From the Center for Molecular Biology of Oral
Diseases, University of Illinois at Chicago, Chicago, Illinois 60612, § Sanofi Recherche, Ligne Hémobiologie, 195 route
d'Espagne, 31036 Toulouse Cedex, France, and the ¶ Department of
Veterinary Medical Chemistry, Swedish University of Agricultural
Sciences, Box 575, S-75123, Uppsala, Sweden
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ABSTRACT |
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To determine the role of individual saccharide residues of a specific heparin pentasaccharide, denoted DEFGH, in the allosteric activation of the serpin, antithrombin, we studied the effect of deleting pentasaccharide residues on this activation. Binding, spectroscopic, and kinetic analyses demonstrated that deletion of reducing-end residues G and H or nonreducing-end residue D produced variable losses in pentasaccharide binding energy of ~15-75% but did not affect the oligosaccharide's ability to conformationally activate the serpin or to enhance the rate at which the serpin inhibited factor Xa. Rapid kinetic studies revealed that elimination of the reducing-end disaccharide marginally affected binding to the native low-heparin-affinity conformational state of antithrombin but greatly affected the conversion of the serpin to the activated high-heparin- affinity state, although the activated conformation was still favored. In contrast, removal of the nonreducing- end residue D drastically affected the initial low-heparin-affinity interaction so as to favor an alternative activation pathway wherein the oligosaccharide shifted a preexisiting equilibrium between native and activated serpin conformations in favor of the activated state. These results demonstrate that the nonreducing-end residues of the pentasaccharide function both to recognize the native low-heparin-affinity conformation of antithrombin and to induce and stabilize the activated high-heparin-affinity conformation. Residues at the reducing-end, however, poorly recognize the native conformation and instead function primarily to bind and stabilize the activated antithrombin conformation. Together, these findings establish an important role of the heparin pentasaccharide sequence in preferential binding and stabilization of the activated conformational state of the serpin.
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INTRODUCTION |
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Antithrombin, a member of the serpin (serine proteinase inhibitor) superfamily of proteins, is the primary physiological inhibitor of blood coagulation proteinases, especially thrombin and factor Xa (for reviews, see Refs. 1 and 2). Antithrombin inhibits these proteinases by forming tight, possibly covalent, complexes in which an exposed reactive center loop of the inhibitor is bound at the enzyme active site in the manner of a substrate. The rate of this inhibition is slow in comparison with most other serpin-proteinase reactions. However, the inhibition rate greatly increases up to several thousand-fold in the presence of the glycosaminoglycan, heparin (1, 2). This property of heparin is responsible for the widespread use of the polysaccharide as an anticoagulant drug.
The extent to which heparin accelerates antithrombin-proteinase reactions is dependent on a unique pentasaccharide sequence (DEFGH1; see Fig. 1), present in about one-third of naturally occurring heparin chains (3-6). Heparin molecules containing this sequence bind antithrombin with high affinity and induce an activating conformational change in the inhibitor (7-10). Those polysaccharide chains that lack this sequence bind antithrombin with ~1000-fold lower affinity and only weakly activate the inhibitor (11). Whereas the antithrombin conformational change is necessary and sufficient for the inhibitor to accelerate the inactivation of factor Xa, it is not sufficient for accelerated thrombin inhibition (12). The latter acceleration additionally requires a longer heparin chain to bridge the proteinase and the inhibitor in a ternary complex (12-16).
The heparin binding site of antithrombin has been tentatively mapped to residues of helix A, helix D, and the N-terminal region of the inhibitor, which are contiguous in the x-ray structure (1, 2, 17-21). Structure-activity studies of variants of the pentasaccharide have additionally revealed key functional groups in the oligosaccharide responsible for tight binding to the inhibitor. Four anionic groups, namely two N-sulfates and two O-sulfates in the glucosamine residues D, F, and H of the pentasaccharide (Fig. 1) are critical for complex formation and are thought to make ionic interactions with basic residues in the heparin binding site of antithrombin (6, 14, 22, 23, 31, 41). Additionally, nonionic interactions make a considerable contribution (about 60% at physiological pH and ionic strength) to the total binding energy (12). The pentasaccharide binds to antithrombin in a two-step process, in which a low-affinity recognition complex is first formed, which then induces an activating conformational change in the inhibitor, leading to a high-affinity complex (10, 12). Structural and functional studies suggest that the activating conformational change in antithrombin involves structural changes in the heparin binding site, which transform the reactive center loop from a partially buried to a fully exposed conformation (17, 21, 24-26).
