From the Departments of Immunology and Vascular Biology, The Scripps Research Institute, La Jolla, California
Received for publication, May 31, 2000, and in revised form, November 13, 2000
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ABSTRACT |
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Serine protease activation is typically
controlled by proteolytic cleavage of the scissile bond, resulting in
spontaneous formation of the activating
Ile16-Asp194 salt bridge. The initiating
coagulation protease factor VIIa (VIIa) differs by remaining in a
zymogen-like conformation that confers the control of catalytic
activity to the obligatory cofactor and receptor tissue factor (TF).
This study demonstrates that the unusual hydrophobic Met156
residue contributes to the propensity of the VIIa protease domain to
remain in a zymogen-like conformation. Mutation of Met156
to Gln, which is found in the same position of the highly homologous factor IX, had no influence on the amidolytic and proteolytic activity
of TF-bound VIIa. Furthermore, the mutation did not appreciably stabilize the labile Ile16-Asp194 salt bridge
in the absence of cofactor. VIIaGln156 had increased
affinity for TF, consistent with a long range conformational effect
that stabilized the cofactor binding site in the VIIa protease domain.
Notably, in the absence of cofactor, amidolytic and proteolytic function of VIIaGln156 were enhanced 3- and 9-fold,
respectively, compared with wild-type VIIa. The mutation thus
selectively influenced the catalytic activity of free VIIa, identifying
the Met156 residue position as a determinant for the
zymogen-like properties of free VIIa.
Allosteric regulation of catalytic activity of the serine protease
factor VIIa (VIIa)1 is
utilized as a mechanism to control the initiation of the coagulation pathways (1). VIIa circulates in the blood plasma as zymogen as well as
the cleaved two-chain enzyme (2), but proteolytic function only ensues
upon binding to its cell surface receptor and catalytic cofactor tissue
factor (TF). TF has two distinct effects that regulate proteolysis by
VIIa. First, TF provides affinity for macromolecular substrate by
contributing to an extended exosite together with the bound VIIa
The low catalytic activity of free VIIa results from a zymogen-like
conformation of the enzyme. Upon zymogen cleavage, serine protease
domains typically undergo a conformational ordering of loop segments,
termed the activation domain (7), resulting in the formation of an
activating canonical salt bridge of Asp194 with the newly
generated amino-terminal Ile16. In the absence of cofactor,
VIIa shows an increased susceptibility of the amino terminus to
chemical modification (8), indicating exposure of Ile16
that can result from an alternative conformation or increased flexibility and disorder in the activation pocket of free VIIa. The
structural determinants for the propensity of VIIa to stay in a
zymogen-like conformation have not been investigated. Available structures of free and TF-bound VIIa (9-13) did not provide
mechanistic insight, because in each case the active site of VIIa was
occupied with inhibitors that are known to stabilize the
Ile16-Asp194 salt bridge (14) and restrict
conformational flexibility in the VIIa protease domain (15). Mutational
studies also failed to elucidate the basis for the labile enzyme
conformation, because the approaches taken mainly probed the active
enzyme in the TF·VIIa complex (16).
This study investigates the role of residue Met156 in
maintaining the zymogen-like conformation of VIIa. This residue is
located within the activation pocket, covering Ile16 upon
amino-terminal insertion (9). The conformation of the 156 side chain
can influence the catalytic activity of serine protease domains. In the
case of tissue plasminogen activator (tPA) (17, 18) and vampire bat
plasminogen activator (19), Lys156 can substitute for
Ile16 to form an activating salt bridge with
Asp194, resulting in efficient catalysis in the absence of
zymogen cleavage. However, Lys at this position is found in a large
number of serine proteases without conferring catalytic activity in the
zymogen precursors, indicating that multiple interactions within the
activation pocket determine the activation state of serine protease
domains. Although Lys or other hydrophilic side chains are predominant in serine proteases that undergo spontaneous ordering of the activation pocket upon zymogen cleavage, VIIa has a Met residue in the 156 position. We hypothesized that the side-chain property of
Met156 is one of the determinants that interfere with the
acquisition of full catalytic activity of VIIa upon zymogen cleavage.
This study demonstrates that replacement of Met156 with
Gln, the side chain found in factor IX, had little effect on the
activity of TF-bound VIIa. However, free VIIaGln156 had
enhanced catalytic function toward macromolecular and small peptidyl
substrates. These experiments thus identify the first residue side
chain that is one of the determinants for the zymogen-like conformation
of the VIIa protease domain.
