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Glycobiology Pages 569-577  

NMR investigations of protein-carbohydrate interactions: refined three-dimensional structure of the complex between hevein and methyl [beta]-chitobioside
Introduction
Results
Materials and methods
Acknowledgments
References

NMR investigations of protein-carbohydrate interactions: refined three-dimensional structure of the complex between hevein and methyl [beta]-chitobioside

NMR investigations of protein-carbohydrate interactions: refined three-dimensional structure of the complex between hevein and methyl [beta]-chitobioside

Juan Luis Asensio, Francisco Javier Cañada, Marta Bruix1, Carlos González1, Noureddine Khiar, Adela Rodríguez-Romero2, Jesús Jiménez-Barbero3

Instituto de Química Orgánica General, CSIC, Madrid, Spain, 1Instituto de Estructura de la Materia, CSIC, Madrid, Spain and 2Instituto de Química, UNAM, Ciudad Universitaria, México D.F. 04510, México

Received on September 29, 1997; revised on October 29, 1997; accepted on October 30, 1997

The specific interaction of hevein with GlcNAc-containing oligosaccharides has been analyzed by 1H-NMR spectroscopy. The association constants for the binding of hevein to a variety of ligands have been estimated from 1H-NMR titration experiments. The association constants increase in the order GlcNAc-[alpha](1->6)-Man < GlcNAc < benzyl-[beta]-GlcNAc < p-nitrophenyl-[beta]-GlcNAc < chitobiose < p-nitrophenyl-[beta]-chitobioside < methyl-[beta]-chitobioside < chitotriose. Entropy and enthalpy of binding for different complexes have been obtained from van't Hoff analysis. The driving force for the binding process is provided by a negative [Delta]H0 which is partially compensated by negative [Delta]S0. These negative signs indicate that hydrogen bonding and van der Waals forces are the major interactions stabilizing the complex. NOESY NMR experiments in water solution provided 475 accurate protein proton-proton distance constraints after employing the MARDIGRAS program. In addition, 15 unambiguous protein/carbohydrate NOEs were detected. All the experimental constraints were used in a refinement protocol including restrained molecular dynamics in order to determine the highly refined solution conformation of this protein-carbohydrate complex. With regard to the NMR structure of the free protein, no important changes in the protein nOe's were observed, indicating that carbohydrate-induced conformational changes are small. The average backbone rmsd of the 20 refined structures was 0.055 nm, while the heavy atom rmsd was 0.116 nm. It can be deduced that both hydrogen bonds and van der Waals contacts confer stability to the complex. A comparison of the three-dimensional structure of hevein in solution to those reported for wheat germ agglutinin (WGA) and hevein itself in the solid state has also been performed. The polypeptide conformation has also been compared to the NMR-derived structure of a smaller antifungical peptide, Ac-AMP2.

