Man [alpha]1-2 Man [alpha]-OMe-concanavalin A complex reveals a balance of forces involved in carbohydrate recognition

Davina N. Moothoo, Booma Canan, Robert A.Field and James H.Naismith1

Centre for Biomolecular Sciences, Purdie Building, The University, St. Andrews KY16 9ST, UK

Received on August 3, 1998; revised on October 7, 1998; accepted on October 11, 1998

We have determined the crystal structure of the methyl glycoside of Man [alpha]1-2 Man in complex with the carbohydrate binding legume lectin concanavalin A (Con A). Man [alpha]1-2 Man [alpha]-OMe binds more tightly to concanavalin A than do its [alpha]1-3 and [alpha]1-6 linked counterparts. There has been much speculation as to why this is so, including a suggestion of the presence of multiple binding sites for the [alpha]1-2 linked disaccharide. Crystals of the Man [alpha]1-2 Man [alpha]-OMe-Con A complex form in the space group P212121 with cell dimensions a = 119.7 Å, b = 119.7 Å, c = 68.9 Å and diffract to 2.75Å. The final model has good geometry and an R factor of 19.6% (Rfree = 22.8%). One tetramer is present in the asymmetric unit. In three of the four subunits, electron density for the disaccharide is visible. In the fourth only a monosaccharide is seen. In one subunit the reducing terminal sugar is recognized by the monosaccharide site; the nonreducing terminal sugar occupies a new site and the major solution conformation of the inter-sugar glycosidic linkage conformation is adopted. In contrast, in another subunit the non reducing terminal sugar sits in the so called monosaccharide binding site; the reducing terminal sugar adopts a different conformation about its inter-sugar glycosidic linkage in order for the methyl group to access a hydrophobic pocket. In the third subunit, electron density for both binding modes is observed. We demonstrate that an extended carbohydrate binding site is capable of binding the disaccharide in two distinct ways. These results provide an insight in to the balance of forces controlling protein carbohydrate interactions.

Key words: carbohydrate conformation/Con A saccharide complex/crystal structure/molecular recognition

Introduction

The recognition of carbohydrates by proteins is known to play a vital role in many biological processes and as such present an attractive target for therapeutic intervention (Dwek, 1996). Interactions between proteins and ligands are governed by a number of factors including binding site characteristics, buried surface area, hydrogen bonding and van der Waals interactions, reorganization of solvent structure and the conformation of the oligosaccharide. In order to deconvolute these contributions to binding, protein-carbohydrate interactions are the focus of much thermodynamic (Bains et al., 1992; Mandal et al., 1994; Toone, 1994; Chervenak and Toone, 1995) and structural work (for recent reviews, see Elgavish and Shaanan, 1997; Loris et al., 1998). The ubiquity of carbohydrates presents a specificity problem which must be overcome in order to design useful therapeutics, and so it is desirable to achieve an atomic level understanding of the binding behavior of proteins and their ligands. Molecular level information is obtainable via structural studies of the protein and its ligands complemented by information gained from other biophysical techniques such as titration microcalorimetry (Bains et al., 1993). The lectins are proteins which specifically recognize oligosaccharides; they weakly bind monosaccharides, but show increased affinities for higher order saccharides (Mandal et al., 1994). The legume lectin concanavalin A (Con A), the most well studied of all lectins, provides a powerful model system for the study of protein-carbohydrate interactions. The thermodynamics of its interactions with carbohydrates are currently under active investigation by several groups (Mandal et al., 1994; Toone, 1994; Chervenak and Toone, 1996; Weatherman et al., 1996). Crystal structures have been determined of Con A complexed with methyl [alpha]-d-mannopyranoside (Derewenda et al., 1989; Naismith et al., 1994), methyl [alpha]-d-glucopyranoside (Harrop et al., 1996), [alpha]-Man-(1-3)-[[alpha]-Man-(1-6)]-Man (Naismith and Field, 1996; Lorris et al., 1996), and [beta]-GlcNAc-(1-2)-[alpha]-Man-(1-3)-[[beta]-GlcNAc-(1-2)-[alpha]-Man-(1-6)]-Man (Moothoo and Naismith, 1998) to 2.0Å, 2.3Å, and 2.7Å, respectively. These structures have characterized the monosaccharide binding site, highlighted the role of a structurally conserved water molecule in the binding site, identified distortions in both protein and carbohydrate on binding, and have rationalized the binding of Con A to the core of glycoprotein N-linked glycans. In order to further probe the balance of forces contributing to protein-carbohydrate interactions, we have determined the structure of Con A with methyl 2-O-([alpha]-d-mannopyranosyl)-[alpha]-d-mannopyranoside (Man [alpha]1-2 Man [alpha]-OMe).