The mechanism of heparin activation of antithrombin is as yet ill-defined at the molecular level. Although residues in the pentasaccharide and in antithrombin that are critical for their high-affinity interaction are known, it has not been elucidated which of these residues are involved in the initial recognition and in the subsequent allosteric activation of the serpin. To assess the roles of individual pentasaccharide residues in the two-step activation mechanism, we have studied the effect of truncating pentasaccharide residues at either the reducing or the nonreducing-end (see Fig. 1) on the ability of the oligosaccharide to bind and induce the conformational change in antithrombin. These effects have been correlated with the ability of the truncated pentasaccharides to activate the inhibitor for rapid inhibition of factor Xa. In agreement with preliminary studies of antithrombin interactions with these oligosaccharides (38), the present study establishes that the nonreducing- end trisaccharide, DEF, of the pentasaccharide is capable of fully activating antithrombin and, consequently, that the reducing-end residues are not essential for this activation, although they stabilize the activated conformation. The study further demonstrates a critical role of the pentasaccharide sequence in preferential binding of the activated conformational state of the serpin, in keeping with the pentasaccharide functioning as a classical allosteric activator.
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MATERIALS AND METHODS |
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Proteins--
Human antithrombin was purified from outdated
plasma as described previously (15, 27). Molar concentrations of the
inhibitor were calculated from absorbance measurements at 280 nm using
a molar absorption coefficient of 37,700 M1
cm
1 (28). Human factor Xa was prepared by activation of
purified factor X, followed by purification on SBTI-agarose, as
described previously (29). Factor Xa preparations were predominantly
the
-form as judged by SDS-polyacrylamide gel electrophoresis and were >90% active by comparisons of active site and protein
concentrations (29).
Oligosaccharides-- The methyl glycosides of pentasaccharide and pentasaccharide variants were synthesized, and their structures were confirmed as described previously (30, 31, 42). A 2-10 mM solution of each variant was prepared based on weight in distilled, deionized water. Stoichiometric titrations of high concentrations of antithrombin (at least 10 times the KD) with DEFGH, DEFGH', and DEFG* (Fig. 1), monitored by the endogenous fluorescence enhancement, showed fair agreement with the weight concentrations. The former concentrations were used for these tighter binding oligosaccharides, whereas concentrations based on weight were used for all other oligosaccharides.
Experimental Conditions-- All experiments were conducted at 25 °C and in 20 mM sodium phosphate buffer, containing 0.1 mM EDTA and 0.1% (w/v) PEG 8000, adjusted to the required pH. In the absence of added salt, the ionic strengths of buffers at pH 6.0 and 7.4 were 0.025 and 0.05, respectively. Sodium chloride was added to concentrations of 100, 250, 350, 500, 600, and 700 mM to achieve higher ionic strengths. Because of the reported decrease in stability of the inhibitor activity at lower pH (32), antithrombin solutions in pH 6.0 buffer were prepared by freshly diluting the protein (>100-fold) from concentrated stocks in pH 7.4, I 0.15 buffer. No significant losses in inhibitor activity were noted over the time frame of the experiments performed in this study.
Spectroscopic Studies-- Fluorescence emission spectra of antithrombin and its complexes with oligosaccharides were obtained at 25 °C in pH 6.0, I 0.025 buffer with 1 µM antithrombin and at least 5 µM oligosaccharide. The spectra were recorded with an SLM 8000 spectrofluorometer in the ratio mode at 1-nm wavelength intervals with excitation at 280 nm (4-nm bandpass), an emission bandpass of 2 nm, and 10-s integrations of the fluorescence signal at each wavelength. Corrections for Raman bands and any background signal from the buffer were made by subtracting buffer spectra. Control experiments showed no significant fluorescence of the variant oligosaccharides alone at concentrations used in the formation of complexes. The corrected spectra consisted of signal from both the free (<20%) and bound antithrombin. The normalized fluorescence spectrum of each complex was calculated by subtracting the expected spectrum of free antithrombin, based on measured dissociation constants for complex formation, and scaling up the resultant spectrum to 1 µM complex.