Key words: hevein/protein-carbohydrate interactions/ conformation/NMR

Introduction

The understanding of how oligosaccharides are recognized (Lasky, 1992; Dwek, 1996; Gabius and Gabius, 1996) by the binding sites of lectins, antibodies, and enzymes is currently a topic of major interest. Detailed information on the three-dimensional structure of protein-carbohydrate complexes has usually been obtained from x-ray crystallography data (Vyas, 1991; Weis and Drickamer, 1996) and modeling (Imberty et al., 1993), since the usually high molecular weight of lectins has prevented their direct studies by means of NMR spectroscopy. However, NMR may also provide information about the driving forces behind protein-carbohydrate interactions in solution (Peters and Pinto, 1996; Richardson et al., 1997). Hevein is a chitin-binding protein that is present in laticifers of the rubber tree (Hevea brasiliensis). It has been shown that hevein inhibits the growth of several chitin-containing fungi. Therefore, it has been suggested that hevein plays a major role in the protection of plants from attack by a wide range of potential pathogens, including fungi (Beintema, 1994; Gidrol et al., 1994). From a structural point of view, hevein is a small, single chain protein of 43 amino acids especially rich in glycines and cysteins (Rodriguez et al., 1986), whose structure has independently been solved in the solid state by x-ray at 0.28 nm resolution (Rodriguez-Romero et al., 1991), and in solution by NMR methods both in water (Asensio et al., 1995a) and in dioxane/water (Andersen et al., 1993), in the last few years. The topology of hevein in solution (Andersen et al., 1993; Asensio et al., 1995a) differs significantly from that observed in the crystal (Rodriguez-Romero et al., 1991). However, it closely resembles the solid state structures of the different domains of wheat germ agglutinin (WGA) (Wright, 1990, 1992) and the solution structure of a smaller polypeptide chain isolated from Amaranthus caudatus, Ac-AMP2 (Martins et al., 1996). According to the x-ray studies (Wright, 1990, 1992), the four domains of WGA have similar three-dimensional structures and, indeed of primary structure, hevein shows a 56% sequence identity to the C domain of WGA. In addition, it has been recently proposed that hevein is involved in the coagulation of latex (Gidrol et al., 1994) by interacting with a 22 kDa glycoprotein through binding to a N-acetyl-glucosamine (GlcNAc). A previous report of our group (Asensio et al., 1995a) demonstrated that hevein binds chitobiose and chitotriose with millimolar affinity and that the binding process is enthalpy driven (Asensio et al., 1995a). This recognition process is not calcium-dependent, in contrast with the proposal made by Gidrol et al. (1994). We also described the interaction between hevein and chitobiose in structural terms, using a NMR-derived three dimensional structure of the protein (Asensio et al., 1995a). Following our studies on the interaction of hevein with chitin-derived oligosaccharides, within a global program directed to the study of protein-carbohydrate interactions in solution (Rivera-Sagredo et al., 1991, 1992; Solis et al., 1993, 1994; Asensio et al., 1995a,b; Espinosa et al., 1996a,b), we now report on the determination of the association constants between hevein and a variety of N-acetyl glucosamine containing oligosaccharides by using NMR spectroscopy. In addition, the thermodynamic parameters for the methyl [beta]-chitobioside-hevein interactions have been obtained. We also present a highly refined NMR structure (0.055 nm backbone rmsd over residues 3-41) of the molecular complex between hevein and methyl [beta]-chitobioside in water, based on the accurate analysis of 475 protein-protein and 15 protein/carbohydrate NOEs. It has to be mentioned that, although a few examples of solution structures of glycoproteins have been recently solved (Wyss et al., 1995; de Beer et al., 1996; Dwek, 1996; Mer et al., 1996; Weller et al., 1996; Gervais et al., 1997) to the best of our knowledge, this example represents the first case of a highly refined NMR three-dimensional structure of a noncovalent protein/carbohydrate complex in solution (Xu et al., 1995; Johnson et al., 1996; Richardson et al., 1997). Finally, the differences in binding constants have been explained in terms of the three-dimensional structure of the complexes.

Results

Ligand binding studies

In a first step, the 1D 1H-NMR spectrum of hevein was monitored in the presence of increasing amounts of GlcNAc-containing oligosaccharides. This protocol is a way to verify the existence of complexes between hevein and the corresponding carbohydrates, and, in addition, the alterations in chemical shifts may be used to determine the equilibrium association constants, Ka values. The observed effects on the chemical shifts and line broadening indicates that the interaction is basically fast in the chemical shift NMR time scale. The association constant values for GlcNAc, chitobiose, and chitotriose binding to hevein have been already reported (Asensio et al., 1995a). We herein have also determined the association constants for the binding of methyl [beta]-chitobioside, benzyl [beta]-chitobioside, p-nitrophenyl [beta]-glucosaminide, and p-nitrophenyl [beta]-chitobioside, following the protocol described in Materials and methods. In all cases, both the backbone NH and the highest field H[gamma] proton of Gln20, which are fairly well resolved signals, were followed as a function of the concentration of ligand added to determine the binding constant values (Figure 1). The individual fits of the data had high correlation coefficients.


Figure 1. Comparison of part of the 1H NMR spectrum of 0.5 mM hevein obtained in the presence of p-nitrophenyl-N-acetyl-[beta]-glucosaminide in 1H2O (A) with that obtained for the free protein (B) (3.8 mM) at 303 K and pH 5.6.

