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A complex of the synthetic tetrasaccharide AGA*IM[GlcN,6-SO3-α(1–4)-GlcA-β(1–4)-GlcN,3,6-SO3-α(1–4)-IdoA-αOMe] and the plasma protein antithrombin has been studied by NMR spectroscopy. 1H and 13C chemical shifts, three-bond proton–proton (3JH-H) and one-bond proton–carbon coupling constants (1JC-H) as well as transferred NOEs and rotating frame Overhauser effects (ROEs) were monitored as a function of the protein : ligand molar ratio and temperature. Considerable changes were observed at both 20 : 1 and 10 : 1 ratios (AGA*IM : antithrombin) in 1H as well as 13C chemical shifts. The largest changes in 1H chemical shifts, and the linewidths, were found for proton resonances (A1, A2, A6, A6′, A1*, A2*, A3*, A4*) in GlcN,6-SO3 and GlcN,3,6-SO3 units, indicating that both glucosamine residues are strongly involved in the binding process. The changes in the linewidths in the IdoA residue were considerably smaller than those in other residues, suggesting that the IdoA unit experienced different internal dynamics during the binding process. This observation was supported by measurements of 3JH-H and 1JC-H. The magnitude of the three-bond proton–proton couplings (3JH1-H2 = 2.51 Hz and 3JH4-H5 = 2.23 Hz) indicate that in the free state an equilibrium exists between 1C4 and 2S0 conformers in the ratio of ≈ 75 : 25. The chair form appears the more favourable in the presence of antithrombin, as inferred from the magnitude of the coupling constants. In addition, two-dimensional NOESY and ROESY experiments in the free ligand, as well as transferred NOESY and ROESY spectra of the complex, were measured and interpreted using full relaxation and conformational exchange matrix analysis. The theoretical NOEs were computed using the geometry of the tetrasaccharide found in a Monte Carlo conformational search, and the three-dimensional structures of AGA*IM in both free and bound forms were derived. All monitored NMR variables, 1H and 13C chemical shifts, 1JC-H couplings and transferred NOEs, indicated that the changes in conformation at the glycosidic linkage GlcN,6-SO3-α(1–4)-GlcA were induced by the presence of antithrombin and suggested that the receptor selected a conformer different from that in the free state. Such changes are compatible with the two-step model [Desai, U.R., Petitou, M., Bjork, I. & Olson, S. (1998) J. Biol. Chem.273, 7478–7487] for the interaction of heparin-derived oligosaccharides with antithrombin, but with a minor extension: in the first step a low-affinity recognition complex between ligand and receptor is formed, accompanied by a conformational change in the tetrasaccharide, possibly creating a complementary three-dimensional structure to fit the protein-binding site. During the second step, as observed in a structurally similar pentasaccharide [Skinner, R., Abrahams, J.-P., Whisstock, J.C., Lesk, A.M., Carrell, R.W. & Wardell, M.R. (1997) J. Mol. Biol.266, 601–609; Jin, L., Abrahams, J.-P., Skinner, R., Petitou, M., Pike, R.N. & Carrell, R.W. (1997) Proc. Natl Acad. Sci. USA94, 14683–14688], conformational changes in the binding site of the protein result in a latent conformation.
