(Received for publication, June 2, 1995; and in revised form, July 28, 1995)
From the
The interaction of fibrinogen
A1-50-
-galactosidase fusion protein with the slow and
fast forms of thrombin was studied and compared to thrombin-fibrinogen
interaction under identical solution conditions. At equilibrium, the
affinity of the fusion protein for the slow form of thrombin is 3 times
higher than its affinity for the fast form. The fusion protein and
fibrinogen have the same affinity for the fast form. On the other hand,
the affinity of the fusion protein for the slow form of thrombin is 40
times tighter than that of fibrinogen. In the transition state, binding
of the fusion protein has the same properties as fibrinogen, with the
fast form showing higher specificity. The N-terminal fragment of the
fibrinogen A
chain thus contains residues that are responsible for
the preferential binding of the fusion protein to the slow form at
equilibrium and to the fast form in the transition state. If this
fragment binds to thrombin in a similar way for fibrinogen and the
fusion protein, then the N-terminal domains of the B
and
chains of fibrinogen, that are not present in the fusion protein, must
play a key role in the binding of fibrinogen to thrombin at
equilibrium. These chains may destabilize binding to the slow form by
nearly 2.4 kcal/mol, thereby favoring binding of fibrinogen to the fast
form. We propose that the three chains of fibrinogen play different
roles in the thrombin-fibrinogen interaction, with the A
chain
containing residues for preferential binding to the fast form in the
transition state and the B
and
chains containing residues
that destabilize binding to the slow form at equilibrium.
Thrombin is an allosteric serine protease involved in blood
coagulation. The enzyme exists in two forms, slow and fast(1) ,
that have been targeted toward anticoagulant and procoagulant
activities (2) . The fast form preferentially cleaves
fibrinogen, while the slow form cleaves protein C with higher
specificity. The molecular basis of preferential binding of fibrinogen
to the fast form (3) has been discussed in connection with the
interaction of thrombin with the potent natural inhibitor
hirudin(4) . Hirudin and fibrinogen bind to thrombin by making
contacts with residues located not only in and around the catalytic
pocket, but also in the fibrinogen binding loop. This loop is separate
from the catalytic pocket and is homologous to the Ca binding loop of trypsin and
chymotrypsin(5, 6, 7, 8) . Either
molecule binds preferentially to the fast form with a free energy
difference of about 1.7 kcal/mol(3, 4) . Energetic
mapping of hirudin binding (4) and recent site-directed
mutagenesis studies (
)have led to the conclusion that most
of the preferential binding of hirudin to the fast form arises from
interaction with residues in and around the catalytic pocket. The
fibrinogen binding loop provides only a small contribution to the
coupling with the allosteric transition. Whether a similar conclusion
can be drawn in the case of the structural origin of preferential
binding of fibrinogen to the fast form of thrombin remains to be
established.
A useful model substrate for thrombin is the tripartite
protein consisting of residues 1-50 of the fibrinogen A
chain linked by a 59-residue segment of collagen to Escherichia
coli
-galactosidase(10) . The central domain of
fibrinogen, represented by the CNBr fragment containing A
chain
residues 1-51, B
chain residues 1-118, and
residues 1-78, makes contact with both the catalytic pocket and
the fibrinogen binding loop in thrombin (11) . Because the
tripartite fusion protein lacks the N-terminal domains of the B
and
chains of fibrinogen, it represents a simplified model for
studying molecular recognition events involved in the
thrombin-fibrinogen interaction. Specifically, comparative studies of
the fusion protein and fibrinogen may shed light on the relative
contribution of the three chains of fibrinogen. Functional studies
indicate that the FpA (
)consisting of residues 1-16 of
the A
chain is cleaved from the fusion protein by thrombin at a
rate comparable to that of fibrinogen(12) . This result
suggests that the B
and
chains may have little influence on
the interaction of the A
chain with thrombin. However,
perturbation of the N-terminal portion of the B
chain of
fibrinogen often results in impaired clotting
activity(13, 14, 15) , and interaction of all
three chains with the fibrinogen binding loop seems to be crucial for
the correct hydrolysis of fibrinogen by
thrombin(11, 16) . In an attempt to dissect the
contribution of A
1-50 from the N-terminal portion of the
three fibrinogen chains, we have decided to explore the interaction of
the fusion protein with the slow and fast forms of thrombin at
equilibrium and in the transition state. Comparison of the results with
those obtained with fibrinogen under identical solution conditions
indicates that the B
and
chains play an important role in
the preferential binding of fibrinogen to the fast form by
destabilizing binding to the slow form. The fibrinogen A
chain, on
the other hand, contains all the structural epitopes for preferential
cleavage by the fast form. Therefore, the N-terminal portions of the
three chains of fibrinogen seem to have been targeted toward different
roles in molecular recognition of this substrate by the slow and fast
forms of thrombin.