The association constant values shown in Table I indicate that methyl [beta]-chitobioside binding is favored with respect to the reducing disaccharide, chitobiose. In addition, the substitution of the methyl group by an aromatic p-nitrophenyl moiety increases the binding [sim]3-fold. Regarding the monosaccharides, benzyl [beta]-chitobiose is bound with an affinity similar to that of the reducing parent compound, GlcNAc, while the p-nitrophenyl derivative is bound with millimolar affinity, just below that measured for chitobiose itself.

Table I. Association constants (Ka, M-1) for the binding of different N-acetyl glucosamine-containing oligosaccharides to hevein, as determined by NMR spectroscopy
Sugar 25°C 30°C 35°C 40°C
GlcNAc-[alpha](1->6)Man - ND - -
GlcNAca - 30 ± 15 - --
(GlcNAc)2 620 ± 50 464 ± 20 381 ± 19 337 ± 24
(GlcNAc)2+EDTA 584 ± 94 - - -
(GlcNAc)2+Ca2+ - 454 ± 23 - -
GlcNAc p-NO2Ph 220 ± 14      
(GlcNAc)2 p-NO2Ph 3690 ± 830      
GlcNAc OBn 40 ± 15      
(GlcNAc)2 OMe 1225 ± 72 1069 ± 54 882 ± 25 647 ± 37
(GlcNAc)3 11,558 ± 2000b 8693 ± 738 6938 ± 488 5675 ± 311
The results are the average values obtained for two different protons (Gln20 NH, H[gamma] ) through nonlinear regression fitting of the corresponding chemical shift variation as function of sugar concentration. Errors are given as standard deviation of these mean values or as the standard deviation of the adjusted parameter after the fitting, whichever is larger. Previous data for GlcNAc, chitobiose and chitotriose (Asensio et al., 1995a) are also shown. ND, No detected significant binding.
aThe low degree of saturation attained even at ligand/hevein ratio > 40 precludes a more precise determination of the binding constant.

In a further step, the titration experiments for methyl [beta]-chitobioside were performed as a function of temperature, and the variation of Ka values was analyzed to give the equilibrium thermodynamic parameters, [Delta]H0 and [Delta]S0, from a van't Hoff plot of ln(Ka) vs. 1/T. (Figure 2). For this disaccharide, the slope of the line afforded a [Delta]H0 of -32.1 kJ/mol. The entropy of binding, [Delta]S0, was found to be -53.5 J mol-1 K-1. For chitobiose and chitotriose, the corresponding values (Asensio et al., 1995a) were [Delta]H0 of -31.3 and -36.4 kJ/mol and [Delta]S0 of -52.5 and -45.1 J mol-1 K-1. Since the derivation of thermodynamic parameters from a van't Hoff analysis assumes that the heat capacity is not temperature dependent, the magnitudes of these numbers should be regarded with caution, although the values are high enough to conclude that their signs are correct and that, therefore, the association processes are enthalpy driven.


Figure 2. Titration plot for methyl [beta]-chitobioside binding to hevein at pH 5.6. Plot of [Delta][delta] for Q20NH proton as a function of sugar concentration at 303 K.

The obtained thermodynamic values are in agreement with those previously described reported for the binding of different oligosaccharides to WGA (Bains et al., 1992) and for other lectin-carbohydrate complexes (Bundle and Young, 1992; Toone, 1994). In spite of a large enthalpy of binding, the dissociation constants measured for hevein binding to disaccharides are in the millimolar range, since the entropy of binding highly disfavors the association. Although the origin of this enthalpy-entropy compensation phenomenon remains an open question (Lemieux, 1989, 1996; Carver et al., 1991; Searle and Williams, 1992), it has been reported (Kronis and Carver, 1985a,b), for this magnitude and sign of [Delta]S0 and [Delta]H0, that hydrogen bonding and van der Waals forces should be the most important factors that stabilize the complex. The observed negative entropy of binding could arise from rigidification of the sugar and/or the protein lateral chains (Carver et al., 1991; Vyas, 1991; Searle and Williams, 1992) or by reorganization of the water structure (Lemieux, 1996). As an example, the conformational entropy (Cumming and Carver, 1987) calculated from the probability distribution of conformers of chitobiose, using the AMBER/Homans force field (Homans, 1990) at 300 K amounts to 8.8 kJ/mol (Asensio et al., 1995a; Espinosa et al., 1996c). Although only qualitative, this number indicates that the freezing of this ligand upon binding would represent an entropy loss of about this magnitude.