Proteins--
Wild-type and mutant VIIa were expressed in
Chinese hamster ovary cells grown in suspension culture in serum-free
medium supplemented with vitamin K3. Mutant and wild-type
recombinant VII was immunoaffinity purified with a
calcium-dependent monoclonal antibody, autoactivated to
VIIa at 4 °C, followed by ion exchange chromatography on MonoQ, as
described previously (20). Factor X was purified from plasma with a
final monoclonal antibody affinity step to reduce contamination by
plasma VII (16). Full-length recombinant human TF, produced from insect
cells, was reconstituted into 30% phosphatidylserine/70% phosphatidylcholine (PCPS), as described previously (21). The soluble
extracellular domain of TF (TF-(1-218)) was expressed in
Escherichia coli and refolded from inclusion bodies (22). The Kunitz-type inhibitor 5L15 selected for VIIa specificity by phage
display (23) was kindly provided by Dr. George Vlasuk (Corvas
International, San Diego, CA).
Functional Assays--
Kinetic parameters for factor X
activation were determined at fixed concentration (200 pM)
of TF reconstituted in PCPS (TF/PCPS) and excess wild-type or mutant
VIIa (1 nM) in Hepes buffer saline (HBS, 10 mM
Hepes, 150 mM NaCl, pH 7.4), 5 mM
CaCl2, 0.2% bovine serum albumin. After a brief incubation
at 37 °C to allow TF·VIIa complex formation, factor X (8 nM to 1 µM) was added, and factor Xa was
quantified with the chromogenic substrate Spectrozyme FXa (American
Diagnostica, Greenwich, CT) in samples quenched with 100 mM
EDTA. Initial rates of factor Xa generation, based on calibration curves made with purified Xa, were fitted to the Michaelis-Menten equation using least squares regression analysis. For the determination of the proteolytic activity of free VIIa, 250 nM VIIa was
incubated with 1 µM factor X at 37 °C in the presence
or absence of 100 µM PCPS, followed by determination of
factor Xa generation by chromogenic assay. Kinetic parameters for
chromogenic substrate hydrolysis (Chromozym tPA, Roche Molecular
Biochemicals) were determined at fixed enzyme concentration (60 nM in the absence of TF or 30 nM in the
presence of 120 nM TF-(1-218)) with varying concentrations
of substrate (0.02-2 mM) in Tris-buffered saline (TBS, 20 mM Tris, 150 mM NaCl, pH 8.0), 5 mM
CaCl2, and 0.2% bovine serum albumin at ambient
temperature. Initial rate data were fitted to the Michaelis-Menten
equation using least squares regression analysis.
VII mutants were expressed in transient transfection experiments, and
expression levels were determined by immunoassay. Proteolytic activities of mutants were analyzed in a functional assay at 37 °C
in HBS, 5 mM CaCl2, and 0.2% bovine serum
albumin. A fixed concentration of TF/PCPS (5 pM) was
saturated with increasing concentrations of mutant or wild-type VIIa,
followed by addition of 50 nM factor X and determination of
factor Xa generation by amidolytic assay. The maximum rate of Xa
generation as a measure of the proteolytic function of the mutant VIIa
in complex with TF was calculated based on calibration curves made with
purified Xa.
Carbamylation of Ile16 in VIIa--
Chemical
modification of the amino-terminal Ile16 of wild-type or
mutant VIIa was performed at ambient temperature according to Higashi
et al. (8). Free VIIa (4 µM) or VIIa (1 µM) in complex with TF-(1-218) (4 µM) was
reacted with 0.2 M KCNO in HBS, 5 mM CaCl2 for various times. Samples were withdrawn and diluted
25- or 60-fold for free or TF-bound VIIa, respectively, and the
residual amidolytic activity was determined with 0.7 mM
Chromozym tPA. Rates of inactivation were calculated from a plot of the
residual activity (in percent of the initial activity)
versus incubation time.
Inhibition of VIIa by Antithrombin III/Heparin--
To analyze
the time dependence of inhibition of free VIIa (100 nM) or
VIIa (50 nM) in complex with TF-(1-218) (250 nM) by antithrombin III/heparin, wild-type and mutant VIIa
were reacted with 0.5 µM antithrombin III (Hematologic
Technologies) in the presence of 5 units/ml unfractionated heparin
(Elkins-Sinn), at 37 °C in TBS, 5 mM CaCl2,
and 0.2% bovine serum albumin. After defined times (2-60 min) samples
were diluted into the chromogenic substrate Chromozym tPA (1.5 mM), and the residual amidolytic activity was immediately
determined in a kinetic microplate reader.
