From the Department of Medicine, Saint Louis
University School of Medicine, St. Louis, Missouri 63104, the
¶ Department of Biochemistry, Michigan State University, East
Lansing, Michigan 48824, and the
Department of Biochemistry and
Biophysics, University of Rochester School of Medicine,
Rochester, New York 14642
Received for publication, December 26, 2000
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ABSTRACT |
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The physiologic activator of factor X consists of
a complex of factor IXa, factor VIIIa, Ca2+ and a
suitable phospholipid surface. In one study, helix 330 (162 in
chymotrypsin) of the protease domain of factor IXa was implicated in
binding to factor VIIIa. In another study, residues 558-565 of the A2
subunit of factor VIIIa were implicated in binding to factor IXa. We
now provide data, which indicate that the helix 330 of factor IXa
interacts with the 558-565 region of the A2 subunit. Thus, the ability
of the isolated A2 subunit was severely impaired in potentiating factor
X activation by IXaR333Q and by a helix replacement mutant
(IXahelixVII in which helix 330-338 is replaced by that of
factor VII) but it was normal for an epidermal growth factor 1 replacement mutant (IXaPCEGF1 in which epidermal growth
factor 1 domain is replaced by that of protein C). Further, affinity of
each 5-dimethylaminonaphthalene-1-sulfonyl (dansyl)-Glu-Gly-Arg-IXa (dEGR-IXa) with the A2 subunit was determined from its ability to
inhibit wild-type IXa in the tenase assay and from the changes in
dansyl fluorescence emission signal upon its binding to the A2 subunit.
Apparent Kd(A2) values are:
dEGR-IXaWT or dEGR-IXaPCEGF1 ~100
nM, dEGR-IXaR333Q ~1.8 µM, and
dEGR-IXahelixVII >10 µM. In additional
experiments, we measured the affinities of these factor IXa molecules
for a peptide comprising residues 558-565 of the A2 subunit. Apparent
Kd(peptide) values are: dEGR-IXaWT
or dEGR-IXaPCEGF1 ~4 µM, and
dEGR-IXaR333Q ~62 µM. Thus as compared with
the wild-type or PCEGF1 mutant, the affinity of the R333Q mutant for
the A2 subunit or the A2 558-565 peptide is similarly reduced. These
data support a conclusion that the helix 330 of factor IXa interacts
with the A2 558-565 sequence. This information was used to model the
interface between the IXa protease domain and the A2 subunit, which is
also provided herein.
Physiologic blood clotting begins by exposure of blood to tissue
factor (TF)1 at an injury
site and formation of the complex between TF and plasma factor VIIa.
The TF·VIIa complex formed activates both factors IX and X (1, 2).
Factor IXa thus generated forms a stoichiometric complex with factor
VIIIa and also activates factor X in the presence of Ca2+
and a suitable phospholipid (PL) surface (1, 2). The role of factor
VIIIa in this complex is to increase the kcat by
several orders of magnitude while PL primarily reduces the
Km for the substrate factor X.
Human factor IX circulates in blood as a single chain protein of 415 amino acids. Upon activation (by factor XIa/Ca2+ or
TF·VIIa/Ca2+), two peptide bonds in factor IX are cleaved
with resultant formation of a serine protease, factor IXa, and release
of an activation peptide (3). Factor IXa is composed of a light chain
consisting of residues 1-145 and a heavy chain consisting of residues
181-415 of native factor IX, that are held together by a single
disulfide bond (4, 5). The light chain of factor IXa consists of an amino-terminal Factor VIII is synthesized as a single chain molecule containing
several domains (A1-A2-B-A3-C1-C2) (9), with a molecular mass of ~300
kDa (10, 11). The A domains are homologous to the ceruloplasmin domains
and to the A domains of factor Va (12), whereas the C domains are
homologous to the galactose lipid binding domain and to the regions
within neuraminidase (13). Factor VIII circulates as a divalent metal
ion-dependent, noncovalent heterodimer resulting from
proteolytic cleavage at the B/A3 junction that generates a heavy chain
(A1-A2-B) and a light chain (A3-C1-C2). This procofactor form is
cleaved by thrombin at Arg372-Ser373,
Arg740-Ser741, and
Arg1689-Ser1690 to yield factor VIIIa, a
heterotrimer composed of A1, A2, and A3-C1-C2 subunits (14, 15). The A1
and A3-C1-C2 subunits remain associated with a divalent metal
ion-dependent linkage, whereas the A2 subunit is weakly
associated with the A1 and A3-C1-C2 dimer (16, 17). Although intact
factor VIIIa is required for maximal enhancement of factor IXa
activity, recent results demonstrate that the isolated A2 subunit
stimulates factor IXa activity by ~100-fold (18).
Ca2+-dependent assembly of factor IXa and
factor VIIIa on a suitable PL surface is essential for hemostasis since
defects or deficiency in the proteins result in severe bleeding
diatheses, namely hemophilia A (factor VIII deficiency) or hemophilia B
(factor IX deficiency) (Ref. 12; see the hemophilia A mutation data base and the Haemophilia B data base of point mutations and short additions and deletions, both available via the World Wide Web). In this assembly, the Ca2+-loaded form of the Gla domain of
factor IXa binds to PL (21), whereas
EGF13/EGF2 region(s) and the
protease domain are thought to interact with the A3 and A2 subunits of
factor VIIIa, respectively (18, 22). Factor VIIIa in this assembly
anchors to the PL surface via its C2 domain (13), and binding of factor
X to the IXa/VIIIa complex may be partly mediated through the A1
subunit of factor VIIIa (23).