Structure calculation from NOE data

The assignment of the 1H-NMR spectrum of the hevein/methyl chitobioside complex in water was based on our previous data for hevein itself (Asensio et al., 1995a). Minor differences were observed in the 1H-NMR chemical shifts of the sample with regard to those previously studied.

These assignments have allowed the calculation of the three dimensional structure of the hevein/methyl [beta]-chitobioside complex in water solution. Regarding the polypeptide chain, a set of 475 constraints (63 intraresidue, 120 sequential, and 221 medium to long range) was unambiguously assigned, and converted into distance constraints as described in Materials and methods. Starting from 300 randomized conformations and the DIANA program, a group of 20 structures with target function values below 0.0158 nm2 were obtained (see Table II). The use of GLOMSA (Güntert et al., 1991) allowed the stereospecific assignments of 22% of the prochiral [beta]-methylenes. These 20 best structures from DIANA were submitted to further refinement through a simulated annealing protocol (see Materials and methods) by using the AMBER force field (Weiner et al., 1984) as implemented in Discover 2.9. The average backbone rmsd of the refined structures was 0.055 nm while the heavy atom rmsd was 0.116 nm (residues 3-41; Figure 3, Table II). The backbone of the calculated structures is extremely well ordered between residues 16-41 (rmsd 0.025 nm), while the convergence is relatively small between residues 6-15 and the C-terminal 42-43 fragment. The structures have very small deviation from ideal geometry and reasonable nonbonded contacts. The two strand antiparallel [beta]-sheet and the [alpha]-helical regions previously encountered for free hevein (Asensio et al., 1995a) are clearly observable. The three-dimensional structure of the protein within the complex in water solution at pH 5.7 is very similar to that previously reported by us for free hevein in water (Asensio et al., 1995a) and Andersen et al. (1993) in water-dioxane at two different pH values, and again markedly different (root mean square deviation, 0.269 nm, residues 16-41) from the x-ray structure (Rodriguez-Romero et al., 1991). This is an unusual case when comparing structures obtained by x-ray diffraction on single crystals and by NMR on protein solutions (Billeter, 1992). Nevertheless, it has to be mentioned that the definition of the polypeptide chain described herein by use of a complete matrix relaxation approach (rmsd 0.055 nm) is much higher than that found previously by us and by Andersen et al. (1993), which was about 0.1 nm. Therefore, the protein-carbohydrate interaction contacts and their nature may be more easily deduced.


Figure 3. Molscript (Kraulis, 1991) plot of the 20 simulated structures derived for the protein backbone in the methyl [beta]-chitobioside/hevein complex.

Before obtaining the three dimensional structure of the complex in solution, the sugar resonances had to be assigned. This process was accomplished by comparison of the chemical shifts (Table III) with those of the free sugar (Espinosa et al., 1996c), and using a protocol which consisted of recording several HMQC spectra at a variety of protein/sugar ratios. It was found that several sugar protons drastically changed their chemical shifts, therefore indicating their interaction with different amino acid residues. Protein-sugar NOEs were detected by comparing NOESY, HMQC, and TOCSY spectra acquired, under different conditions, for the free protein and for the complex (Table IV). Intermolecular sugar-protein NOEs were negative, indicating the binding of the disaccharide within the recognition site. The obtained disaccharide conformation is in agreement with the calculated global minimum for chitobiose (Espinosa et al., 1996c) by means of the AMBER/Homans force field (Homans, 1990).