Human -thrombin was purified and tested for activity as
described(1, 10, 17) . The chromogenic
substrate S2238 was purchased from Chromogenix (Molndal, Sweden). The
fusion protein was expressed, purified, and tested for activity as
described(10, 12) . The release of FpA from the fusion
protein was quantified by reverse-phase HPLC (18) using a Vydac
C
column. Elution was carried out at a flow rate of 1
ml/min, with a gradient containing 25 mM
Na
HPO
/NaH
PO
buffer at
pH 6.0 (solvent A) and 50% acetonitrile in solvent A (solvent B).
Optimal separation was obtained using a 30-min linear gradient to 40%
of solvent B. The effluent was monitored at 206 nm. A molar absorption
coefficient for FpA was determined by calibration curves. The slow and
fast forms of thrombin were studied under experimental conditions of 5
mM Tris, 0.1% PEG, pH 8.0, at 25 °C, in the presence of
200 mM ChCl (slow form) or NaCl (fast form). Progress curves
for the release of FpA were analyzed using the expression for
first-order kinetics shown in , where e
is the active thrombin concentration, t is time, k
and K
refer to
the hydrolysis of FpA, [F] is the concentration of FpA at
time t, and [F]
is the asymptotic
concentration of FpA.
This value was consistent with the concentration of fusion protein estimated from SDS-polyacrylamide gel electrophoresis analysis of the purified protein.
The equilibrium constant for the binding of
the fusion protein to thrombin was measured using the viscogenic method
introduced for the study of fibrinogen binding and described in detail
elsewhere(19) . In the viscogenic method, the K for the hydrolysis of FpA from
fibrinogen is measured as a result of the competition of the hydrolysis
of S2238 by thrombin as a function of fibrinogen concentration. Values
of K
determined as a function of the
relative viscosity of the solution are analyzed using the expression
shown in , where
is the ratio between the acylation
and dissociation rates,
is the ratio between acylation and
deacylation, and
is the relative viscosity of the
medium.
The value of the equilibrium dissociation constant K is obtained in a plot of K
versus
as
the extrapolation of K
for
0. The viscogenic method yields information
on the equilibrium components of the binding interaction (K
), as well as on the rate-limiting
events during the catalytic conversion of the substrate leading to the
release of FpA (
and
). The value of K
for the release of FpA from the fusion protein was derived
from the competitive effect on the hydrolysis of S2238. Measurements of K
were then carried out as a function of
relative viscosity, with thrombin either in slow or fast form. The
results were expressed in units of [FpA] to allow a direct
comparison between the fusion protein, containing 4 FpA
fragments/molecule, and fibrinogen, containing 2 FpA
fragments/molecule.
The results of the fusion protein binding to the slow and
fast forms of thrombin are shown in Fig. 1as a plot of Kversus
.
The data obey a straight line in the plot, over the range of relative
viscosity values examined, suggesting that
0 in . This implies that deacylation and diffusion of the FpA
away from the catalytic pocket of thrombin occur on a time scale much
faster than acylation, in either the slow or fast forms. The value of
in either form indicates that the fusion protein behaves as a
``sticky'' substrate, with the dissociation rate being
comparable to acylation. This situation is seen for the cleavage of FpA
from fibrinogen by the fast form, but not by the slow form(3) .
The extrapolation of K
for
0 in Fig. 1gives the value of K
for the fusion protein binding to the slow and fast forms.
Unlike the case seen for fibrinogen(3) , the fusion protein
preferentially binds to the slow form with an affinity nearly 3 times
higher. The affinity for the fast form is about the same as that seen
for fibrinogen. In the slow form, on the other hand, the fusion protein
binds to thrombin with an affinity 40 times higher compared to
fibrinogen (see Table 1).
Figure 1:
Experimental values of K(in units of FpA concentration) for the
release of FpA from the fusion protein as a function of the relative
viscosity
. The data obey a straight line as
predicted by for
0. The value of the
equilibrium dissociation constant can be derived from the plot as the
extrapolation of K
for
0. Experimental conditions are: 5 mM Tris, 0.1% PEG, pH 8.0, 25 °C, and either 200 mM ChCl
(
) or 200 mM NaCl (
). Continuous lines were drawn
according to with values of K
listed in Table 1,
= 0 and
=
2.2 ± 0.9 (
), or
= 0 and
= 1.4
± 0.3 (
). Note the higher affinity when thrombin is in the
slow form.