In the IXa/VIIIa complex, residues 558-565 of the A2 subunit of factor
VIIIa are thought to bind to the protease domain of factor IXa (24).
Further, helix 330-338 (c162-170) of the protease domain of factor
IXa has been shown to bind to factor VIIIa (25). However, whether or
not helix 330-338 of factor IXa binds to the A2 subunit or more
specifically to its 558-565 peptide region is not known. The present
study is designed to address this issue. The kinetic, fluorescence, and
inhibition data indicate that helix 330 (c162) of factor IXa most
likely interacts with the 558-565 region of the A2 subunit. An
interface model between the helix 330-338 of factor IXa and the A2
558-565 region was then constructed. Other spatially nearby residues
in factor IXa as well as those in the A2 subunit that could contribute
to the interface were noted. The role of these residues in this
interaction is discussed.
Reagents--
Benzoyl-Ile-Glu-Gly-Arg-p-nitroanilide
(S-2222) was purchased from Helena Laboratories.
Dansyl-Glu-Gly-Arg-chloromethyl ketone (dEGR-ck) was obtained from
Calbiochem. Phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, recombinant hirudin, and fatty acid-free bovine serum albumin (BSA) were obtained from Sigma. PL vesicles containing 75% phosphatidylcholine and 25% phosphatidylserine were
prepared by the method of Husten et al. (26) and used for all experiments except for the fluorescence emission studies. For
fluorescence experiments, PL vesicles comprised 40%
phosphatidylcholine, 20% phosphatidylserine, and 40%
phosphatidylethanolamine and were prepared using octyl glucoside as
described (27). Recombinant factor VIII preparations
(Kogenate®) were a gift from Drs. Lisa Regan and Jim Brown
of Bayer Corp.. Purified recombinant factor VIII was also a generous
gift from Debra Pittman of the Genetics Institute. Normal plasma factor IX (IXNP) and factor X were isolated as described (6), and factor Xa was prepared as outlined (28). Purified human factor XIa and
Proteins--
The Kogenate® concentrate was
fractionated to separate factor VIII from albumin following S-Sepharose
chromatography as outlined (29). Factor VIIIa was prepared from factor
VIII using thrombin and subsequently purified using CM-Sepharose
chromatography (30), and the A2 subunit and A1/A3-C1-C2 dimer were
separated by Mono S chromatography (15). The A2 subunit was further
purified using an anti-A2 immunoaffinity column (15). The purified A2
subunit was essentially homogeneous (>95% pure), as judged by
SDS-PAGE. For some experiments, proteins were concentrated using a
Microcon concentrator (Millipore, 10-kDa cut-off). The concentration of the A2 subunit was determined by the Coomassie Blue dye binding method
of Bradford (31). Wild-type factor IX (IXWT), as well as
mutants IXR333Q (a point mutant in which R333 is replaced
by Gln), IXVIIhelix (a replacement mutant in which helix
330-338 (c162-170) is replaced by that of factor VII), and
IXPCEGF1 (a replacement mutant in which EGF1 domain is
replaced by that of protein C), were constructed, expressed, and
purified as described previously (25, 32, 33). Purified proteins were
homogeneous on SDS-PAGE and contained normal Gla content (25, 32).
Preparation of dEGR-ck-inhibited Factor IXa Proteins--
Each
factor IX protein was activated at 200 µg/ml by factor XIa (2 µg/ml) for 90 min. The buffer used was TBS, pH 7.5 (50 mM
Tris, 150 mM NaCl, pH 7.5), containing 5 mM
CaCl2. SDS-PAGE analysis revealed full activation of each
factor IX to factor IXa without degradation in the autolysis loop (34).
dEGR-IXaWT and various dEGR-IXa mutant proteins free of
dEGR-ck were obtained as described previously (34).
Activation of Factor X by Each Factor IXa Protein in the Presence
of Ca2+ and PL--
For these studies, each factor IX was
activated with factor XIa/Ca2+ as described above. For
factor X activation studies, the concentration of factor IXa was kept
at 20 nM and the buffer used was TBS/BSA (TBS with 200 µg/ml BSA) containing 5 mM CaCl2. The
concentrations of PL used were 10, 25, 50, and 100 µM in
different sets of experiments. The concentration of factor X at each PL
concentration ranged from 25 nM to 3 µM. The
activations were carried out for 5-15 min, and the amount of factor Xa
generated was measured by hydrolysis of S-2222 as described previously
(25, 34). The Km and kcat
values were obtained using the program GraFit from Erithacus Software.
Determination of EC50 of Interaction of factor IXa
Proteins with the A2 Subunit--
The EC50 (functional
Kd) of binding of each factor IXa protein with the
A2 subunit was measured essentially as described previously for its
interaction with the intact factor VIIIa (25, 34). For these
experiments, concentrations of factor IXa and factor X were kept
constant, and the rates of formation of factor Xa were determined at
increasing concentrations of the A2 subunit. Reaction mixtures
contained 5 nM factor IXa, 250 nM factor X, 25 µM PL, and various concentrations of the A2 subunit in
TBS/BSA, pH 7.5 containing 5 mM CaCl2.
