(Received for publication, July 19, 1995)
From the
Thrombin is an allosteric serine protease existing in two forms,
slow and fast, targeted toward anticoagulant and procoagulant
activities. The slow fast transition is induced by Na
binding to a site contained within a cylindrical cavity formed by
three antiparallel
-strands of the B-chain
(Met
-Tyr
,
Lys
-Tyr
, and
Val
-Gly
) diagonally crossed by the
Glu
-Glu
strand. The site is shaped
further by the loop connecting the last two
-strands and is
located more than 15 Å away from the catalytic triad. The cavity
traverses through thrombin from the active site to the opposite surface
and contains Asp
of the primary specificity site near its
midpoint. The bound Na
is coordinated octahedrally by
the carbonyl oxygen atoms of Tyr
, Arg
,
and Lys
, and by three highly conserved water molecules in
the D-Phe-Pro-Arg chloromethylketone thrombin. The sequence in
the Na
binding loop is highly conserved in thrombin
from 11 different species and is homologous to that found in other
serine proteases involved in blood coagulation. Mutation of two Asp
residues flanking Arg
(D221A/D222K) almost abolishes the
allosteric properties of thrombin and shows that the Na
binding loop is also involved in direct recognition of protein C
and antithrombin.
Thrombin is a serine protease playing a key role in
hemostasis(1, 2) . The enzyme is allosteric and exists
in two forms, slow and fast (3) . The slow form plays an
anticoagulant role because it activates protein C (PC) ()more specifically, generating a potent inhibitor of blood
coagulation; the fast form has a procoagulant role because it cleaves
fibrinogen with higher specificity, promoting the formation of a blood
clot(4) . The slow
fast transition is triggered by the
specific binding of a Na
ion(3, 5) .
Other monovalent cations are less effective and bind with much lower
affinity. Under physiological conditions of Na
concentration, pH, and temperature, the slow and fast forms are
almost equally populated (3) and help maintain the balance
between procoagulant and anticoagulant activities that is crucial for
effective hemostasis. Furthermore, the slow
fast equilibrium in vivo is under conditions where allosteric regulation is
most efficient and can be exploited in much needed therapeutic
treatments of thrombotic and bleeding
disorders(2, 6) .
Specific monovalent cation
effects in proteins appear to be widespread and have been documented
for a long time(7, 8) . In the realm of blood
coagulation, these effects were first reported for factor Xa (9) and then for thrombin (10) and activated
PC(11) . Monovalent cations can act as allosteric effectors,
influencing the overall structure of an enzyme, or as cofactors that
interact directly with substrates. Detailed structural information on
the molecular basis underlying these effects has recently emerged for
enzymes with specific requirement for
K(12, 13, 14) . In the case
of Na
specific effects, as seen for thrombin,
identification of the bound Na
ion in the crystal
structure is complicated by the comparable electron density of a water
molecule and this monovalent cation. For this reason, the exact
position of the Na
binding site of thrombin has
remained elusive, notwithstanding a number of crystallographic
structures of the enzyme complexed with various ligands and inhibitors
that have been reported in the presence of NaCl (15) . Given
the crucial physiological effect of Na
on
thrombin(4) , identification of the Na
binding
site should reveal key aspects of structure-function relationships for
this important enzyme.
Crystals of thrombin inhibited at the active site with PPACK
were grown from sodium phosphate and PEG 8000 in the presence of NaCl
as described previously(16, 17) . The mother liquor
solution containing PPACK-thrombin crystals was exchanged with
K ion solution of identical composition replacing
Na
with K
ions (0.1 M potassium phosphate buffer, pH 7.3, 32% PEG 8000, 0.187 M KCl), by adding a 2-µl aliquot to a 10-µl hanging drop and
withdrawing 2 µl from the other side of the drop. This was done
four times over 2 days. To complete the
Na
-K
exchange, the crystals were
transferred directly into 1 ml of the potassium solution. The
Na
-Rb
exchange was accomplished using
a capillary soaking method (18) with the potassium phosphate
solution containing RbCl instead of KCl. The crystal was placed in a
capillary tube used to mount crystals for diffraction purposes. Then 20
µl of the potassium phosphate solution was placed over the crystal,
upon which was layered 20 µl of a similar solution that contained
1.88 M RbCl, followed by another 20-µl layer of the
potassium phosphate solution. Diffusion was allowed to take place for
90 h before removing the solution and mounting the crystal in a usual
manner. The final concentration of the Rb
ion in the
capillary is estimated to be 0.625 M. The x-ray diffraction
pattern of the Na
ion exchanged crystal was measured
with an R-AXIS imaging plate detector and used to determine and refine
the Rb
ion PPACK-thrombin structure (R = 0.19 without solvent structure).
The double mutation D221A/D222K was obtained by amplification of the BglII/EcoRI sequence of pNUT-hII (19) using appropriate primers for the polymerase chain reaction. The double mutant was expressed in baby hamster kidney cells (BHK21) as prethrombin-1 using the pNUT vector constructed with the bovine factor V signal peptide and a 12-amino acid HPC4 epitope(20) . The prethrombin-1 mutant preceded by a 12-amino acid HPC4 epitope was purified on an HPC4 Affi-Gel 10 column. The zymogen form was activated, and the thrombin form was purified and tested for activity as described (21) .