Table II. Statistics for hevein at different stages of refinement
  N. of structures Superim. range Backbone RMSD Heavy Atom RMSD Target function NOE constraint violation information
            Number >0.2 Åa Maximumb
Diana 20 3-41 0.53 ± 0.16 1.09 ± 0.16 1.58 ± 0.25 8 0.34 ± 0.04
Structures     0.18-0.95 0.74-1.59 1.17-2.01 5-12 0.23-0.38
Diana 20 16-41 0.37 ± 0.11 0.90 ± 0.15 -    
Structures     0.14-0.63 0.56-1.26      
SA 20 3-41 0.55 ± 0.16 1.16 ± 0.19 - 5.4 0.26
Structuresc     0.22-0.92 0.78-1.58   3-8 0.23-0.32
SA 20 16-41 0.25 ± 0.06 0.86 ± 0.13 -    
Structures(c)     0.13-0.40 0.53-1.20      
aThe upper number shows the average number of violations per structure (number of violations / number of structures). The lower number describes the range of the number of violations.
bThe upper number shows the average maximum violation. The lower number describes the range of the maximum violation.
cAmber energies for SA structures are within a range of -214.97 and -255.83 kcal/mol.

Table III. Chemical shifts ([delta], ppm) of the sugar protons when free in solution and when bound to hevein
Proton Free Bound
H-1 4.45 4.26
H-2 3.73 3.72
H-3 3.71 3.65
H-4 3.61 3.53
H-5 3.49 3.47
H-6ª 3.88 3.83
H-6b 3.68 3.63
H-1[prime] 4.54 4.54
H-2[prime] 3.76 3.74
H-3[prime] 3.58 3.58
H-4[prime] 3.49 3.45
H-5[prime] 3.49 3.47
H-6ª[prime] 3.94 3.92
H-6b[prime] 3.76 3.70
O-Me 3.51 3.46

Table IV. Intermolecular NOEs detected for the hevein/methyl [beta]-chitobioside complex
Proton pair Proton pair Proton pair Proton pair
O-Me/Trp21 indol NH H-6a/Trp 21 indol NH H-2[prime]/Trp 23 indol NH Ac[prime]/Tyr 30 H-[delta][prime]
O-Me/Trp21 H-4 H-3/Trp23 H-2 H-4[prime]/Trp 23 indol NH Ac[prime]/Ser 19 H-[alpha]
O-Me/Trp21 H-5 H-6a[prime]/Trp23 H-4 H-4[prime]/Trp 23 H-7 Ac[prime]/Ser 19 H-[beta]1
Ac/Trp21 H-5 H-2[prime]/Tyr 30 H-[delta] Ac[prime]/Tyr 30 H-[delta] Ac[prime]/Trp 21 H-2
Ac and Ac[prime] refer to the methyl group of the acetamide moieties of the reducing and nonreducing ends of the disaccharide, respectively.

The location of the binding site, already deduced for the hevein/chitobiose complex (Asensio et al., 1995a) was confirmed by the presence (Table IV) of 15 unambiguous intermolecular protein-carbohydrate NOEs (Figure 4). These NOEs, as well as a hydrogen bond between the hydroxyl group of Ser19 and the carbonyl group of the nonreducing GlcNAc residue, were included as upper bound constraints in a simulated annealing protocol to determine the three-dimensional structure of the hevein-chitobiose complex. The hydroxyl group of Ser19 moves downfield upon addition of carbohydrate to the NMR tube containing the protein and, finally, broadens and disappears under the noise level. The resulting three-dimensional structure of the protein/carbohydrate complex is fairly well defined and indicates that the protein experiences only slight changes in its conformation when interacting with the disaccharide. The backbone maintains the same topology, and minor movements are observed in the lateral chains of the amino acids which form the binding site (Figure 5). In particular, Tyr30 lateral chain, which was fairly well defined in the free structure stays in the same orientation when complexed to methyl chitobioside, and the rmsd decreases in a noticeable way (rmsd for all heavy atoms, 0.015 nm). The rmsd for the lateral chain atoms of Trp21 and Ser19 is similar (0.018 and 0.016 nm, respectively), while those for the other Trp residue directly implicated in the binding is even better (0.010 nm for Trp23). Both GlcNAc residues and the O-methyl group make interactions with several protein lateral chains. The nonreducing acetamido methyl group shows non polar contacts with two aromatic residues: Tyr30 and Trp21, and, in addition, there are important hydrogen bonds which confer stability to the complex: one between the nonreducing sugar acetamido group and Ser19 and a second one involving C3-OH and Tyr30. An additional interaction is observed between the less polar [alpha]-face of the reducing GlcNAc moiety and the plane of the indole ring of Trp21. Additional evidence for the existence of stacking interaction between the lateral chain of Trp21 with the reducing end came from the observation of strong shielding of several protons of the indole ring of Trp21 in the complex of hevein with p-nitrophenyl-GlcNAc, and from the upfield shifting of the O-methyl group of methyl chitobioside in the presence of hevein with respect to that measured for the free sugar. The conformation around the glycosidic linkage of the disaccharide (rmsd for all heavy atoms, 0.019 nm) is reasonably well defined, since, according to the simulated annealing protocol, [Phi] angle oscillates between 40 and 52°, while [Psi] ranges between 5 and -10°. Additional differences between the solution and solid state structures are also evident from the superimposition of the binding sites. The x-ray structure (Figure 6a) cannot accommodate the nonreducing sugar in the pocket provided by Tyr30, Ser19, Trp21, and Trp23, unless a drastic change in the orientation of the lateral chains takes place. However, the comparison of the three-dimensional structure of the polypeptide chain of hevein with the B domain of WGA produces a fair agreement (rmsd 0.060 nm) over residues 16-32 (Figure 6b). Recently, the solution conformation of a small peptide, AcAMP II, isolated from Amaranthus caudatus has been reported. Although smaller, this peptide presents also a high degree of sequence homology with hevein. In fact, the rmsd deviation of the backbone atoms (residues 12-32) (Figure 6c) between hevein and AcAMP II amounts to only 0.100 nm. Finally, similar deductions, regarding the role of the aromatic Tyr and Trp residues of hevein and other related proteins for the binding of chitobiose have been recently found by using CINDP methods (Siebert et al., 1997).