Reactions were carried out at 37 °C for 5-20 min and stopped by
adding 1 µl of 500 mM EDTA. The amount of factor Xa
generated was determined by S-2222 hydrolysis as described previously
(25, 34). The EC50 was obtained by fitting the data to a
single-site ligand binding equation (Equation 1) by non-linear
regression analysis using the program GraFit from Erithacus Software.
Determination of the Apparent Kd(A2) of Binding of
dEGR-IXa Proteins to the A2 Subunit--
The
apparent Kd (termed
Kd(A2)) for binding of each dEGR-IXa protein to
the A2 subunit was determined by its ability to inhibit factor
IXaWT:A2 subunit interaction in the tenase complex as
described previously for intact factor VIIIa (25, 34). The reactions
were carried out as described for the EC50 experiments
above, except that the dEGR-IXa and IXaWT were mixed prior
to addition of the A2 subunit; this ensured steady state conditions.
Mixtures contained 100 nM IXaWT, 30 nM A2 subunit, 250 nM factor X, 25 µM PL, and various concentrations of dEGR-IXa proteins in
TBS/BSA, pH 7.5 containing 5 mM CaCl2. The
IC50 (concentration of inhibitor required for 50%
inhibition) was determined by fitting the data to IC50
four-parameter logistic equation of Halfman (35) given below.
Fluorescence Quenching of the Dansyl Moiety in dEGR-IXa by the A2
Subunit--
Effect of the A2 subunit on the emission intensity of the
dansyl moiety in each dEGR-IXa protein was determined using the SLM AB2
spectrofluorometer. Each reaction mixture contained 220 nM
dEGR-IXa in 20 mM Hepes, pH 7.2, 100 mM NaCl, 5 mM CaCl2, 0.01% Tween, 200 µg/ml BSA, and
100 µM PL vesicles. The excitation wavelength was 340 nm
(slit width, 8 nm) and the emission wavelength was 540 nm (slit width,
8 nm). First, blank values (in triplicate) were obtained for the buffer
containing PL. dEGR-IXa was then added, and the emission intensity in
the absence of the A2 subunit was recorded. Each reaction mixture was
subsequently titrated with the A2 subunit, and the emission readings
(in triplicate) were obtained at each point. The fluorescence emission
intensity at each point was corrected for increases in the reaction
volume prior to analysis of the data. The volume of added A2 subunit did not exceed 10% of the total volume. Data are presented as F/F0, where F0
is the emission intensity in the absence of A2 subunit and F
is the intensity at a given A2 subunit concentration.
Determination of the Apparent Kd(peptide) of binding
of each factor IXa to the A2 558-565 peptide--
The
apparent Kd (termed
Kd(peptide)) for binding of each factor IXa to
the A2 558-565 peptide was determined by its ability to inhibit the
respective IXa:A2 subunit interaction, as measured by reduction in the
rate of factor X activation in the tenase system. The reaction mixtures
for both IXaWT and IXaPCEGF1 contained 100 nM factor IXa, 30 nM A2 subunit, 250 nM factor X, and 25 µM PL in TBS/BSA, pH 7.5, with 5 mM CaCl2. The reaction mixture for
IXaR333Q contained 300 nM factor IXa instead of
100 nM used for IXaWT or IXaPCEGF1;
concentrations of other components were the same. The amount of factor
Xa generated was determined by hydrolysis of S-2222. The
IC50 values were obtained using Equation 2. Here,
y is the rate of factor Xa formation in the presence of a
given concentration of the A2 558-565 peptide represented by
x, and a is the maximum rate of factor Xa
formation in the absence of the A2 peptide. Equation 3 was then used to
obtain the apparent Kd(peptide) values. Here,
A is the concentration of IXaWT,
IXaPCEGF1, or IXaR333Q, and EC50 is
the apparent Kd(A2) for the respective IXa:A2
subunit interaction.
Molecular Modeling--
The three (A1, A2, and A3) domains in
factor VIIIa are homologous to the three respective domains in
ceruloplasmin (12, 38). The A1, A2, and A3 domains of factor VIIIa were
modeled using the coordinates of each respective domain of
ceruloplasmin (39). Each domain was modeled using the homology model
building module from Biosym/MSI (San Diego, CA), as well as the
Swiss-Model server using the optimize mode (40, 41). The two approaches used in building the homology models resulted in minor differences between the structure of each of the A subunits. However, the structure
pertaining to the loop containing the 310 helical turn involving residues 558-565 as well as other nearby interface regions of the A2 subunit implicated in binding to factor IXa were invariant between the two models. Further, the Biosym/MSI models of all three A
subunits were similar to those published previously by Pemberton
et al. (12). Thus, we used the coordinates of Pemberton et al. (Ref. 12; see hemophilia data base, available
on-line) in building the interface between the A2 subunit and the
protease domain of factor IXa. Details are provided under "Results
and Discussion."
Activation of Factor X by Various Factor IXa Proteins in the
Presence of Only Ca2+ and PL--
The kinetic constants
for the activation of factor X were obtained by various factor IXa
proteins in the presence of Ca2+ and several concentration
of PL. This analysis was performed to establish whether or not the
factor IXa proteins under investigation bind to Ca2+ and PL
normally and possess a functional active site. The kinetic constants
obtained under these conditions in the absence of factor VIIIa are
listed in Table I. All mutants activated
factor X normally, and the specificity constant
(kcat/Km) for each mutant at different PL
concentrations did not differ appreciably from that observed for
IXaWT or IXaNP. The increase in
Km values at higher concentrations of PL for WT or
for a given mutant may reflect binding of factor IXa and factor X to
different PL vesicles (42, 43). Further, our Km and
kcat values are in close agreement with the
earlier published data (18, 43). Consistent with earlier observations
(43), we also observed a slight increase in kcat
for each factor IXa protein at higher concentrations of PL.