Equilibrium Binding Studies-- Equilibrium dissociation constants for antithrombin-oligosaccharide complexes were determined by titrating the oligosaccharide into a solution of antithrombin and monitoring the increase in intrinsic protein fluorescence accompanying the interaction, as described previously (9, 27). Antithrombin concentrations were in the range of 1-2 times the KD, except for KD values >1 µM, where the concentration was ~1 µM. The increase in fluorescence signal with increasing oligosaccharide concentration was fit to the quadratic equilibrium binding equation, assuming a 1:1 binding stoichiometry (9, 27).
The nonionic and ionic contributions to the total pentasaccharide binding energy were resolved by analyzing the NaCl concentration dependence of the observed dissociation constant (KD, obs) according to the equation (12, 16, 33), log KD, obs = log KD, nonionic + ZRapid Kinetic Studies--
The rate of oligosaccharide binding
to plasma antithrombin was measured in pH 6.0, I 0.025 buffer at 25 °C in an Applied Photophysics stopped-flow fluorometer,
as described previously (10, 12). Pseudo-first-order conditions in
which the oligosaccharide and antithrombin molar concentrations were
maintained at a ratio of at least 5:1 and in most cases 10:1 were
employed. The interaction was monitored from the increase in intrinsic
protein fluorescence with an excitation wavelength of 280 nm and an
emission filter that transmitted light only at wavelengths above 310 nm. Excitation slits corresponded to an 8-nm bandpass. The fluorescence
traces were acquired for about 10 half-lives and could be
satisfactorily fit by a single exponential function for DEFGH, DEFG*,
DEF, and DEFGH' interactions, which provided the amplitude of the
fluorescence change and the observed pseudo-first-order rate constant,
kobs. The traces for variant EFGH" interacting
with antithrombin could only be fit satisfactorily by a double
exponential function that yielded two pseudo-first-order rate constants
and two fluorescence amplitudes. Typically, 12-18 fluorescence traces
were acquired for each set of concentrations and averaged.
Factor Xa Inhibition Studies--
The accelerating effects of
oligosaccharides on the kinetics of antithrombin inhibition of factor
Xa were measured under pseudo-first-order conditions. A fixed 10 nM concentration of factor Xa was incubated with 2-12
µM antithrombin and oligosaccharide in pH 6.0 buffer in a
50-µl total reaction volume. The oligosaccharide concentration was
typically less than 20 nM, except for EFGH" and FGH,
which were present in the 20-240 nM range, and at least
three different oligosaccharide concentrations were examined. After
incubation for various times, reactions were quenched with 950 µl of
100 µM Spectrozyme FXa in 20 mM sodium
phosphate buffer, containing 100 mM sodium chloride, 0.1 mM EDTA, 0.1% (w/v) PEG 8000, at pH 7.4. The residual
factor Xa activity was then measured from the initial rate of substrate
hydrolysis at 405 nm. The observed pseudo-first-order rate constant
(kobs) at each oligosaccharide concentration was determined from the exponential decay of factor Xa activity (27). The
second-order rate constant for factor Xa inhibition by antithrombin alone (kuncat) and those for the inhibition by
antithrombin-oligosaccharide complexes (kH) were
obtained by least-squares analysis of the linear dependence of
kobs on antithrombin-oligosaccharide complex concentration according to the equation, kobs = kuncat × [AT]o + kH × [H]o × ([AT]o/(KD + [AT]o)), where
[AT]o and [H]o are the total concentrations of
antithrombin and heparin oligosaccharide, respectively, and KD is the dissociation constant of the complex (27). The expression [H]o × [AT]o/(KD + [AT]o), in this equation represents the concentration of
the antithrombin-oligosaccharide complex, because under the
experimental conditions, [AT]o
[AT]free.
The fractional saturations achieved were 98-100% for DEFGH, DEFGH',
and DEFG*; 50% for DEF; 22-45% for EFGH"; and 15-52% for FGH
based on measured KD values for
antithrombin-oligosaccharide interactions in Table I.