Figure 4. Schematic view of the protein carbohydrate NOEs unambiguously detected for the methyl [beta]-chitobioside/hevein complex.


Figure 5. Superimposition of 10 NMR-derived structures of the binding site of the hevein-methyl [beta]-chitobioside complex.

Figure 6. (A) Superimposition of the hevein-methyl [beta]-chitobioside complex solution structure and free hevein crystal structure. (B) Superimposition of the hevein-methyl [beta]-chitobioside complex solution structure and WGA-B crystal structure. (C) Superimposition of the hevein-methyl [beta]-chitobioside complex solution structure and AcAMP II structure.

The interactions described above have also been observed in the crystal structures of WGA-chitobiose (Wright, 1984) and WGA-sialyllactose (Wright, 1990). Both van der Waals and polar interactions contribute to the complexation process, stabilizing the orientation of the sugar rings through the formation of hydrogen bonds and stacking interactions with aromatic side chains. Therefore, the structural view obtained in solution perfectly agree with the deductions from the equilibrium thermodynamic parameters. In the solid state, x-ray crystallography has also demonstrated that these interactions are key features for the establishment of protein-carbohydrate complexes (Vyas, 1991). The variations in binding constants in solution can now be explained in structural terms: The minimum sugar size which can be bound by hevein is the monosaccharide, which is stabilized by non polar forces involving Trp23 and Tyr30, and by hydrogen bonds involving Ser19 and the hydroxyl group of Tyr30. The binding of the [beta] anomer is probably preferred over the [alpha] analog, since for this anomer, the hydroxyl group would make unfavored contacts with Trp23. The binding constant for p-nitrophenyl [beta]-GlcNAc derivative is one order of magnitude higher than that of the free monosaccharide, due to the fixing of the [beta]-anomeric configuration and to the additional stabilization provided by the interaction of the Trp21 indol ring and the p-nitrophenyl moiety of the sugar. On the other hand, the benzyl derivative has a binding constant similar to that of the reducing sugar, indicating that the orientation of the phenyl ring is not appropriated for the stabilizing aromatic/aromatic interactions. In addition, due to the high flexibility of the benzyl derivative, lost of entropy upon binding can also be claimed as responsible for the small association constant experimentally observed. Despite the mixture of anomers present in chitobiose and the fact that only the [beta]-anomer is effectively bound by hevein (Asensio et al., 1995a), the binding constant of chitobiose is higher than that of p-nitrophenyl [beta]-GlcNAc. Probably, both entropic effects, due to the intrinsically higher flexibility of the p-nitrophenyl derivative with respect to the disaccharide, as well as better van der Waals interactions provided by the three-dimensional shape of the pyranoid chair, may account for the better binding. The use of methyl [beta]-chitobioside improves the binding. Both the fixing of the anomeric configuration in the favored [beta]-orientation, and the presence of additional nonpolar interactions between the O-methyl group and the extended surface of Trp21 are probably the key factors in this case. A further increase in binding is observed when p-nitrophenyl [beta]-chitobioside is employed. Although the corresponding changes in energy (between methyl [beta]- and p-nitrophenyl [beta]-chitobioside) are small, the aromatic/aromatic interaction which exits in this case is expected to be favored over the O-methyl/aromatic interaction which takes place for methyl [beta]-chitobioside. Finally, the binding constant found for chitotriose is the highest of all of those measured herein. With respect to p-nitrophenyl [beta]-chitobioside, and as also observed above for the comparison between chitobiose and p-nitrophenyl [beta]-GlcNAc, the binding is better for the trisaccharide, due to the better van der Waals contacts which are established between the extended surface of Trp21 indol ring and the pyranoid chair. In addition, the flexibility of the p-nitrophenyl derivative will also be higher than that of the trisaccharide and, therefore, the better association constant measured in this case may also have a component of entropic origin.