Cumulatively, our data presented in Table I indicate that the factor
IXa mutants under investigation interact with Ca2+ and PL
normally. Further, in the absence of factor VIIIa, activation of factor
X by these IXa mutants is not impaired.
A2 Subunit-mediated Enhancement of Factor X Activation by Various
Factor IXa Mutants--
In this section, we evaluated the ability of
the A2 subunit to augment factor X activation by various factor IXa
mutants. These data are presented in Fig.
1. The presence of the A2 subunit in the
reaction mixtures enhanced the factor X-activating activity of
IXaPCEGF1 to the same extent as that of IXaWT.
However, the ability of the A2 subunit to potentiate the activity of
IXaR333Q was severely impaired, and it was essentially
absent for IXaVIIhelix. Next, we determined the
EC50 (functional Kd) values for interaction of each factor IXa protein with the A2 subunit using Eq. 1.
Fitting the data to a single-site binding model yielded an apparent
Kd(A2) of 257 ± 31 for both
IXaWT and IXaPCEGF1; for IXaR333Q
or IXaVIIhelix, it could not be calculated. These data
indicate that the helix 330 (c162) of factor IXa interacts with the A2
subunit of factor VIIIa.
In further experiments, we measured the EC50 values for
interaction of IXaWT and of IXaPCEGF1 with the
A2 subunit using different concentrations of factor X ranging from 50 nM to 5 µM. These data are presented in Fig.
2. At each concentration of factor X, the concentration of factor IXa was held constant at 5 nM and
the rate of factor Xa generation was determined in the presence of increasing concentrations of the A2 subunit. The EC50
values ranged from ~280 nM at lower concentrations of
factor X (<150 nM) to ~200 nM at higher
concentrations of factor X (>1 µM) for both IXaWT and IXaPCEGF1. Our functional
Kd (EC50) values ranging from 200 to 280 nM for the interaction of IXaWT (or
IXaPCEGF1) and the A2 subunit employing different factor X
concentrations are consistent with the EC50 values obtained
earlier using similar conditions for IXaNP and the A2
subunit (18). From these observations, we conclude that factor X does
not appreciably influence the functional Kd of
IXa:A2 subunit interaction. This is in contrast to the results obtained
using factor VIIIa where factor X reduces the functional
Kd of IXa:VIIIa interaction by ~10-fold (34).
These results support previous observations that the A1 subunit of
factor VIIIa interacts with factor X (23). More importantly, our data
with the IXaPCEGF1 mutant indicate that the EGF1 domain of
factor IXa does not interact with the A2 subunit of factor VIIIa.
Determination of Apparent Kd(A2 Values for the
Interaction of A2 Subunit with dEGR-IXa Proteins--
Here, we
investigated the steady state inhibition of IXaWT:A2
subunit interaction by different dEGR-IXa proteins. These data are
presented in Fig. 3. The IC50
values were obtained using Equation 2, and the respective apparent
Kd(A2) values were obtained using Equation 3.
dEGR-IXaWT and dEGR-IXaPCEGF1 interacted with the A2 subunit with a similar Kd(A2) of ~100
nM, whereas dEGR-IXaR333Q interacted with the
A2 subunit with a Kd(A2) of ~1.8
µM and dEGR-IXaVIIhelix failed to compete
with factor IXaWT up to 12 µM concentration.
The apparent Kd(A2) (~100 nM)
obtained from the inhibition data (Fig. 3) and EC50 values
(200-280 nM) obtained from the potentiation of factor X activation data (Figs. 1 and 2) for the factor IXaWT and
IXaPCEGF1 are in close agreement with each other. Of
significance is the observation that the mutations in the helix 330 (c162) of the protease domain of factor IXa severely impairs its
interaction with the A2 subunit.
Effects of the A2 Subunit on the Fluorescence Emission of dEGR-IXa
Proteins--
Since dansyl emission is quite sensitive to its
environment, we examined the changes in dansyl emission intensity
(excitation wavelength, 340 nm; emission wavelength, 540 nm) of
dEGR-IXa proteins in the presence of increasing concentrations of the
A2 subunit. Reaction mixtures contained 220 nM amounts of
each dEGR-IXa protein, 100 µM PL, and various
concentrations of the isolated A2 subunit. The results are presented in
Fig. 4. For IXaWT or
IXaPCEGF1, a dose-dependent decrease in the
fluorescence emission of the dansyl probe was observed. However, little
if any change in the emission intensity was observed when the A2
subunit was titrated into the reaction mixtures containing factor
IXaR333Q or IXaVIIhelix. A nonlinear least
squares fitting of the data for IXaWT and
IXaPCEGF1 to a bimolecular association model yielded a
plateau value of 0.59 ± 0.05 for
F/F0 and an apparent
Kd(A2) value of 82 ± 18 nM for
each protein. Further, the Kd(A2) for IXaR333Q or IXaVIIhelix could not be
calculated. These results suggest that the isolated A2 subunit
interacts equivalently with IXaWT and
IXaPCEGF1, and similarly modulates the emission of the active site-labeled dansyl probe. The apparent
Kd(A2) value of ~82 nM for factor
IXaWT or IXaPCEGF1 obtained using the fluorescence quenching measurements is in agreement with the values obtained from steady state inhibition experiments. Consistent with the
data presented in Figs. 1 and 3, these fluorescence results suggest
that the factor IXaR333Q and IXaVIIhelix
mutants are severely impaired in their interactions with the A2
subunit.