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RESULTS |
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Characterization of the Binding of Truncated Pentasaccharides
to Antithrombin by Intrinsic Protein Fluorescence
Changes--
The series of truncated pentasaccharides shown
in Fig. 1 were used to assess the role of
individual saccharide residues in the allosteric activation of
antithrombin. This series consisted of tetrasaccharide DEFG* and
trisaccharide DEF, representing reducing-end truncations, and
tetrasaccharide EFGH" and trisaccharide FGH, representing
nonreducing-end truncations. These two groups of oligosaccharides
contained individual saccharides whose structure was slightly modified
from those in pentasaccharide DEFGH, as denoted by the primes and
asterisks, due to the difficult synthesis of sufficient quantities of
the systematic series of truncated pentasaccharides. Extensive
structure-activity studies of pentasaccharide DEFGH have shown that
such structural modifications minimally affect pentasaccharide binding
and activation of antithrombin in most cases (41,
42).2 For the one case in
which a structural modification did significantly enhance
pentasaccharide activity, namely the addition of a
3-O-sulfate on saccharide H in EFGH", a reference
pentasaccharide containing this structural modification (DEFGH') served
as a control (42), thereby allowing the effect of the D residue to be
determined.
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Origin of the pH-dependent Change in Pentasaccharide
Binding Affinity--
To determine whether the higher pentasaccharide
affinity for antithrombin at pH 6.0 than at pH 7.4 was due to
additional charge-charge interactions, the dissociation constant for
the antithrombin-pentasaccharide interaction was measured as a function
of the NaCl concentration both at pH 7.4 and 6.0. The log
KD, obs increased linearly with
increasing log [Na+] both at pH 7.4 and 6.0 (not shown),
in accordance with the expected behavior of the protein-heparin
interaction (12, 16, 33). The slopes of these lines indicated that
5.2 ± 0.1 and 4.0 ± 0.2 charge-charge interactions
contributed to the binding at pH 6.0 and 7.4, respectively, whereas the
intercepts gave values of 4.92 ± 0.05 and
4.58 ± 0.02 for log KD,nonionic at these respective pH
values (see "Materials and Methods"). The values obtained at pH 7.4 are similar to those reported previously (12). These results indicate
that the increase in pentasaccharide binding affinity when the pH is
lowered from 7.4 to 6.0 results primarily from ~one additional ionic
interaction at pH 6.0, which is presumably made by residue D.
Rapid Kinetic Studies of DEFGH, DEFG*, and DEF Binding to Antithrombin-- The kinetics of binding of oligosaccharides DEFGH, DEFG*, and DEF to antithrombin was studied by continuously monitoring the protein fluorescence changes accompanying their binding by stopped-flow fluorimetry under pseudo-first-order conditions in pH 6.0 buffer. Binding was observable in the stopped-flow time frame as a monophasic exponential process in all cases. The dependence on oligosaccharide concentration of the observed pseudo-first-order rate constant (kobs) for binding of these oligosaccharides to antithrombin is shown in Fig. 3. In all cases, a progressive saturation of kobs with increasing oligosaccharide concentration was observed, indicative of a two-step binding process, in which an initial binding of the oligosaccharide to antithrombin induces a subsequent conformational change in the inhibitor (induced-fit pathway in Scheme 1) (10, 12). The data of Fig. 3 were satisfactorily fit by the rectangular hyperbolic Equation 1, which characterizes the induced conformational change pathway (10).
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(Eq. 1) |
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Rapid Kinetic Studies of DEFGH', EFGH', and FGH Binding to
Antithrombin--
The observed pseudo-first-order rate constant,
kobs, for binding of pentasaccharide DEFGH' to
antithrombin at pH 6.0, I 0.025, 25 °C progressively
increased to a limiting value with increasing concentration of
oligosaccharide (Fig. 4A),
similar to the pattern observed for binding of oligosaccharides DEFGH,
DEFG*, and DEF to antithrombin. Fitting of this data by Equation 1 for
the induced conformational change binding mechanism resulted in a
K1 value of 1.2 ± 0.4 µM, a
k2 value of 1210 ± 230 s
1,
and a k
2 value indistinguishable from 0. k
2 was estimated to be ~10
5
s
1 based on measured values of K1
and k2 and an approximated value for
KD, obs (Table I). The
K1 and k2 parameters for
DEFGH' are very similar to those for heparin pentasaccharide, DEFGH,
indicating that the greater affinity of DEFGH' than of DEFGH for
antithrombin is due to a reduced k
2 value.
Both 3-O-sulfation of reducing end residue H and deletion of
this residue therefore affect the same kinetic parameter. Replacement
of the N-sulfate of residue H in DEFGH' with an
O-sulfate had no effect on the binding kinetics (not
shown).
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(Eq. 2) |
Competitive Binding of FGH and DEFG* to Antithrombin--
To
determine whether FGH
bound to the same site on antithrombin as the
pentasaccharide, competitive binding experiments were performed.