Concluding remarks

The interaction of hevein with GlcNAc-containing oligosaccharides has been described in structural terms, making use of a highly refined NMR three-dimensional structure. We have shown that the binding process is enthalpy driven and that both hydrogen bonds and van der Waals forces contribute to the stability of the complexes in water solution. The binding site is fairly well defined in the free protein and experiences only minor movements upon complexation. Hevein is thus a small but fairly adequate model to study protein-carbohydrate interactions in solution. Further investigations directed to the location of long-life water molecules in the surroundings of the binding site in order to explore their role in the association process as well as the study of several hevein analogs are now in progress.

Supplementary material available

The coordinates of the complex will be deposited in the Brookhaven data bank and are available from the authors upon request. NOE data and Ramachandran plots are also available from the authors on request.

Materials and methods

Hevein was isolated from Hevea brasiliensis latex as previously described (Rodriguez et al., 1986) and used without further purification. Methyl [beta]-chitobioside was prepared in our laboratory from chitobiose using standard methods. All other carbohydrates were purchased from Sigma Chemical Co.

NMR spectroscopy

NMR experiments. 1H-NMR spectra were recorded in 85:15 1H2O:2H2O on Bruker AMX-600 and Varian Unity 500 MHz spectrometers at 27° and 37° C. The 2D-experiments were acquired using 1.6 mM solutions, while the 1D spectra were obtained with 0.25 or 0.50 mM samples.

600 MHz experiments. The TOCSY (Bax and Davis, 1985) and NOESY (Kumar et al., 1980) experiments were obtained in the phase sensitive mode using the TPPI method (Marion and Wüthrich, 1983) for quadrature detection in F1. Typically, a data matrix of 512 * 2K points was used to digitize a spectral width of 7800 Hz. Eighty scans were used per increment with a relaxation delay of 1 s. Prior to Fourier transformation, zero filling was used in F1 to expand the data to 1K * 2K. Baseline correction was applied in both dimensions. The corresponding shift was optimized for the different spectra. The TOCSY spectra were carried out using MLEV-17 (Bax and Davis, 1985) during the 60 ms of isotropic mixing period. The NOESY experiments were performed with mixing times of 50, 125, 200, and 275 ms.

500 MHz experiments. One bond proton-carbon correlation experiments were performed using the HMQC sequence at 299 K. A relaxation delay of 1 s was used, with 128 scans per increment. A data matrix of 256 * 1K points was used. HMQC experiments were performed for five different protein/ligand ratios in order to follow the shifting of the sugar protons upon binding to the lectin.