Determination of Apparent Kd Peptide Values for Binding
of Factor IXa Proteins to the A2 558-565 Peptide--
The data
presented thus far strongly indicate that the A2 subunit interacts with
residues of the helix 330 (c162) of factor IXa. Previous studies also
suggest that residues 558-565 of the A2 subunit are involved in
binding to factor IXa (18). However, it is not known whether the
558-565 peptide region of the A2 subunit represents the site of direct
interaction with the helix 330 of factor IXa. We investigated this
possibility by measuring the affinity of the A2 558-565 peptide for
IXaWT, IXaPCEGF1, and IXaR333Q. These data are presented in Fig. 5. The
A2 558-565 peptide inhibits the interaction of IXaWT and
IXaPCEGF1 with similar IC50 values of ~8
µM.4 However,
the A2 558-565 peptide inhibited the IXaR333Q:A2 subunit interaction with an IC50 value of ~70 µM,
which is ~9-fold higher than the value obtained for IXaWT
or IXaPCEGF1 (Fig. 5). We next used the Cheng and Prusoff
relationship (36, 37) to obtain apparent
Kd(peptide) values for each factor IXa protein. These apparent Kd(peptide) values along with the
changes in Gibbs free energy are listed in Table
II. The peptide bound to
IXaWT and IXaPCEGF1 with an apparent
Kd of ~4 µM and to
IXaR333Q with an apparent Kd of ~62
µM. Thus, the affinity of the A2 558-565 peptide for
IXaWT and IXaPCEGF1 is similar, whereas it is
reduced ~15-fold for the IXaR333Q.
Notably, comparison of the data presented in Figs. 3 and 5 reveal that
the increase in apparent Kd(A2) or
Kd(peptide) for IXaR333Q is similar
as compared with the apparent Kd(A2) or
Kd(peptide) obtained for IXaWT (or
IXaPCEGF1). Further, the difference in
Modeling of the Interface between the Protease Domain of Factor IXa
and the A2 Subunit of Factor VIIIa--
Based upon the preceding
information, we modeled the interface between the protease domain of
factor IXa (Ref. 5, Protein Data Bank code 1RFN) and the A2 subunit
(see "Experimental Procedures") by bringing together the helix 330 of factor IXa and the 310 helical turn in residues 558-565
of the A2 subunit and maximizing the interaction among the charged
residues. Emphasis was also given for interactions involving hydrogen
bonds and hydrophobic contacts. An important guiding principle in the
construction of this interface model was that the Gla domain of factor
IXa and the C2 domain of factor VIIIa must be oriented such that each may contact the PL surface. To achieve this, the A2 structure (along
with the A1 and A3 subunits) was rotated and translated as a rigid
body. The principle approach used was that described earlier by
Tulinsky and co-workers (44) in building the prothrombin model from the
structures of prothrombin fragment 1 and the fragment 2-thrombin
complex. Minor adjustments in the side chains of both proteins were
also made. All residues in the interface of both proteins were checked
for distances to ensure no improper contacts (45). The interface model
that resulted from this approach is shown in Fig.
6A. In this display, the Gla
domain of factor IXa and the C2 domain of factor VIIIa are projecting
away from the viewer.
In addition to the A2 558-565 region and the factor IXa 330-338
region, other spatially nearby regions that may play important roles in
the interaction of A2 subunit with the protease domain were also noted.
The details of the composite interface region are shown in Fig.
6B. It appears that electrostatic forces might play a
significant role in the interaction between the A2 subunit and the
protease domain, and an electrostatic potential for the interface
calculated using the program GRASP (46) is shown in Fig. 6C.
Further, in addition to the electrostatic interactions outlined in Fig.
6, hydrophobic and polar uncharged interactions between
Thr343 (c175) and Tyr345 (c177) of factor IXa
and His444 of the A2 subunit were observed. Moreover, a
hydrogen bond between Asn258 (c93) of factor IXa and
Ser709 of the A2 subunit could also be formed. Importantly,
a significant hydrophobic patch involving Ile566 and
Met567 in the A2 subunit and Ile298 (c129B),
Tyr295 (c128), Phe299 (c130),
Phe302 (c133), Phe378 (c208), and
Phe98 (EGF2 domain) in factor IXa was noted. Thus, it
appears that the hydrogen bonds as well as the hydrophobic and
electrostatic interactions all play important roles in the proposed
interface between factor IXa and the A2 subunit. In this context, an
apparent Kd(A2) of ~100 nM
observed for this interaction reflects the net change in free energy
involved in making and breaking such bonds.
A factor IXa-interactive site comprising residues 484-509 in the A2
subunit that was identified using a monoclonal antibody (47) does not
appear to contact the protease domain in our interface model. However,
it should be noted that, in a previous study (48), Lollar et
al. concluded that this same monoclonal antibody does not
interfere with the IXa:VIIIa interaction. The reason(s) for the
differing results obtained in the two studies (47, 48) is not fully
understood. Further, in the proposed interface model shown in Fig.