Surface Plasmon Resonance Analysis--
Binding constants for
wild-type and mutant VIIa were analyzed using a BIAcore 2000 instrument
(Amersham Pharmacia Biotech Biosensor). A noninhibitory anti-TF
antibody (TF9-10H10) was directly immobilized by amino-coupling to an
activated dextran matrix for capture of full-length recombinant TF, as
described previously (24). TF was injected to saturate the antibody,
and association data were collected from injections of five
concentrations (25 nM to 1 µM) of VIIa in
HBS, 5 mM CaCl2, 0.005% surfactant P20, and 3 mM CHAPS. Binding kinetics in the presence of the
Kunitz-type inhibitor 5L15 was determined by premixing VIIa with 10 µM 5L15. Dissociation data were collected for 250 s
after return to buffer flow, and the chip surface was regenerated with
pulses of 0.1 M EDTA and 4 M MgCl2.
Dissociation of TF from the antibody could not be detected over a 6-h
period under the standard buffer conditions, and the measured
dissociation upon injection of VIIa thus reflects dissociation of the
TF·VIIa complex, rather than the release of TF from the immobilized
antibody. Association and dissociation constants
(kon and koff) were
determined using the software provided by the manufacturer.
Mutational Analysis of Position 156 in VIIa--
As a first step
to define the role of the 156 residue position in catalytic activity of
VIIa, we characterized the proteolytic function of various side chain
replacements in transient transfection experiments. Maximum rates of
factor Xa generation were determined by saturating a fixed
concentration of phospholipid-reconstituted TF with the goal of
defining the permissive mutations that do not interfere with amidolytic
and proteolytic function of TF-bound VIIa. Changing the 156 position to
Lys, as found in the highly homologous factor X, resulted in diminished
function. In contrast, a Gln side chain, as found in factor IX of all
species and in the majority of chymotrypsin-like serine proteases,
allowed for normal or slightly enhanced proteolytic function, as
compared with wild-type VIIa (Table I).
Negatively charged residues, such as Glu and Asp, were consistently
less well tolerated than their respective amide counterparts. In the
case of Asn, ~60% of wild-type activity was retained. Only
Gln at 156 produced a fully functional VII molecule, whereas a number
of smaller side chain replacements, such as Ala, Val, or Ser, showed
significantly reduced proteolytic function. Thus, replacement of the
hydrophobic Met156 side chain by the more hydrophilic side
chain Gln is the only permissive mutation that allows for normal
proteolytic function of the TF·VIIa complex.
Effect of the Met156 to Gln Mutation on TF
Binding--
VIIGln156 was stably expressed,
purified, and autoactivated at 4 °C. Autoactivation of the mutant
appeared to proceed somewhat faster than wild-type VII, but the
purified protein showed electrophoretic mobility and >95% conversion
to the two-chain enzyme indistinguishable from wild-type VIIa. Binding
of mutant and wild-type VIIa to antibody-captured full-length TF was
analyzed by surface plasmon resonance measurements. Although the
mutation had only a minor <2-fold effect on the association rate,
VIIaGln156 dissociated from TF with a significantly lower
rate (Table II), indicating that the
cofactor binding site is in a conformation that allows for tighter
binding to TF. Active site occupancy of wild-type VIIa by the
Kunitz-type inhibitor 5L15 is known to tighten the binding with TF by
slowing the dissociation rate (24). Although 5L15 increased affinity
for wild-type VIIa 4-fold, only marginal changes were observed for
VIIaGln156. Amidolytic function of mutant or wild-type VIIa
were blocked >99% by the inhibitor under the experimental conditions,
excluding a loss of inhibitor binding to the mutant as the reason for a lack in the change of binding kinetics. Thus, the conformation of the
mutant's protease domain, independent of active site occupancy, appeared to be in a higher affinity state for TF.
Normal Cofactor-mediated Stabilization of the Amino-terminal
Insertion--
Although free VIIa displays a labile enzyme
conformation with the Enhanced Amidolytic Activity of VIIaGln156 in the
Absence of Cofactor--
The higher affinity binding of
VIIaGln156 to TF may reflect conformational changes in the
cofactor binding site that are associated with increased catalytic
function of the mutant in the absence of cofactor. To address this
issue, the catalytic activities of wild-type or mutant VIIa were
analyzed with small peptidyl substrates. The catalytic efficiency of
hydrolysis of Chromozym tPA by free VIIaGln156 was
increased 3-fold compared with wild-type VIIa (Table
III), indicating that the Gln side chain
stabilizes a more active conformation of the VIIa protease domain.