The sulfated glycosaminoglycan heparin and some heparin-derived oligosaccharides are known for their anticoagulant properties mediated by a serine protease inhibitor, the plasma protein antithrombin. The need to understand these biological properties at the molecular level has stimulated both theoretical and experimental studies involving analysis of the structure of both saccharides and the plasma protein during the binding process [1–7]. It has been shown [8,9] that the most important features of the antithrombin-binding site lie in the specific pentasaccharide sequence [–GlcN,6-SO3-α(1–4)-GlcA-β(1–4)-GlcN,3,6-SO3-α(1–4)-IdoA2-SO3-α(1–4)-GlcN,6-SO3-α- (AGA*IA)]. Systematic chemical modifications of this pentasaccharide, as well as of other heparin-derived oligosaccharides, have been used to analyse the effect of individual monosaccharide units and functional groups on the biological activity of these compounds . It was found that most sulfate and carboxylate groups are important in the activation of antithrombin. For example, the loss of a single carboxylate can lead to a decrease (more than 90%) in the antifactor Xa activity. Comparable changes in activity can occur when one of the N-sulfate groups is lacking in the glucosamine residue structure. Computer modelling of heparin-derived oligosaccharides, complexed with antithrombin, suggests that the presence of the pentasaccharide can induce elongation of helix D in antithrombin . Recently, an analysis of the crystal structure of the antithrombin–pentasaccharide complex provided direct evidence that the formation of the complex is accompanied by a change in the conformation of the active site of antithrombin . In the latent conformation, helix D is unkinked and the side chains of three of the binding residues are hydrogen-bonded to other regions of the molecule. In the active conformation, helix D was observed as slightly kinked and the side chains of the most important binding residues are not stabilized by hydrogen-bonding and are thus readily available to form ionic interactions with the heparin saccharides.
Despite the progress in understanding the mechanism of heparin-induced potentiation of antithrombin and the role of amino acids in the receptor site during the complexation, many phenomena are still not clear and require further analysis. In order to understand the binding processes at the molecular level and the thermodynamics of complexation, a knowledge of possible changes in the three-dimensional structure of the ligand in solution during the binding process and the role of the charged groups involved in the weak interaction with the antithrombin-binding residues appears to be very important.
NMR spectroscopy, together with molecular modelling, is often a major tool used to study protein–ligand binding processes; transferred NOE experiments can provide information on ligand structure in both the free and the bound state when a ligand exchanges rapidly, compared with the time scale of spin-lattice relaxation rates [12–16]. Such experiments have proved useful for analysing the conformation in several protein–carbohydrate systems [17–25]. Nevertheless, in addition to data based on NOE analysis, important information on the ligand structure can be obtained from J-couplings and chemical shifts. In fact proton chemical shifts can be affected by the presence of protein, thus the cited variables can provide quite a detailed picture of the influence of the receptor on the individual atoms in the ligand molecule. Furthermore, it is possible to monitor the torsion angle changes on glycosidic linkages through variations of both proton and carbon shieldings as well as one-bond proton–carbon coupling constants.
The present study deals with the analysis of the synthetic tetrasaccharide GlcN,6-SO3-α(1–4)-GlcA-β(1–4)-GlcN,3,6-SO3-α(1–4)-IdoA-αOMe (AGA*IM, Fig. 1) in the free state and in the presence of antithrombin. AGA*IM represents a good model for the interaction of other heparin-derived oligosaccharides with high affinity for antithrombin, and the results are discussed and compared with data obtained in kinetic studies on the same system . The lower binding affinity of the compound allowed the use of NOE and rotating frame Overhauser effect (ROE) transfer experiments at various temperatures. Thus, the NMR analysis was based on the measurement of 1H and 13C chemical shifts, proton–proton and proton–carbon coupling constants as well as transferred NOEs and ROEs. The data provide information on the behaviour of heparin-derived oligosaccahride during the binding process, and describe the three-dimensional structure of this molecule in the presence of antithrombin.
Figure 1. Conformational structure of the synthetic tetrasaccharide AGA*IM.