Human -thrombin was purified and tested for
activity as described (3, 21) . Recombinant
desulfatohirudin variant 1 (Revasc(TM)) was obtained from Ciba-Geigy
Pharmaceuticals (Horsham, United Kingdom). The concentration of hirudin
was determined by amino acid analysis. Human fibrinogen, PC,
antithrombin (AT), and rabbit lung thrombomodulin (TM) were from
Hematologic Technologies (Essex Junction, VT). Heparin was from Sigma.
The release of fibrinopeptides A and B (FpA and FpB), binding of
hirudin, and cleavage of PC by the slow and fast forms of wild-type
thrombin and the ARK mutant were studied and analyzed as
described(4, 5) . Inhibition of thrombin activity by
AT was studied using the procedure of Olson (22) and was
analyzed using KINSIM and FITSIM (23, 24) to obtain
the apparent second-order rate constant for formation of the
thrombin-antithrombin complex (k
in Table 2). Experimental conditions were: 5 mM Tris, 0.1%
PEG, pH 8.0, 25 °C, and 0.2 M NaCl for the fast form, or
0.2 M choline (Ch
) chloride for the slow
form. The concentration of TM was 10 nM, and heparin was used
at concentrations of 1 unit/ml.
The Na binding site of thrombin has been
unequivocally identified here by exchanging Na
with
Rb
ions in crystals of PPACK-thrombin. The site
displays octahedral coordination involving Tyr
,
Arg
, Lys
, and three highly occupied water
molecules (Fig. 1). It is located more than 15 Å away from
the catalytic triad of thrombin and lies near Asp
of the
primary specificity site within a cylindrical cavity (26) formed by three antiparallel
-strands of the B-chain
(Met
-Tyr
,
Lys
-Tyr
, and
Val
-Gly
) diagonally crossed by
Glu
-Glu
and shaped by the loop
connecting the last two
-strands (Fig. 2). The cavity
traverses through thrombin from the active site to the opposite surface
with Asp
near its midpoint. Very significantly, the
channel is filled with about 20 water molecules, most of which are
conserved in different thrombin structures. Furthermore, they are
linked among each other by a complicated hydrogen bonding network,
which also links up to the peptide chain of the protein and appears to
stabilize the cavity. A Na
ion and a water molecule
are practically equivalent in an electron density map. Examination of
different crystal structures of thrombin reveals an octahedrally
coordinated water molecule at the Na
site in over 20
of them, most with exceptionally small contacts to the central water
molecule (2.2-2.4 Å).
Figure 1:
The Na binding site of
PPACK-thrombin. Electron density of PPACK-thrombin at 2.5 Å
resolution in blue (dotted) contoured at 1.20
(A. Tulinsky, unpublished results); Rb
ion difference
electron density at same resolution in red at 4
,
calculated using Fourier coefficients of the form:
(
F
-
F
)exp(-i
),
where
F
is the observed
structure amplitude of Rb
ion soaked PPACK-thrombin,
F
that of PPACK-thrombin, and
the phase angle of PPACK-thrombin without water
molecules included; distal water is designated WAT in correct
sixth octahedral position, but about 3.4 Å from Na
position. The average Na
-O distance is 2.9
Å. Numbering is based on topological similarities with
chymotrypsin (25) .
Figure 2:
MOLSCRIPT plot (27) of the B-chain
of human -thrombin, derived from the coordinates of the
thrombin-hirudin complex(26) , with the inhibitor removed. The
residues of the catalytic triad are shown at the interface between the
two six-stranded
-barrels. The N terminus of the chain is at the bottom, and the C terminus is at the end of the
-helix at
the top. Important structural domains of the enzyme are noted
as follows: 1, Na
binding loop; 2,
autolysis loop; 3, fibrinogen binding loop; 4,
heparin binding site. The Na
ion is shown as a redball in the loop connecting the last two
-strands of
the B-chain terminating into the heparin binding site. Binding of
Na
to this loop induces the slow
fast
transition and controls allosterically both the activity (3) and specificity (4) of the enzyme. The highly
conserved fragment Cys
-Gly
of this
loop (see Table 1) is in yellow.
The sequence
Cys-Gly
involving the Na
binding loop and part of the last
-strand of the B-chain is
highly conserved in thrombin from 11 different species (Table 1),
thereby supporting the fundamental role of Na
binding
in thrombin function. While Asp
is the only variable
residue, Arg
is replaced by a Lys in the
hagfish(28) . Comparison with analogous sequences in other
serine proteases (Table 1) indicates a high degree of homology,
especially with factors IX and X and to a lesser extent with plasmin,
PC, and factor VII. Homology is weaker between thrombin and pancreatic
serine proteases like trypsin, chymotrypsin, and elastase. Hence, the
architecture of the Na
binding site of thrombin and
the allosteric regulation based on the slow
fast equilibrium may
be paradigmatic of a molecular strategy shared by other members of the
blood coagulation cascade, like factors VII, IX, X, and PC, but not by
pancreatic serine proteases. Proteases active in the blood might have
evolved to exploit Na
-dependent allosteric transitions
to accomplish their multifunctional roles, taking advantage of the
availability of this monovalent cation in the extracellular
environment.