Tetrasaccharide DEFG* was used as the competitor because of the much
greater affinity of DEFGH for antithrombin than of FGH
at pH 6.0. Equilibrium binding of DEFG* to antithrombin in the absence and
presence of fixed concentrations of FGH
at ~1 and ~4 times the
KD for the FGH
-antithrombin interaction was
monitored from the ~20% greater increase in inhibitor fluorescence produced by DEFG* than by FGH
binding (Table I). The apparent dissociation constant for DEFG* binding to antithrombin increased from
34 ± 3 nM in the absence of FGH
to values of
89 ± 6 and 181 ± 13 nM in the presence of
FGH
concentrations of 13.3 and 43.3 µM, respectively.
These measured KD values were indistinguishable from the calculated KD values of 75 ± 10 and 167 ± 20 nM that were expected if FGH
was
acting as a competitive inhibitor of DEFG* binding to antithrombin (see
"Materials and Methods").
Accelerating Effects of Truncated Pentasaccharides on Factor Xa
Inhibition by Antithrombin--
The second-order rate constant for the
uncatalyzed inhibition of factor Xa by antithrombin
(kuncat) was reduced by ~15-fold at pH 6.0 from that at pH 7.4 (Table III),
consistent with the increased levels of catalytically inactive serine
proteinase resulting from protonation of histidine 57 of the catalytic
triad (36). Second-order rate constants for the accelerated inhibition
of factor Xa by antithrombin-oligosaccharide complexes
(kH) at pH 6.0 were evaluated from the slopes of
the linear dependence of the pseudo-first-order inhibition rate
constant on the concentration of antithrombin-oligosaccharide complex
(see "Materials and Methods") and are presented in Table III.
Antithrombin complexes with DEFGH, DEFG*, and DEF showed
indistinguishable kH values of 4-5 × 104 M1 s
1,
representing about 300-fold rate enhancements, indicating that deletion
of reducing-end residues G and H did not affect the ability of the
oligosaccharides to activate antithrombin. Similar
kH values for antithrombin complexes with DEF
and DEFGH were also found at pH 7.4 (Table III). Complexes of DEFGH'
and EFGH" with antithrombin resulted in kH
values similar to that of the antithrombin-DEFGH complex, indicating no
effect of H residue 3-O-sulfation or D residue deletion on
antithrombin activation. In contrast, binding to antithrombin of
trisaccharide FGH
significantly reduced kH to
~10% of the pentasaccharide accelerated rate constant, indicating that removal of both residues D and E from the nonreducing-end greatly
compromised the ability to activate antithrombin.
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DISCUSSION |
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We have elucidated the role of individual saccharide residues of a specific heparin pentasaccharide in the allosteric activation of the serpin, antithrombin, by determining the effects of deleting pentasaccharide residues on this activation. These effects had to be analyzed at a lower ionic strength and pH than was used in past studies to reliably measure the affinities and rates of interaction of the truncated pentasaccharides with antithrombin. Such conditions did not appear to alter the activation mechanism, since the interaction of the pentasaccharide with antithrombin under these conditions was characterized by the same two-step binding process, fluorescence enhancement, and acceleration of factor Xa inhibition previously found at physiological ionic strength and pH (10, 12). Notably, the binding energy contribution of residue D was more dominant under these experimental conditions due to an additional electrostatic interaction of residue D with antithrombin. The origin of this additional electrostatic binding energy is presently unclear, since histidine residues close to the heparin binding site do not appear to participate in ionic interactions with heparin at pH 6 (37).
While both reducing and nonreducing-end residues contribute to
pentasaccharide binding energy, our results show that these residues
are not absolutely essential to activate antithrombin for rapid
inhibition of factor Xa. Thus, all truncated pentasaccharides, with the
exception of oligosaccharide FGH, which appears to bind antithrombin
nonproductively (see below), were capable of inducing the tryptophan
fluorescence enhancement associated with antithrombin activation and of
maximally accelerating antithrombin inhibition of factor Xa at
saturation. Previous assertions that residues D and H are essential for
full conformational activation of the serpin (39) were based on
indirect measurements of the rate of factor Xa inactivation at a single
concentration of inhibitor-oligosaccharide complex. In the present
study, direct measurements of factor Xa inactivation over a wide range
of oligosaccharide concentrations approaching inhibitor saturation
clearly show that deletion of residues D or H alone or G and H together
has no significant effect on pentasaccharide activation of
antithrombin.