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The interaction between antithrombin and tetrasaccharide AGA*IM has been analysed using three NMR parameters: chemical shifts, coupling constants and NOEs. Each depends on the structure of the ligand and complex in a different way, thus a detailed picture of this interaction could be obtained. The origins of the effects, which lead to variations in both 1H and 13C shieldings, can be divided into two groups. The major effect is due to changes in electron density caused by the ‘direct’ shielding/deshielding influence of the protein. As seven charged groups (SO3−, COO−) are present in the structure of AGA*IM, strong electrostatic interaction with protein amino acids that take part in the binding process can be expected. Recently, a crystal structure of the complex of a structurally similar pentasaccharide, AGA*IAM, with antithrombin was analysed . The data indicate that the conformational rearrangement in antithrombin causes a significant redistribution of positive charges in the binding site of the protein upon the adoption of a low-affinity latent conformation. In contrast, in the ligand molecule considerable variations in electron density can be expected, particularly in the central A* residue (bearing three charged groups), as the result of electrostatic interaction with antithrombin amino acids. In fact, the largest 1H chemical shift changes were observed for this residue, with practically all the shifts varying upfield or downfield in the presence of antithrombin. The differences in the 1H shifts of A6 and A2 are probably from the same origin in the non-reducing GlcN,6-SO3− residue. The second effect is due to induced conformational change in the ligand molecule upon binding to antithrombin. Both 1H and 13C shieldings depend strongly on the glycosidic linkage conformation, and such changes could be correlated with torsion angles . Thus, chemical shifts of atoms at the glycosidic linkage, such as A1, G4, A1* and A4*, could be affected by conformational change due to variations in the φ, ψ dihedral angles. In particular, 1H A1 and 13C G4 chemical shift variations agreed well with the differences in other NMR parameters, and suggested conformational change at the GlcN,6-SO3–GlcA glycosidic linkage during the binding process.
In several NMR studies dealing with protein–carbohydrate interactions [20,48–50], considerable spin-diffusion effects were observed to influence the magnitude of the transferred NOEs. Such mediated effects were caused by magnetization transfer via intramolecular (carbohydrate) protons as well as by protein protons. However, in the present study, no mediated effects were observed, suggesting that the protein protons are not in close contact with the protons in the AGA*IM molecule that were analysed by transferred NOEs. This seems to be incompatible with the observed relatively large variations in proton chemical shift, which would indicate quite close antithrombin protons. The above contradictory evidence may stem from the bulky charged SO3− and COO− groups of AGA*IM. Because of these relatively large groups in the tetrasaccharide, the protein protons remain close to the surface of sulfo and carboxyl groups but relatively distant from the anomeric and ring protons (those monitored in two-dimensional transferred NOEs) owing to steric effects. Thus, antithrombin could cause variation in electron density in AGA*IM as the result of electrostatic interaction, with a consequent large variation in proton shieldings. However, the transfer of magnetization during the NOE experiments was not greatly influenced by protein protons. Such electrostatic interactions and the effect of the sulfate and carboxylate groups confirms the importance of these groups on activity of heparin, the pentasaccharide AGA*IAM and other heparin-derived oligosaccharides [2,4,10].
The effect of flexibility and the conformational equilibrium in IdoA on the biological activity of heparin and heparin-derived compounds is a subject of interest [10,37,38]. Both synthetic and crystallographic studies have analysed the possible active conformers of this residue in the bound state. For example, a synthetic analogue of pentasaccharide AGA*IAM, in which an additional OSO3− group was introduced at the reducing-end GlcN,6-SO3 residue and the conformational equilibrium was shifted almost completely towards the 2S0 conformer in the IdoA residue, showed a biological activity that was about twice as high as that of natural heparin . Furthermore, a recent analysis of a derivative of pentasaccharide with an L-IdoA residue in the fixed 1C4 chair form showed very low activity in an antithrombin-mediated anti-Xa assay . This evidence led to the conclusion that the 1C4 conformer is unlikely to be the active one. However, the crystal structure of the heparin-derived hexasaccharide complexed to the basic fibroblast growth factor showed that the IdoA ring adopted multiple conformations in the presence of the protein: one IdoA residue in the hexasaccharide was in the 1C4 chair form, the other one was in the skew 2S0 form , indicating that an even energetically less favourable conformer of the IdoA residue can be the active one. In the present case, 3JH-H coupling constants measured in the IdoA residue in the tetrasaccharide in the free solution suggested that the conformational equilibrium is shifted towards the 1C4 conformer (population of about 75%). A small decrease in coupling constants was observed in the complex with antithrombin, which may indicate a further shift towards the chair form of the pyranose ring. The shift in conformational equilibrium towards the chair form in the IdoA residue suggests a stabilization of the energetically more favoured conformer in the presence of the protein. However, as already mentioned, the exclusive presence of a more stable conformer in the bound state is not always the case, even in structurally very similar systems. Thus, a direct comparison of the present data or extrapolation with other heparin-derived oligosaccharides may not be the most appropriate approach.