6A, the 484-509 region in the A2 subunit is not in close
proximity to the 558-565 interface region and shows no apparent
contacts with factor IXa. However, we cannot exclude the possibility of
a change in the conformation of the A2 subunit upon binding the
protease domain, which may juxtapose (and subsequently involve) this
region. Alternatively, the monoclonal antibody may prevent the
association of the A2 subunit with factor IXa through steric interference.
Analysis of Hemophilia Data Bases--
Of significance is the
observation that numerous mutations in the helix 330 (c162) of factor
IXa cause hemophilia B (see Ref. 25 and the Haemophilia B data base of
point mutations and short additions and deletion), whereas several
mutations in or near factor VIII residues 558-565 result in hemophilia
A (Ref. 12; see hemophilia data base, available on-line).
Arg333 (c165) in our interface model (Fig. 6) interacts
with Glu440 residue of the A2 subunit, and mutations in the
Arg333 (c165) that eliminate the charge (Arg
Mutations in the hydrophobic patch of the interface model are also
known to cause bleeding diathesis. Thus, mutation of Phe378
(c208) to Val or Leu in factor IXa causes hemophilia B (see Haemophilia B data base, available via the World Wide Web), and change of Ile566 to Thr or Arg in the A2 subunit causes hemophilia A
(Ref. 12; see hemophilia data base, World Wide Web). The mutation of
Ile566 to Arg is expected to disrupt the hydrophobic
interaction. However, Thr substitution yields Asn-X-Thr
consensus sequence that leads to a new N-glycosylation site
at Asn564, which could disrupt the Factor IXa:A2 subunit
interaction. Moreover, change of Phe302 (c133) to Ala has
been shown to impair the interaction of factor IXa with factor VIIIa
(19). Mutations of Phe302 (c133) to Ala and
Phe378 (c208) to Val or Leu are expected to diminish the
hydrophobic interactions involving Ile566 and
Met567 of the A2 subunit. The change of Ile566
to Arg in the A2 subunit would have similar consequences.
Concluding Remarks--
Previous studies have indicated that the
helix 330 (c162) of the protease domain (25) and 558-565 region of the
A2 subunit (18) represent important determinants for the interaction of factor IXa and factor VIIIa, respectively. However, it was not known
whether these two regions interact with each other in the IXa:VIIIa
complex. The present study provides evidence that these two regions may
form an interface and interact with each other involving hydrophobic as
well as electrostatic forces (Fig. 6). Modeling of the interface
suggests that other spatially nearby regions may also participate in
the interaction of factor IXa with factor VIIIa. Several mutations in
the proposed interface cause hemophilia A or B and are known to impair
IXa:VIIIa interaction. Thus, our interface model is compatible with the
existing biochemical as well as with the two-dimensional electron
crystallography data of Stoylova et al. (20). However, the
three-dimensional cocrystal structure of the factor IXa protease domain
and A2 subunit will be required to fully establish this view. In light
of these considerations, we emphasize that our interface model is an
interim model subject to refinement as new biochemical and
experimentally determined structural data become available.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-carboxyglutamic acid (Gla)-rich domain (residues 1-40), a short hydrophobic segment (residues 41-46), and two
epidermal growth factor (EGF)-like domains (EGF1 residues 47-84 and
EGF2 residues 85-127), whereas the heavy chain contains the serine protease domain, which features the catalytic triad residues, namely
His221 (c57),2
Asp269 (c102), and Ser365 (c195) (5). The Gla
domain contains several high and low affinity Ca2+-binding
sites, whereas EGF1 and protease domain each contain a high affinity
Ca2+ site (7). For proper binding of factor IXa to PL and
factor VIIIa, all of the Ca2+ sites in factor IXa
must be filled (7, 8).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-thrombin were purchased from Enzyme Research Laboratories (South
Bend, IN). A synthetic peptide corresponding to the A2 subunit residues
558-565 (Ser-Val-Asp-Gln-Arg-Gly-Asn-Gln) was obtained as described
previously (18), and its concentration was determined by amino acid analysis.
V is the rate of formation of factor Xa at a given
concentration of the A2 subunit, denoted by L, and
Vmax is the rate of factor Xa formation by the
factor IXa:A2 subunit complex. EC50 is the functional
Kd defined as the concentration of free A2 subunit
yielding 50% of the Vmax. The background rate of factor Xa generation was obtained by carrying out the reaction in
the absence of the A2 subunit. This represented less than 1% of the
Vmax and was subtracted before data analysis. To
obtain EC50 values as a function of substrate
concentration, a series of experiments were performed in which factor X
was varied from 50 nM to 1 µM.
(Eq. 1)
y is the rate of factor Xa formation in the presence
of a given concentration of dEGR-IXa protein represented by
x, a is the maximum rate of factor Xa formation
in the absence of dEGR-IXa, and s is the slope factor. Each
point was weighted equally, and the data were fitted to Equation 2
using the nonlinear regression analysis program GraFit from Erithacus
Software. The background value represented ~5% of the maximum rate
of factor Xa formation in the absence of dEGR-IXa. To obtain apparent
Kd(A2) values for the interaction of dEGR-IXa
proteins with A2, we used the following equation as described by Cheng
and Prusoff (36) and further elaborated by Craig (37).
(Eq. 2)
A is the concentration of factor IXaWT,
and EC50 is the concentration of factor IXaWT
that gives a 50% maximum response in the absence of the competitor at
a specified concentration of factor X used in the experiment.
(Eq. 3)
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
Kinetic parameters of factor X activation in the absence of factor
VIIIa
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Fig. 1.