However, VIIaGln156 in complex with TF cleaved the
chromogenic substrate indistinguishable from wild-type VIIa. Thus, the
side-chain replacement selectively influenced the catalytic function of
the free enzyme.
Effect of the Met156 to Gln Mutation on Inhibition by
Antithrombin III--
To test whether inhibitor binding to the active
site of VIIaGln156 was also enhanced in comparison to
wild-type VIIa, inhibition of VIIa activity by a fixed concentration of
antithrombin III in the presence of 5 units/ml heparin was studied.
Consistent with previous studies (26, 27), the amidolytic activity of free VIIa was inefficiently inhibited over time by antithrombin III/heparin (Fig. 1).
VIIaGln156 was inhibited at a rate that was approximately
twice as fast as the rate of inhibition of wild-type VIIa (50%
inhibition in 31 ± 3 min for mutant versus 64 ± 12 min for wild-type VIIa, n = 3). Inhibition of
TF·VIIa complexes occurred with significantly faster rates for both
mutant and wild-type VIIa, reaching 50% inhibition at 4.5 ± 1.8 min for mutant versus 6.0 ± 1.9 min for wild-type VIIa
(n = 3). The somewhat more efficient inactivation of
VIIaGln156 in the presence of TF suggests improved
interaction of the serpin with the mutant. This may involve exosite
interactions, because mutation of the 156 position in tPA also
influenced the interaction with plasminogen activator inhibitor 1 (18).
Thus, the enhanced inhibition of free VIIaGln156
by antithrombin III cannot solely be attributed to a stabilized active
conformation of the mutant but may also involve direct inhibitor
binding to the newly introduced Gln side chain.
Increased Proteolytic Function of Free VIIa upon Replacement of
Met156 with Gln--
Factor X activation by free VIIa was
analyzed in the presence and absence of a negatively charged
phospholipid surface (PCPS). In both cases, activation of factor X by
VIIaGln156 was enhanced 9-fold compared with wild-type VIIa
(Table IV). However, only subtle changes
in the activation of factor X by VIIaGln156 in complex with
phospholipid-reconstituted TF were detected (Table IV), both in regard
to Km and kcat. These data
demonstrate that macromolecular substrate binding as well as scissile
bond cleavage are not affected by the mutation after complex formation with the catalytic cofactor and after acquisition of full catalytic activity. In free VIIa, however, the catalytic function toward the
macromolecular substrate is enhanced, consistent with the data for
small substrate hydrolysis that also demonstrated selectively increased
amidolytic activity of free VIIaGln156.
The zymogen-like conformation of VIIa is required to subject the
regulation of VIIa's catalytic activity under strict control of the
cell surface expression of the cofactor TF. The labile enzyme
conformation of VIIa allows for the presence of significant amounts of
the two-chain enzyme in the circulating blood and thus for the
initiation of the coagulation pathways, as soon as the cofactor is
exposed upon disruption of vascular integrity. This mechanism of
triggering an enzyme cascade differs from the activation of the
fibrinolytic system by tPA, which utilizes a catalytically active
zymogen for the initial proteolytic events. Whereas an activating salt
bridge of Lys156 with Ile16 has been defined as
the structural basis for the active zymogen conformation of tPA (17,
18), structural determinants for the labile enzyme conformation of VIIa
remained unclear from previous structural and mutational studies. Here,
we provide evidence that the hydrophobic Met156 residue
side chain within the activation pocket, in part, is responsible for
the zymogen-like conformation of the VIIa protease domain.
Replacement of Met156 by Gln, the side chain found in
factor IX, did not appreciably influence catalytic function of the
TF-bound enzyme toward small chromogenic substrates or the
macromolecular substrate factor X. Thus, the cofactor-stabilized,
active conformation of VIIa does not absolutely require a Met side
chain at this position. Backbone-superimposition of the structures of
porcine factor IXa (28) and VIIa in the TF·VIIa complex (9)
demonstrate that essentially the same space is occupied by the Met and
Gln side chains in the respective proteases, providing a structural
rational why the Gln replacement is permissive for function of the
TF-bound VIIa. However, free VIIaGln156 displayed increased
function compared with wild-type VIIa. First, the mutant had higher
affinity for TF and affinity was not appreciably influenced by active
site inhibitor binding that typically slows dissociation for wild-type
VIIa and for most of the previously characterized mutants (15). The
cofactor binding site of VIIaGln156 thus appears to be
altered, indicating a long range conformational change of the residue
replacement of Met156 by Gln in the activation pocket.