The allosteric nature of thrombin makes it possible to
control its function by interfering with Na binding. A
mutation involving the two Asp residues flanking Arg
(Table 1) in the Na
binding loop was made
to mimic the ARK sequence found in factor Xa, which has a reduced
Na
affinity (K
=
110 mM at 25 °C) compared to thrombin (K
= 22 mM at 25 °C,
see (3) ). The difference may be due to a reduction of the
negative electrostatic potential at the Na
site due to
the presence of Ala
and Lys
, as opposed to
Asp
and Asp
in thrombin. In the crystal
structure(25, 29) , Asp
and Asp
are ion-paired to Arg
and make no contacts with
synthetic substrates(25) , hirudin(26) , and most
likely fibrinogen(30) . This double mutant D221A/D222K (ARK)
has lost the ability to bind Na
selectively (Fig. 3) and cleaves a synthetic substrate with nearly the same
specificity in the presence of Li
,
Na
, K
, or the bulky cation
Ch
. The ARK mutant interacts with fibrinogen and
hirudin with increased specificity in the slow form and reduced
specificity in the fast form (Table 2). The perturbed energetics
originate from an indirect effect, because the mutation impairs
Na
binding and stabilizes a conformation that is
functionally and structurally intermediate between the slow and fast
forms of the wild type. Circular dichroic spectra (Fig. 4)
support this conclusion. The spectra for the slow and fast forms are
significantly different for the wild type, but not for the ARK mutant
that behaves as an intermediate between the slow and fast forms of the
wild type.
Figure 3:
Value of the specificity constant for the
hydrolysis of S2238
(H-D-Phe-pipecolyl-Arg-p-nitroanilide) by wild-type
thrombin and the ARK mutant, as indicated. Experimental conditions are:
5 mM Tris, 0.1% PEG, pH 8.0, 25 °C. The salt concentration
was kept constant at 200 mM with the chloride salts indicated
on the abscissa. The large difference seen between
Na (fast form) and Ch
(slow form) for
the wild type, is not observed in the ARK
mutant.
Figure 4:
Circular dichroic spectra of wild-type
thrombin (,
) and the ARK mutant (
,
) in the
slow (
,
) or fast (
,
) forms. Spectra were
recorded on a Jasco J600A spectropolarimeter under solution conditions
of: 5 mM Tris, 0.1% PEG, pH 8.0, 25 °C, and 0.2 M NaCl for the fast form, or 0.2 M ChCl for the slow form.
The concentration of the wild type and the ARK mutant was 1.5-2.0
µM. The pathlength was 1 cm. The slow and fast forms of
the wild-type enzyme show significant differences in the regions around
204 and 218 nm, that are not seen for the ARK
mutant.
When PC interacts with the ARK mutant, the functional
differences between the slow and fast forms are restored (Table 2). Furthermore, the mutation has the noteworthy effect of
slightly enhancing the catalytic properties of the slow form compared
to the wild type. This result is also observed in the presence of TM.
Protein C presumably makes favorable contacts with Lys of
ARK, and this effect overwhelms the indirect effect of the mutation on
the slow
fast equilibrium. In the absence of a direct effect,
activation of PC by the ARK mutant would be decreased in the slow form
and increased in the fast form compared to the wild type, consistent
with the findings on S2238, fibrinogen, and hirudin. Binding of AT in
the absence of heparin is perturbed exclusively as a result of the
effect of the mutation on the slow
fast equilibrium (Table 2). In the presence of heparin, however, binding of AT is
decreased by more than 10 times. The contacts made by AT with thrombin
are likely different when heparin is bound, as a result of
heparin-induced conformational changes. Favorable contacts made by AT
with Asp
and Asp
of the wild type in the
presence of heparin may explain the loss of binding affinity in the ARK
mutant. Hence, the Na
binding loop of thrombin plays
an important role in the direct recognition of PC and AT in the
presence of heparin.
The architecture of the Na binding site of thrombin reveals important details on the
molecular trigger for the slow
fast transition that contributes
to the conversion of the enzyme from an anticoagulant to a procoagulant
factor(4) . The slow form has increased specificity for
substrates like PC carrying an acidic residue at P3. Even in the
absence of a structure of the slow form, the side chain of
Arg
, which is ion-paired to Glu
in the
fast form(25, 29) , is oriented differently and makes
a water mediated contact with the acidic side chain of the residue at
P3 of substrates like the thrombin platelet receptor peptide (31, 32) and probably PC. The enhanced anticoagulant
activity of the ARK mutant may well be a result of the D222K
substitution, with the side chain of Lys
mimicking or
enhancing synergistically the role of Arg
. Further
mutagenesis analysis of the Na
binding loop will
reveal the exact origin of Na
specificity and the
trigger for the allosteric slow
fast transition.