The release of FpA from the fusion protein upon interaction with the slow and fast forms of thrombin is documented in Fig. 2. The fast form cleaves FpA at a rate 9 times faster. Cleavage obeys first-order kinetics in both the fast and slow forms. This behavior parallels the results seen for the release of FpA from fibrinogen under identical solution conditions(2) . The release of FpA is in this case nearly 7 times faster for the fast form of thrombin. In either thrombin form, fibrinogen is cleaved at a rate that is about 6 times faster compared to the fusion protein.
Figure 2:
Progress curves for the release of FpA
from the fusion protein, as determined by HPLC analysis. The kinetics
is first-order, independent of the allosteric state of the enzyme.
Experimental conditions are: 5 mM Tris, 0.1% PEG, pH 8.0, 25
°C, and () 2.1 nM thrombin, 0.05 µM fusion protein (in FpA units), 200 mM ChCl, or (
)
0.53 nM thrombin, 0.07 µM fusion protein (in FpA
units), 200 mM NaCl. Continuous lines were drawn according to with values for the kinetic parameters listed in Table 1. The value of [F]
in is 0.062 ± 0.003 µM in NaCl and 0.041
± 0.004 µM in ChCl.
Important details on the kinetic mechanism of recognition of
fibrinogen and the fusion protein by the slow and fast forms of
thrombin are revealed by the individual rate constants for substrate
binding (k), dissociation (k
), acylation (k
),
and deacylation (k
). The constants can be
estimated from the data in Table 1and Fig. 1and Fig. 2. The values for fibrinogen can be derived from data
published previously(2, 3) . The results are given in Table 2. The origin of preferential binding of fibrinogen to the
fast form is mostly due to the faster dissociation rate constant of
this substrate from the slow form. In the case of the fusion protein,
dissociation is much faster from the fast form leading to tighter
binding to the slow form. Hence, the B
and
chains
destabilize binding to the slow form by enhancing the dissociation rate
constant. In the absence of these chains, dissociation is faster from
the fast form. The rate constants for fibrinogen binding to the slow
and fast forms are significantly slower than that found for chromogenic
substrates (1) and hirudin(4) , but are nearly 1 order
of magnitude faster than those of the fusion protein. Acylation of
fibrinogen occurs with comparable rate constants in the slow and fast
forms, while acylation of the fusion protein is 20 times faster in the
fast form. Hence, a significant conversion of the enzyme from the slow
to the fast form may be expected before reaching the transition state
for the acylation step in the case of fibrinogen, but not for the
fusion protein.
The preferential interaction with either thrombin
form is quantified by the coupling free energy,
G
. This terms measures the difference in
binding affinity between the fast and slow forms and provides a measure
of the coupling between the binding process and the allosteric slow
fast transition(4) .
If binding of a ligand
involves residues that contribute equally in the slow and fast forms,
then
G
= 0. If binding involves
residues that contribute differently in the slow and fast forms, then
G
0. Hence, the coupling free energy is
a very useful parameter to relate structural components to binding
energetics of the slow
fast transition. The value of coupling free
energy for fibrinogen is -1.7 ± 0.1 kcal/mol(3) .
Fibrinogen binds to the fast form with higher affinity and with a free
energy difference of -1.7 ± 0.1 kcal/mol. The value of
coupling free energy for the fusion protein is 0.7 ± 0.1
kcal/mol (see Table 1). Unlike fibrinogen, the fusion protein
binds to the slow form with higher affinity and the value of
G
is positive. The difference in coupling
free energy between fibrinogen and the fusion protein is 2.4 ±
0.1 kcal/mol. This quantity must be accounted for by the structural
domains of fibrinogen that interact with thrombin and are not present
in the fusion protein. A quantity analogous to
G
can be defined in the transition state as a measure of the change
in the specificity constant between the slow and fast forms (see Table 1). If binding of a substrate in the transition state
involves residues that contribute equally in the slow and fast forms,
then
G
= 0, otherwise
G
0. Since FpA is cleaved from
fibrinogen or the fusion protein with higher specificity if thrombin is
in the fast form, the value of
G
is negative
for both substrates. The similarity of coupling free energies suggests
that the structural components responsible for the stabilization of the
transition state in the fast form relative to the slow form are common
to fibrinogen and the fusion protein.