Effect of the isolated A2 subunit of factor
VIIIa on the rate of activation of factor X by various factor IXa
proteins. Rate of formation of factor Xa by each factor IXa
protein was measured as described under "Experimental Procedures."
The reaction mixtures contained 5 nM factor IXa, 250 nM factor X, and various concentrations of A2 subunit. The
buffer used was TBS/BSA, pH 7.5 containing 25 µM PL and 5 mM CaCl2. The proteins used are:
IXaWT (closed circles),
IXaPCEGF1 (open circles),
IXaR333Q (closed triangles), and
IXaVIIhelix (open triangles). The
data were fitted to a single-site binding equation (Equation 1).
View larger version (10K):
[in a new window]
Fig. 2.
Effect of factor X concentration on the
EC50 (functional
Kd) of the interaction of
A2 subunit with IXaWT or IXaPCEGF1. The
EC50 of the interaction of factor IXaWT
(closed circles) or factor IXaPCEGF1
(open circles) with the A2 subunit was determined
at various concentrations of factor X. Each point (EC50)
shown is the concentration of free A2 subunit (y axis)
providing 50% of the Vmax. Each
EC50 value was obtained from a direct plot (similar to Fig.
1) of factor Xa generation at various concentrations of the A2 subunit
and a constant concentration of factor X. The factor IXa concentration
in each experiment was fixed at 5 nM. The buffer used was
TBS/BSA, pH 7.5, containing 25 µM PL and 5 mM
CaCl2. Factor Xa concentration was measured by S-2222
hydrolysis.
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[in a new window]
Fig. 3.
Abilities of various dEGR-IXa proteins to
inhibit factor IXa:A2 subunit interaction as measured by a decrease in
factor Xa generation in the tenase system. The reaction mixtures
contained 100 nM IXaWT, 30 nM A2
subunit, 250 nM factor X, 25 µM PL, and
various concentrations of dEGR-IXa proteins in TBS/BSA, pH 7.5, containing 5 mM CaCl2. Factor Xa generation was
measured by S-2222 hydrolysis. The value of slope factor, s,
was 0.9 ± 0.1, indicating a single affinity binding site between
the interacting proteins. The curves represent best fit of
the data to the IC50 four-parameter logistic equation
(Equation 2). The proteins used are: dEGR-IXaWT
(closed circles), dEGR-IXaPCEGF1
(open circles), dEGR-IXaR333Q
(closed triangles), and
dEGR-IXaVIIhelix (open triangles).
The inhibition curve obtained with dEGR-IXaNP was similar
to that depicted for dEGR-IXaWT (graph not shown).
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Fig. 4.
Effect of the A2 subunit on the fluorescence
emission intensity of dEGR-IXa proteins. Reactions (160 µl) were
titrated with A2 subunit in buffer containing 20 mM Hepes,
pH 7.2, 100 mM NaCl, 5 mM CaCl2,
0.01% Tween 20, 200 µg/ml BSA, and 100 µM PL vesicles.
Fluorescence emission intensity of each dEGR-IXa (220 nM)
at a given A2 subunit concentration was determined as described under
"Experimental Procedures." Data were fitted to a bimolecular
association model and are presented as
F/F0, where F0
is the emission intensity in the absence and F is the
emission intensity at a given A2 subunit concentration.
dEGR-IXaWT (triangles),
dEGR-IXaPCEGF1 (circles),
dEGR-IXaR333Q (diamonds), and
dEGR-IXaVIIhelix (squares).
View larger version (17K):
[in a new window]
Fig. 5.
Ability of the 558-565 A2 peptide to inhibit
the interaction of various factor IXa proteins with the A2
subunit. The reaction mixture for factor IXaWT
(closed circles) or factor IXaPCEGF1
(open circles) contained 100 nM of
factor IXa protein, 30 nM A2 subunit, 250 nM
factor X, 5 mM CaCl2, and 25 µM
PL in TBS/BSA, pH 7.5. The reaction mixture for factor
IXaR333Q (open squares) contained 300 nM factor IXa instead of 100 nM used for factor
IXaWT or factor IXaPCEGF1; the concentrations
of other components were unchanged. Factor Xa generation was determined
by S-2222 hydrolysis, and the curves represent best fit to
the IC50 four-parameter logistic equation (Equation 2). The
value of slope factor, s, was 0.9 ± 0.1, indicating a
single affinity binding site between the various IXa proteins and the
A2 peptide.
Apparent Kd and Gibbs free energy values for the interaction of
various factor IXa proteins with the A2 subunit and the A2 558-565
peptide
G0 for the interaction of A2 subunit with
IXaWT (or IXaPCEGF1) and IXaR333Q
is 1.72 kcal mol
1 (Table II). This difference
in
G0 is essentially the same as that (1.62 kcal mol
1) obtained for the interaction of A2
peptide with IXaWT (or IXaPCEGF1) and
IXaR333Q. If the A2 558-565 peptide bound to a different
region than the helix 330 of factor IXa, then one would expect it to bind to IXaR333Q with the same affinity as that for
IXaWT. Since this is not the case, our data support a
conclusion that the helix 330 (c162) in factor IXa is most likely in
direct contact with the 558-565 region of the A2 subunit.
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Fig. 6.