Rapid kinetic studies allowed us to distinguish whether defects in
pentasaccharide binding to antithrombin were due to a reduced ability
of the pentasaccharide to bind the native low- heparin-affinity state
of antithrombin or to induce the inhibitor into the activated high-heparin-affinity state (Scheme 1, induced-fit pathway)
(10, 12). Deletion of reducing-end residue H or both G and H from the
pentasaccharide only modestly affected binding to the
low-heparin-affinity state of the serpin but greatly impaired
conformational activation to the high-heparin-affinity-state. This
impairment was due to increases in the rate constant for reversal of
the conformational activation step (k2 in
Scheme 1), which destabilized the activated conformation relative to
the native conformation by as much as ~100,000-fold (Table II). Such
findings suggest that the binding energy of residues G and H is
utilized to form interactions mostly or exclusively with the activated
antithrombin conformation. The loss of such interactions would thus be
expected to increase the rate constant for reversal of the
conformational change, since this rate constant should be inversely
related to the number of interactions that must be disrupted in going
from the activated conformation back to the native conformation.
Although loss of residues G and H greatly destabilized the activated
antithrombin conformation, the conformational equilibrium constant,
k2/k
2, remained large
enough (
8) to still favor the activated conformation (
89%). This
explains why oligosaccharides lacking residue H or both G and H
appeared to fully activate antithrombin for accelerated factor Xa
inhibition when present at saturating levels. While residues G and H of
the pentasaccharide are thus not required for conformational
activation, they are nevertheless critical for stabilizing the
activated conformation induced by residues D, E, and F.
Contrasting with these effects of deleting pentasaccharide reducing-end residues, deletion of the nonreducing-end residue D resulted in an altered mechanism of oligosaccharide binding and activation of antithrombin. This altered binding mechanism was characterized by a preferential binding of the oligosaccharide to the small amount of conformationally activated antithrombin in pre-equilibrium with native unactivated antithrombin, which thereby resulted in the unactivated inhibitor being pulled into the activated state (Scheme 1, pre-equilibrium pathway) (34, 35). Binding by this alternative pathway was complete in a rapid kinetic phase associated with a normal fluorescence enhancement and resulted in a normal acceleration of factor Xa inhibition. The observation that a further slow increase in antithrombin fluorescence followed the initial rapid fluorescence change with a rate that was independent of oligosaccharide concentration suggests that a further conformational change is induced in antithrombin following its activation by the variant pentasaccharide lacking residue D (EFGH").
The different pathway and altered mode of EFGH" binding to antithrombin suggests a key role for the D residue in pentasaccharide recognition of the native antithrombin conformation and in anchoring of the pentasaccharide in the heparin binding site of both native and activated inhibitor conformations. Thus, the failure of EFGH" to bind by the induced conformational change mechanism implies a major defect in binding to the native inhibitor conformation. If much of the binding energy of residue D under these conditions is utilized in forming the initial recognition complex, then loss of this residue would be expected to drastically weaken the affinity of this complex. The presence of residues G and H, which are only able to bind the activated antithrombin conformation with significant affinity, would additionally favor selective binding of EFGH" to activated antithrombin in pre-equilibrium with unactivated antithrombin. Since at most 8 kcal/mol of binding energy is utilized in forming the initial recognition complex at pH 6 and since the binding energy contribution of residue D greatly exceeds 8 kcal/mol at this pH (Tables I and II), a significant amount of the binding energy of residue D also appears to be available to enhance the binding of this residue to the activated antithrombin conformation and thereby to assist in stabilizing the activated conformation.