The analysis of transferred NOEs was focused mainly on the characterization of glycosidic linkage conformation. The geometry of the tetrasaccharide, found in the Monte Carlo conformational search, was used to interpret NOEs using the full relaxation matrix approach. In the free state (Fig. 9), the best fit to experimental NOEs was obtained with geometry of φ1, ψ1 (−45°, −30°), φ2, ψ2 (42°, 18°) and φ3, ψ3 (−27°, −48°). The geometry at the glycosidic linkages is comparable with that computed for the pentasaccharide AGA*IAM. This evidence is not surprising as the structure of the A-G-A* part of the molecule is identical. However, in the bound state (Fig. 10), the present data suggest that the receptor probably selected a different conformation (with φ1 = 39°,ψ1 = 12°) at the A-G glycosidic linkage. The difference in conformation at this linkage is also supported by the variation in the other two NMR parameters, the chemical shifts and 1JC-H. Protein-induced conformational changes in the ligand molecule were also observed in other protein–carbohydrate systems [54–56] and support the idea that the bound conformation may not correspond to the one in the free state. In fact, the indications of conformational changes in two other glycosidic linkages, G-A* and A*-I, were observed as well, as discussed in connection with the differences in (A1*)–(I4) and (A1*)–(I3) NOEs as well as chemical-shift changes of A1* and A4*. Detailed quantitative analysis of the mentioned NOEs is not straightforward owing to the complex internal dynamics at the A*-I linkage (the different correlation times for A1*–I3 and A1*–I4 relaxation vectors because of pseudo-rotation of the IdoA residue and the flexibility of the A*-I glycosidic linkage) and will be the subject of further studies. Moreover, the interference of the residual HDO resonance with the G1 proton signal precluded a precise evaluation of transferred NOEs at the G–A* glycosidic linkage. Quantitative analysis using the corcema program allows estimation of Kd and koff. The computed value for Kd was 0.47 mm at 25 °C, which was somewhat larger than that (17 μm) determined by fluorescence emission spectroscopy for the AGA*IM–antithrombin complex, indicating weaker binding . The difference in the computed and experimental Kd values is probably due to the simplified two-state model used to characterize the AGA*IM–antithrombin interaction in the present interpretation of the NOE experiments . The same is valid for the computed values of off-rates which were, respectively, 20 s−1 and 60 s−1, depending on temperature, and are higher than those obtained recently .
Figure 9. Structure of tetrasaccharide GlcN,6-SO3-α(1–4)-GlcA-β(1–4)-GlcN,3,6-SO3-α(1–4)-IdoA-αOMe in the free state. The corresponding dihedral angles at the glycosidic linkages are φ1, ψ1 (−45°, −30°), φ2, ψ2 (42°, 18°) and φ3, ψ3 (−27°, −48°).
Figure 10. Structure of tetrasaccharide GlcN,6-SO3-α(1–4)-GlcA-β(1–4)-GlcN,3,6-SO3-α(1–4)-IdoA-αOMe in the presence of antithrombin. The corresponding dihedral angles at the glycosidic linkages are φ1, ψ1 (39°, 12°), φ2, ψ2 (43°, 14°) and φ3, ψ3 (–27°, –48°).