Interface model between the factor IXa
protease domain and the A2 subunit of factor VIIIa. The
coordinates for the human factor IXa structure are from the
Brookhaven Protein Data Bank (code 1RFN) and the coordinates for the
A1, A2, and A3 subunits (12) of factor VIIIa are based upon homology
models built using ceruloplasmin coordinates (Protein Data Bank code
1KCW). A, schematic representation of the interface model.
Ribbon structure for each protein is depicted. The IXa protease domain
is in light blue, and the EGF2 domain is in
red. The A1 subunit is in yellow, the A2 subunit
is in magenta with residues 484-509 in white,
and the A3 subunit is in cyan with the COOH terminus in
red. The Gla and the EGF1 domains of factor IXa and the C1
and C2 domains of factor VIIIa are not shown. The interface residues of
factor IXa protease domain and of the A2 subunit are shown as CPK
space-filling models. The molecules are oriented such that the Gla
domain of factor IXa and the C2 domain of factor VIIIa are projecting
away from the viewer. The Gla domain in factor IXa and the C2 domain of
factor VIIIa bind to the PL surface. B, detailed interface
between factor IXa protease domain and the modeled A2 subunit. Only the
charged residues that participate in the binding interactions are
depicted. The hydrophobic residues that participate in this interaction
are discussed in the text. The orientation of the molecules is the same
as in A. Chymotrypsin numbering system for the factor IXa
protease domain is used. Corresponding factor IX numbering system are
338 (c170), 332 (c164), 333 (c165), 346 (c178), 403 (c233), 293 (c126),
and 410 (c240). Factor IXa residues are labeled light
blue, and A2 subunit residues are labeled
magenta. C, electrostatic potential between the
factor IXa protease domain and the A2 subunit interface as determined
using the program GRASP (46). Blue represents positive,
red represents negative, and white represents
neutral residues.
Gln or
Leu) cause severe hemophilia B (see Haemophilia B data base of point
mutations and short additions and deletion, available via the World
Wide Web). Further, Asn346 (c178) of factor IXa interacts
with both Lys570 and Glu445 of the A2 subunit,
and a mutation of Asn346 (c178) to Asp causes hemophilia B
(see Haemophilia B data base). Similarly, Arg403 (c233) in
our model interacts with Glu633 of the A2 subunit and
mutations in Arg403 (c233) to Trp or Gln cause hemophilia B
(see Haemophilia B data base). Moreover, Arg338 (c170) of
factor IXa interacts with Asp560 of the A2 subunit, and
mutations in both of these residues result in hemophilia (HAMSTeRS data
base and Haemophilia B data base, both available via the World Wide
Web). In addition, Arg562 contained within the A2 558-565
peptide region is cleaved by activated protein C (17), and factor IXa
selectively protects this site from cleavage (49). In support of this
observation, Arg562 of the A2 subunit along with
Gln561 interacts with Asp332 (c164) in our
interface model and change of Asp332 (c164) to Tyr results
in hemophilia B (see Haemophilia B data base).
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ACKNOWLEDGEMENTS |
---|
We thank Tomasz Heyduk and Jim Kiefer for useful discussions. We also thank Lisa Regan and James Brown of Bayer Corp. and Debra Pittman of the Genetics Institute for providing the recombinant factor VIII, and Jennifer Chandler for excellent technical assistance.
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FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant HL36365 and American Heart Association Grant 9950228N (both to S. P. B.) and by National Institutes of Health Grants HL30616 and HL38199 (to P. J. F.).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: Div. of Hematology and Oncology, Saint Louis University Health Sciences Center, 3635 Vista Ave., P.O. Box 15250, St. Louis, MO 63110-0250. Tel.: 314-577-8499/8854; Fax: 314-773-1167; E-mail: bajajps@slu.edu.
Published, JBC Papers in Press, February 14, 2001, DOI 10.1074/jbc.M011680200
2 For comparison, the factor IX amino acid numbering system is used. The numbers with a prefix c (e.g. c57) in parentheses refer to the chymotrypsin equivalents for the protease domain of factor IXa (6).
3 Although there is a considerable controversy as to the role of EGF1 domain in the interaction of factor IXa and factor VIIIa, at present it cannot be completely ruled out that this domain contains a direct interactive site for factor VIIIa (8).
4 The present IC50 value (~8 µM) for the A2 peptide inhibition of the IXaWT:A2 subunit interaction is 5-fold lower than the IC50 value (~40 µM) obtained from the inhibition studies of the A2 subunit enhancement of IXaNP activity (18). This difference in IC50 values is most likely due to the different concentrations (30 nM in present study versus 240 nM in previous study) of the A2 subunit used in the two studies.
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ABBREVIATIONS |
---|
The abbreviations used are:
TF, tissue factor;
Gla, -carboxyglutamic acid;
EGF, epidermal growth factor;
PL, phospholipid;
BSA, bovine serum albumin;
WT, wild type;
TBS, Tris-buffered saline;
dansyl, 5-dimethylaminonaphthalene-1-sulfonyl;
dEGR-ck, dansyl-Glu-Gly-Arg-chloromethyl ketone;
dEGR-IXa, IXa
inactivated with dEGR-ck;
S-2222, benzoyl-Ile-Glu-Gly-Arg-p-nitroanilide;
NP, normal plasma;
Kd(A2), dissociation constant for dEGR-IXa and
the A2 subunit;
Kd(peptide), dissociation
constant for factor IXa and the A2 558-565 peptide;
TBS, Tris-buffered
saline;
PAGE, polyacrylamide gel electrophoresis.
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REFERENCES |
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