Further truncation of the pentasaccharide from the nonreducing-end
produced yet different effects on oligosaccharide binding and
activation of antithrombin. A key observation was that the kinetics of
FGH binding were complete within the ~1.5-ms dead time of the
stopped-flow instrument. Such behavior suggests that FGH
cannot be
binding by the pre-equilibrium mechanism observed for EFGH" binding,
since the rate of binding by this pathway should have been limited by
the 6 s
1 rate constant for conformational activation of
antithrombin in the absence of bound oligosaccharide. It follows that
FGH
must be binding to antithrombin by the induced conformational
change pathway. This could happen if FGH
bound nonproductively to
the DEF sites of native antithrombin involved in recognizing the
pentasaccharide. Binding of FGH
in the pentasaccharide site was
confirmed from the observation that the trisaccharide competes with
tetrasaccharide DEFG* for binding to this site. Nonproductive binding
of FGH
to the DEF interaction sites of native antithrombin would be
compatible with the glucosamine/hexuronic acid specificity of the DEF
sites. Such nonproductive binding to unactivated antithrombin would be favored because of the sizable binding energy available from the D and
possibly also the F interaction sites (6, 23) in the unactivated
conformation together with the unfavorable energy requirement for
activating antithrombin to allow productive binding. Moreover, such
nonproductive binding would explain the reduced ability of FGH
to
activate antithrombin, as judged both from a decreased protein
fluorescence enhancement (~10%) and lower enhancement of the rate of
factor Xa inhibition (about 10% that of the pentasaccharide). The
extent of this reduced activation is reminiscent of the reduced
activating effect of low-affinity heparin that lacks the
pentasaccharide activating sequence (11). The more favorable,
nonproductive mode of binding of FGH
to antithrombin may also
explain why so little binding energy is lost when residue E is deleted
as compared with the deletion of residue D.
Together, the results of the present and past studies favor the model
for pentasaccharide binding and activation of antithrombin depicted in
Fig. 5. In this model, binding of the
rigid nonreducing end residues D, E, and F (40) occurs first to the
native antithrombin conformation, with residue D making a primary
contribution to the binding energy, although residues E and F may also
contribute. Residues G and H make very weak or no interactions with
this conformation. Both modeling studies (43) and a preliminary x-ray
structure of the antithrombin-pentasaccharide complex (44) suggest that this initial binding of residues D, E, and F is at the C-terminal end
of helix D with the reducing-end oriented toward the N terminus of this
helix. Residues D, E, and F then induce antithrombin to undergo an
activating conformational change in which the inhibitor reactive center
loop is exposed to allow rapid inhibition of factor Xa (21, 25). The
activated conformation produces a complementary fit of saccharides D,
E, and F in the heparin binding site, which enhances their binding to
the activated conformation and thereby stabilizes this conformation. In
particular, more favorable interactions of the unique
3-O-sulfate of residue F in the activated conformation may
provide the driving force for this conformational change, since loss of
the 3-O-sulfate mostly abolishes conformational activation
(39). However, increased interactions of residues D and E with the
activated antithrombin conformation are also likely, given the
substantial binding energy resulting from the interaction of residue D
and the decreased activation resulting from nonproductive binding of
oligosaccharide FGH in the DEF interaction sites. Further
stabilization of the activated conformation results from the generation
of additional complementary sites of interaction for the more flexible
reducing-end residues G and H in this conformation (40). Conformational
changes in the reducing- end saccharides, in particular the
conformationally flexible iduronate residue G, may be required to align
the charges in these residues for an optimal fit with the activated
conformation (40). According to our model, the pentasaccharide
functions as a classical allosteric modifier by preferentially binding
and stabilizing the activated antithrombin conformation. Unique to this
model are the different roles of nonreducing and reducing-end
pentasaccharide residues in recognizing the native low-heparin-affinity
state and in preferentially binding and stabilizing the activated
high-heparin-affinity state.
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ACKNOWLEDGEMENT |
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We thank Peter Gettins of the University of Illinois at Chicago for critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant HL 39888 (to S. T. O.), Swedish Medical Research Council Grant 4212 (to I. B.), and American Heart Association of Metropolitan Chicago Senior Research Fellowship (to U. R. D.).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: Center for
Molecular Biology of Oral Diseases, University of Illinois at Chicago, Room 530E Dentistry (M/C 860), 801 S. Paulina Street, Chicago, IL
60612. Tel.: 312-996-1043; Fax: 312-413-1604.
1 The DEFGH designation of the heparin pentasaccharide originates from it being a part of a larger decasaccharide fragment, designated ABCDEFGHIJ, which was earlier isolated from heparin and later shown to contain the minimal pentasaccharide activating sequence (45).
2 Structure-activity studies of the natural pentasaccharide, DEFGH, have shown that replacement of N-sulfates with O-sulfates or methylation of -OH groups minimally affects pentasaccharide binding to antithrombin or activation of the inhibitor toward factor Xa (41).
3
This relation is valid when
k2
k2. See
Footnote b in Table II.
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REFERENCES |
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