The determination of the three-dimensional structure of AGA*IM in the bound state is an important step toward understanding the interaction mechanism of heparin-derived oligosaccharides with antithrombin at the molecular level and the thermodynamics of the binding process. Both modelling and crystallographic studies have focused on analysing the interaction of pentasaccharide AGA*IAM with antithrombin [2,3,6,10,11,57,58]. One fundamental conclusion of these studies was the importance of specific SO3− and COO− groups in the structure of the oligosaccharide. The lack of a single group can considerably decrease the biological activity , underlining the importance of their involvement in the binding process, particularly the electrostatic interaction with amino acid residues in the binding site. As found in the present study, the 6-O-SO3− group in the GlcN,6-SO3− residue, the COO− group in GlcA,N-SO3− and 3-SO3− groups in GlcN,3,6-SO3− residues create a sequence of four negative charges in the molecule in the free state (Fig. 9), in which the distances between the sulfur atom in the 6-SO3− group in the GlcN,6-SO3− residue and sulfur atom in the 3-SO3− group in the GlcN,3,6-SO3− residue was 5.8 Å and the distance between the sulfur atom in the 6-SO3− group in the GlcN 6-SO3− residue and the carboxylic carbon in the GlcA COO− residue was 4.5 Å. However, in the bound state (Fig. 10), the distribution of negatively charged groups was different, a consequence of the variation in glycosidic-linkage conformation evident from the interatomic distances. The distance between the sulfur atoms in the 6-SO3− group in the GlcN, 6-SO3− residue and the 3-SO3− in the GlcN,3,6-SO3− was 7.5 Å whereas the distance between sulfur and carbon atoms (6-SO3− group in the GlcN,6-SO3− residue and carboxylate carbon in COO− in GlcA) was 6.7 Å. Thus, the orientation of the charged groups on the non-reducing end of AGA*IM is different in the presence of antithrombin. Such spatial distribution would appear to fit the structure of the antithrombin binding site better, probably increasing the strength of the electrostatic interaction with the amino acid residues. This picture of the binding process is compatible with the recently proposed two-step model [2,6] describing the interaction of heparin-derived oligosaccharides with antithrombin, with a minor extension: in the first step a complex between ligand and receptor is formed, accompanied by conformational changes in the tetrasaccharide AGA*IM, creating a complementary three-dimensional structure to fit the active conformation of the protein-binding site. During the second step, as observed in a structurally similar pentasaccharide , conformational changes in the binding site of the protein result in a relaxed state.
An analysis of the tetrasaccharide–antithrombin complex, using both 1H and 13C chemical shifts, homo- and heteronuclear coupling constants and relaxation measurements, led to a relatively detailed picture of this interaction.
The chemical shifts suggest considerable antithrombin shielding/deshielding effects, predominantly influencing atoms linked to charged groups, owing to strong electrostatic interaction. The effect was pronounced in GlcN,3,6-SO3 as well as in GlcN,6-SO3 residues, particularly for A1, A2, A6, A6′, A1*, A2*, A3* and A4*.
Protein-induced changes at the glycosidic linkage could be qualitatively monitored by the variations in chemical shifts and one-bond proton–carbon coupling constants.
An analysis of the magnitude of proton–proton and proton–carbon coupling constants in IdoA residue indicated that the conformational equilibrium is most likely shifted towards the 1C4 chair form in the presence of antithrombin.
NOE analysis in the free and the bound states revealed that the conformation at the glycosidic linkage between the GlcN,6-SO3 and GlcA residues changed during the binding process. This change was supported by the variations in two other NMR parameters. Changes in the three-dimensional structure also resulted in a different charge distribution in the AGA*IM molecule in the presence of antithrombin, which was due to the altered orientation of the SO3− and COO− groups.
The NMR experimental data confirmed the crucial role of charged sulfate and carboxylate groups in heparin-derived tetrasaccharide during the binding process with antithrombin.
The data presented are compatible with the two-step model [2,6] describing the complexation of heparin-derived saccharides with antithrombin. However, a minor modification in the first step is introduced: a complex between the ligand and the receptor is formed, accompanied by a conformational change in the ligand molecule. The driving force for such a change in ligand conformation appears to be a strong electrostatic interaction between AGA*IM and antithrombin. During the second step conformational changes [2,11] in the binding site of the protein result in latent conformation of antithrombin.