NMR titration experiments

The binding of carbohydrates to hevein was monitored by recording monodimensional 500 MHz 1H-NMR spectra of a series of samples with variable sugar concentration (6-11 different concentrations). The concentration of hevein during the experiments was held constant. The hevein sample was prepared by dissolving the protein in 1.0 ml buffer (85:15, 1H2O:2H2O, 100 mM sodium chloride, 13.5 mM sodium acetate, pH 5.6). The concentration of hevein was calculated from the UV absorbance at 280 nm, by using an extinction molar coefficient of [epsis] = 12660 (Andersen et al., 1993). The 1D-NMR spectrum for the sample with the highest ligand/protein ratio was recorded by dissolving the corresponding sugar ([sim]15 mM) in 0.5 ml of the hevein solution described above. The sample with the other 0.5 ml of this hevein solution was used to obtain the 1H-NMR chemical shifts of the free-sugar hevein sample ([delta]free). The titration curve was built by adding small aliquots of the sugar-concentrated hevein solution over the free-sugar protein sample as described previously (Asensio et al., 1995a).

Addition of a 20-fold excess of GlcNAc-[alpha]-(1->6)-Man did not modify the chemical shifts of hevein, thus eliminating the possibility of nonspecific binding of chitobiose derivatives with hevein.

Thermodynamic equilibrium parameters, [Delta]S0, [Delta]H0, for the hevein-methyl [beta]-chitobioside association were determined from van't Hoff plots (Kronis and Carver, 1985a,b), in which the association constants were determined at 25, 30, 35, and 40°C. Since the use of van't Hoff plots makes the approximation that the heat capacity does not depend on temperature, these results should be considered as only semiquantitative.

Structure calculations

Accurate calculations of upper and lower limits for proton-proton distances from NOESY cross-peak intensities at four mixing times were obtained with the iterative complete relaxation matrix algorithm MARDIGRAS (Borgias and James, 1990). Isotropic tumbling motion was assumed for the protein, and two different overall correlation times of 2 and 3 ns were tested. In addition, two different starting model structures were used. Therefore, a maximum set of 16 distances were obtained for every cross peak (4 mixing times by 2 correlation times by 2 model structures). The upper limit for structure calculation was that corresponding to the largest theoretical value provided by MARDIGRAS. Disulfide linkages were included as distance constraints between S-S (0.20 nm < r < 0.21 nm) and between C[beta]-S (0.30 nm < r < 0.31 nm). Distance geometry calculations were performed on a Silicon Graphics Indigo computer using the program DIANA (Güntert et al., 1991) and the REDAC (Güntert and Wüthrich, 1991) strategy. Stereospecific assignment of a 22% of the prochiral methylene groups was accomplished with the program GLOMSA (Güntert et al., 1991). A set of 475 (112 intraresidue, 152 sequential, and 211 medium and long range) constraints were used in the final round of calculations, besides several structurally meaningless constraints. The 15 experimentally observed protein-carbohydrate NOEs were added as upper-bound constraints to deduce the three dimensional structure of the hevein/methyl [beta]-chitobioside complex. The calculated (from the relaxed energy surfaces) global minimum of the disaccharide (Espinosa et al., 1996c) was used as starting geometry for the bound carbohydrate, as previously described (Asensio et al., 1995a).

The 20 best DIANA structures in terms of target function were submitted to a simulated annealing protocol (Scheek et al., 1989) with the AMBER force field (Weiner et al., 1984) as implemented into the DISCOVER 2.9 program (Biosym Technologies, USA). After an initial restrained energy minimization (REM) with 500 conjugate gradient iterations, the structures were heated up to 1100 K in 2 ps and, at this temperature, 2 ps of restrained molecular dynamics (RMD) was performed. The structures were then subjected to a cooling procedure in which the temperature was decreased in 100 K every 2 ps until a temperature of 100 K was reached. At this temperature, 4 ps of RMD was carried out. The final structures were energy minimized (REM) using 1000 conjugate gradient iterations.

Acknowledgments

Financial support from DGICYT (Grant PB-96-0833) is gratefully acknowledged. We thank Prof. Martín-Lomas for his interest and support throughout this work. J.L.A. thanks Europharma S.A. for a fellowship. A.R.-R. thanks the Direccion General de Asuntos del Personal Academico (Mexico), Proyecto IN200489. We also thank Dr. Martins and Verheyden for providing the coordinates of AcAMP-II and Dr. Siebert and his colleagues for a copy of the CINDP manuscript.

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3To whom correspondence should be addressed at: Departamento de Química Orgánica Biológica, Instituto de Química Orgánica General, CSIC, Juan de la Cierva 3, 28006 Madrid, Spain

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