Second, VIIaGln156 had higher amidolytic and proteolytic
activity in the absence of cofactor. Amidolytic activity was enhanced
3-fold, although activation of factor X was increased 9-fold in the
presence or absence of phospholipid. The difference in enhancement may
result from an additional ordering of regions that influence
macromolecular substrate scissile bond cleavage, but not the hydrolysis
of p-nitroanilide chromogenic substrates, which bind
primarily to the S1-S3 subsites of the catalytic cleft.
Because stabilization of the amino-terminal
Ile16-Asp194 salt bridge is generally
considered to be the major determinant for the activation state of the
VIIa protease domain, it was surprising that chemical modification of
the amino-terminal Ile16 in free VIIaGln156
was little different from wild-type VIIa. However, we have found poor
correlation of cofactor-mediated catalytic enhancement and susceptibility of the amino terminus to chemical modification in a
number of other VIIa mutants (29). Most importantly, certain mutations
in the TF binding interface of the VIIa protease domain resulted in a
complete failure to show protection of the amino terminus upon cofactor
binding, but TF was still able to enhance amidolytic activity of these
VIIa mutants by >10-fold. Stabilization of the insertion of the amino
terminus, as measured by the susceptibility to chemical modification,
is thus not absolutely required for changes in catalytic function of
VIIa to occur.
The local environment at the 156 position in VIIa differs from
other homologous coagulation factors such as factor IXa. Although porcine factor IXa has a hydrophobic Ile residue in the 154 position along with a pair of charged residues at position 21 (Asn) and 156 (Gln), VIIa has a charged Glu154 and a pair of hydrophobic
residues, i.e. Val21 and Met156
(Fig. 2A). One or two water
molecules (not shown) are consistently resolved between the
Glu154 and Met156 side chain in structures of
the TF·VIIa complex (9, 10). Fig. 2B shows
Met156 replaced by Gln in one possible rotamer position
that orients the amide of the Gln side chain toward Glu154.
The low catalytic function of acidic side chain replacement mutants and
the high functional activity of amide counterparts (Table I) indirectly
supports the notion that such an orientation of the introduced
Gln156 is most compatible with optimal activity. The charge
complementarity of the Gln156 and Glu154 side
chains may stabilize the local conformation of this region in free
VIIaGln156, possibly involving the coordination of a water
molecule between the two side chains. The activating effect of the
Gln156 mutation may thus be mediated through an effect on
the Glu154 position rather than the adjacent amino-terminal
insertion.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-carboxyglutamic acid-rich (Gla) domain. This exosite is a binding
site for the factor X Gla-domain (3-5). Second, TF enhances catalytic
activity by allosteric effects on the VIIa protease domain. In the
absence of cofactor, VIIa has only very low catalytic activity toward
small peptidyl substrate mimetics and TF stimulates the amidolytic of
VIIa up to 100-fold (1). However, macromolecular substrate factor X scissile bond catalysis is enhanced >1000-fold (6), indicating that
cofactor-induced conformational changes may influence extended macromolecular substrate recognition regions in addition to the S1-S3
subsite2 that is probed by the small substrates.
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
Factor X activation by VIIa Met156 replacement mutants
Kinetics of binding to TF
4.
-amino group of Ile16 susceptible
to chemical modification, cofactor binding induces structural
rearrangements resulting in a protected amino terminus. Because
carbamylation of Ile16 in VIIa by reaction with KNCO
inactivates VIIa (8, 25), the decrease in the amidolytic activity can
be used to determine the rate of modification of the amino terminus as
measure for the stability of the Ile16-Asp194
salt bridge. Carbamylation of free enzyme showed a similar rate of
inactivation of VIIaGln156 versus wild-type VIIa
(6.3 ± 0.4 versus 7.5 ± 0.3% loss of initial activity/10 min), demonstrating that the residue replacement was not
sufficient to completely order the activation pocket and to stabilize
the Ile16-Asp194 salt bridge. In addition, the
rate of inactivation of the TF-bound mutant was also indistinguishable
from wild-type VIIa (1.1 ± 0.1 versus 1.4 ± 0.1% loss of initial activity/10 min). Thus, the Gln replacement for
Met156 does not appreciable influence the stability of the
Ile16-Asp194 salt bridge in free or TF-bound enzyme.
