C. T.,
and Johnson, A. E.
(1993)
J. Biol. Chem.
268,
2373-2379[Abstract/Free Full Text]
|
27.
|
Miletich, J. P.,
and Broze, G. J., Jr.
(1990)
J. Biol. Chem.
265,
11397-11404[Abstract/Free Full Text]
|
28.
|
Grinnell, B. W.,
Walls, J. D.,
and Gerlitz, B.
(1991)
J. Biol. Chem.
226,
9778-9785
|
29.
|
Esmon, N. L.,
DeBault, L. E.,
and Esmon, C. T.
(1983)
J. Biol. Chem.
258,
5548-5553[Free Full Text]
|
30.
|
Gerlitz, B.,
and Grinnell, B. W.
(1996)
J. Biol. Chem.
271,
22285-22288[Abstract/Free Full Text]
|
31.
|
Lentz, S. R.,
Chen, Y.,
and Sadler, J. E.
(1993)
J. Biol. Chem.
268,
15312-15317[Abstract/Free Full Text]
|
32.
|
Liu, L.,
Vu, T. H.,
Esmon, C. T.,
and Coughlin, S. R.
(1991)
J. Biol. Chem.
266,
16977-16980[Abstract/Free Full Text]
|
33.
|
Vincenot, A.,
Gaussem, P.,
Pittet, J. L.,
Debost, S.,
and Aiach, M.
(1995)
FEBS Lett.
367,
153-157[CrossRef][Medline]
[Order article via Infotrieve]
|
34.
|
Yang, L.,
Manithody, C.,
and Rezaie, A. R.
(2002)
Biochemistry
41,
6149-6157[CrossRef][Medline]
[Order article via Infotrieve]
|
35.
|
Rezaie, A. R.,
and Esmon, C. T.
(1995)
Biochemistry
34,
12221-12226[Medline]
[Order article via Infotrieve]
|
36.
|
Rezaie, A. R.,
He, X.,
and Esmon, C. T.
(1998)
Biochemistry
37,
693-699[CrossRef][Medline]
[Order article via Infotrieve]
|
37.
|
Le Bonniec, B. F.,
and Esmon, C. T.
(1991)
Proc. Natl. Acad. Sci. U. S. A.
88,
7371-7375[Abstract]
|
38.
|
Ye, J.,
Esmon, N. L.,
Esmon, C. T.,
and Johnson, A. E.
(1991)
J. Biol. Chem.
266,
23016-23021[Abstract/Free Full Text]
|
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