The fusion protein is the first substrate to be found to bind
with higher affinity to the slow form of thrombin. All substrates,
effectors, and inhibitors studied previously have been reported to bind
to the fast form with higher affinity. Such is the case of fibrinogen (3) , hirudin and its C-terminal fragment(4) ,
thrombomodulin(2) , and a variety of synthetic substrates and
inhibitors(1, 4) . Although the fusion protein binds
preferentially to the slow form at equilibrium, FpA is cleaved by the
fast form with higher specificity. This result bears directly on
thrombin-fibrinogen interaction. Fibrinogen binds preferentially to the
fast form (3) and is cleaved by the fast form with higher
specificity(2) . This observation suggests that the structural
components responsible for preferential binding to the fast form at
equilibrium are also involved in the preferential stabilization of the
transition state in this form. The results for the fusion protein,
however, demonstrate that different structural domains of fibrinogen
may control molecular recognition at equilibrium and at the transition
state. In addition, the results reinforce the notion that the
N-terminal domains of the B and
chains of fibrinogen play a
key role in molecular recognition of the natural substrate. These
chains presumably destabilize binding to the slow form. The binding
affinities of the fusion protein and fibrinogen are the same when
thrombin is in the fast form, but differ by a factor of 40 when
thrombin is in the slow form. If the sequence 1-50 of the A
chain present in the fusion protein makes contacts with thrombin as the
analogous sequence in the fibrinogen molecule, then this sequence must
bind to the slow form with higher affinity. Preferential binding to the
fast form, as seen in the case of fibrinogen, would result from
contacts made by the N-terminal domains of the B
and
chains
with thrombin. These contacts would provide 2.4 kcal/mol toward the
stabilization of the fast form. Alternatively, the B
and
chains may constrain the A
chain of fibrinogen such that the
contacts with thrombin are not analogous to those in the fusion
protein.
The lack of structural information on the
thrombin-fibrinogen complex makes assignment of residues involved in
recognition very difficult. A structure of thrombin covalently bound to
the fragment 1-16 of the fibrinogen A chain has documented
the expected contacts with primary recognition subsites in the
catalytic pocket, along with hydrophobic contacts with the aryl binding
site of thrombin and Gly-216(8) . A seemingly important salt
bridge between Arg-173 of thrombin and Glu-11 of fibrinogen has also
been reported in the crystal structure. This assignment, however, may
be questionable since the side chain of Arg-173 is disordered. The E11A
replacement in the fusion protein is without
effect(11, 12) , but the E11G mutation in fibrinogen
Mitaka II impairs thrombin binding(20) . The mutation R173E of
thrombin decreases the release of FpA and FpB from fibrinogen by a
factor of 3 and 2, respectively,
which is an effect too
small to be assigned to the lack of an important salt bridge
interaction. The fibrinogen binding loop provides a significant portion
of the binding free energy (19) , but contributes very little
to the value of
G
(4) .
This is because residues of the fibrinogen binding loop
contribute almost equally to binding in the slow and fast forms.
Mutagenesis studies of thrombin have indicated that Lys-60f, located
strategically in between the catalytic pocket and the fibrinogen
binding loop, may play a significant role in the recognition of
fibrinogen since the mutant K60fE has a reduced clotting
activity(9) . This residue is also important in the binding of
hirudin (5) . The portion of the NSDK of fibrinogen interacting
with the region of thrombin surrounding Lys-60f may hold the key to
unravel the contribution of the B
and
chains to the
destabilization of binding to the slow form.
The significant
differences seen at equilibrium between fibrinogen and the fusion
protein disappear in the transition state. Preferential binding to the
fast form in the transition state must originate from contacts made
with residues within the catalytic moiety and the recognition subsite
Asp-189 of thrombin. All the molecular components responsible for the
preferential interaction are contained in the A1-50, with no
apparent contribution from the N-terminal domains of the B
and
chains. The three chains in the NSDK of fibrinogen appear to have
different roles in the recognition mechanism. The A
chain contains
the structures required for recognition by the fast form in the
transition state, while the B
and
chains contain the
structures that destabilize binding to the slow form at equilibrium. In
this model the B
and
chains act as intramolecular allosteric
effectors of the A
chain. They destabilize binding to the slow
form, inducing the slow
fast transition, which in turn
facilitates binding of the A
chain in the transition state and the
release of FpA by the fast form.
The observation that the fusion
protein binds to the slow form with higher affinity is intriguing and
represents a significant step toward our understanding of the molecular
basis of the slow fast transition of thrombin and the structural
epitopes important for fibrinogen recognition. An important implication
of our results is that synthetic inhibitors tailored after the
1-50 segment of the A
chain of fibrinogen may work as
effective stabilizers of the anticoagulant slow form of thrombin. Such
inhibitors would also be effective in enhancing the enzyme specificity
toward protein C and may reveal key details involved in the interaction
of thrombin with thrombomodulin.