Chromogenic substrate hydrolysis
3.
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Fig. 1.
Inhibition of wild-type VIIa
(squares) or VIIaGln156
(triangles) by antithrombin III/heparin. Residual
amidolytic activity was determined after the indicated times of
incubation of 100 nM free VIIa (filled symbols)
or 50 nM VIIa in complex with 250 nM
TF-(1-218) (open symbols) with 0.5 µM
antithrombin III in the presence of 5 units/ml unfractionated heparin
at 37 °C in TBS, 5 mM CaCl2, and 0.2%
bovine serum albumin.
Proteolytic activity of VIIaGln156
3).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 2.
A, view of the activation pocket of VIIa
in complex with TF (9). The position of the amino terminus of
the protease domain is marked by NH2. Key residues
(positions 21, 154, and 156) that differ between VIIa and other serine
proteases, such as factor IXa, are shown in a surface representation
(at 80% of the actual size). The backbone is shown in a ribbon
representation, colored to highlight residues 16-20 (white)
and 142-153 (yellow), and the atoms of residues 21, 154, and 156 are color-coded: red, oxygen; blue,
nitrogen; green, carbon; yellow, sulfur.
B, replacement of Met156 by one possible rotamer
position of Gln in the structure of VIIa is shown in relation to
residues Glu154 and Val21.
In wild-type VIIa, Ala mutation of Glu154 reduces amidolytic function 3-fold and proteolytic function 6- to 10-fold, displaying some divergence of the mutational effect on amidolytic versus proteolytic function (30). The free Gln156 mutant showed functional enhancement of similar magnitude in both assays, arguing in favor of a function linkage between Glu154 and Gln156 in VIIaGln156. Glu154 together with the adjacent Leu153 side chain are within a "hot spot" that is known to allosterically regulate VIIa's catalytic activity. First, Glu154 is located within the epitope of an inhibitory monoclonal antibody and crucial for the inhibitory allosteric switch induced by this exosite inhibitor (30). Second, a small peptide inhibitor of VIIa binds to an epitope that includes Leu153 (13). This exosite inhibitor influences catalytic function by a major reorientation of the 142-154 "autolysis" loop that also contains specific residues involved in extended macromolecular substrate recognition (20). The 142-154 loop is considered part of the activation domain, and stabilization and ordering of this loop is essential for the zymogen to enzyme transition. Structural studies on prothrombin derivatives (31) further suggest that the 142-154 loop can behave as a partially autonomous region of the activation domain. The enhanced function of VIIaGln156 in the absence of a stabilization of the amino-terminal insertion is thus consistent with a conformational effect on the 142-154 loop through the proximity of Glu154 with Gln156.
This study identifies the first residue side chain that contributes to
the zymogen-like state of free VIIa. Although the single side chain
replacement was insufficient to confer complete spontaneous ordering
and full catalytic activity of the VIIa protease domain, the proposed
stabilization of the 142-154 loop would achieve one of the necessary
conformation orderings in the activation domain. The significantly
improved function of free VIIaGln156 argues that a limited
number of side chains may be responsible for retaining VIIa in the
zymogen-like conformation. Whether changing the local environment of
the activation pocket is sufficient to achieve a fully active VIIa or
whether changes in the unique loop segments that contact TF are also
required remain important questions for further studies.
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ACKNOWLEDGEMENTS |
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We thank Jennifer Royce, Cindi Biazak, and Dave Revak for the assistance in the production and purification of recombinant proteins.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant HL48752 and performed during the tenure of an Established Investigator of the American Heart Association (to W. 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 Immunology,
C204, The Scripps Research Institute, 10550 North Torrey Pines Rd., La
Jolla, CA 92037. Tel.: 858-784-2748; Fax: 858-784-8480; E-mail:
ruf@scripps.edu.
Published, JBC Papers in Press, November 16, 2000, DOI 10.1074/jbc.M004726200
2 S denotes subsite numbering according to Schechter and Berger (32).
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ABBREVIATIONS |
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The abbreviations used are:
VII/VIIa, coagulation factor VII/VIIa;
TF, tissue factor;
Gla domain, -carboxyglutamic acid-rich domain;
PCPS, phosphatidylcholine/phosphatidylserine;
tPA, tissue plasminogen
activator;
HBS, Hepes-buffered saline;
TBS, Tris-buffered saline,
CHAPS,
3-[(3-cholamidopropyl)dimethylamonio]-1-propanesulfonate.
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