Con A binds methyl [alpha]-d-mannopyranoside with an association constant of 0.82 × 104 M-1, corresponding to a total of 5.3 kcal mol-1 of free energy liberated upon binding (Mandal et al., 1994). The free energies of binding [alpha]1-3 and [alpha]1-6 mannobioses and their methylated counterparts are comparable with monosaccharide binding. Binding 2-O-([alpha]-d-mannopyranosyl)-[alpha]-d-mannopyranose (Man [alpha]1-2 Man) liberates 6.3 kcal mol-1 and binding Man [alpha]1-2 Man [alpha]-OMe liberates 7.0 kcal mol-1 of free energy corresponding to both more favorable enthalpic and entropic contributions to the binding energy. The requirements for carbohydrate binding to the so-called monosaccharide binding site of Con A, known as the Goldstein Rules (Goldstein et al., 1974), are free equatorial hydroxyl groups at positions 3 and 4 and a free primary alcohol group in the 6 position. Both sugar rings in Man [alpha]1-2 Man can potentially be recognized by the monosaccharide binding site. It has been proposed that this enhances the probability of binding the carbohydrate and leads to a more favorable entropy contribution to the binding energy (Mandal et al., 1994). On the basis of thermodynamic data, another group proposed that the increased affinity is due to the presence of additional sites for binding the disaccharide (Goldstein, 1975). Neither hypothesis has been confirmed experimentally.

Table I. Data collection and refinement statistics for the Man [alpha]1-2 Man [alpha]-OMe-Con A complex
Crystallographic data collection statistics and refinement statistics
Unique reflections 26,568  
Completeness of data (%)
   Entire resolution range 25.0-2.75 Å 99.7
   Highest resolution shell 2.8-2.75 Å 99.8
Rmerge (I) (%)a
   Entire resolution range 25.0-2.75 Å 10.1
   Highest resolution shell 2.8-2.75 Å 30.9
Average data redundancy
   Entire resolution range 25.0-2.75 Å 3.29
   Highest resolution shell 2.8-2.75 Å 3.28
% of data > 1[sigma]
   Entire resolution range 25.0-2.75 Å 95
   Highest resolution shell 2.8-2.75 Å 88.8
Refinement
   Resolution range (Å)   25.0-2.75
   Rfree (%)b   22.78
   R factor (%)   19.57
   Bond r.m.s. deviation (Å)c   0.008
   Angle r.m.s. deviation (0)c   1.633
   Noncrystallographic symmetry r.m.s. deviation (c[alpha] atoms) (Å) 0.04  
   B-Factor bonded atoms r.m.s. deviation (Å2)d 1.74  
   Ramachandran core/additional/generously allowed (%)e 83.2/16.3/0.5  
   Protein mean B-factor (Å2)d (all)   27.21
   Protein mean B-factor (Å2)d (main chain) 27.02  
   Protein mean B-factor (Å2)d (side chain) 27.42  
   Sugar mean B-factor (Å2)   35.42
   Solvent mean B (Å2)   23.63
   Number of protein atoms   7236
   Number of sugar atoms   74
   Number of solvent molecules   73
   Number of metal ions   8
aRmerge(I) = [Sigma]hkl[Sigma]i ¦Ii - I(hkl)¦/[Sigma]hkl[Sigma]iIi(hkl).
bRfree is calculated on 10% of data excluded during refinement.
cr.m.s. deviation from Engh and Huber ideal values (Engh and Huber, 1991).
dCalculated with MOLEMAN (G. J. Kleywegt, unpublished program). All stereochemically modeled atoms were removed prior to B-factor analysis, all bonded atoms including those in the sugars are included in the calculation of r.m.s. B-factor deviation for bonded atoms.par eCore and additionally allowed regions as defined by PROCHECK (Laskowski et al., 1993). No residues are in the generously allowed or disallowed regions.

Recently the 1.75 Å structure of Man [alpha]1-2 Man complexed to Con A has been determined, in which the nonreducing terminal mannose occupies the monosaccharide binding site (Naismith and Moothoo, unpublished observations) and the reducing mannose occupies the region of the reducing mannose in the trimannoside complex (Naismith and Field, 1996). We have determined the Man [alpha]1-2 Man [alpha]-OMe Con A in complex with Con A to 2.75 Å resolution. Two distinct overlapping modes of binding for the disaccharide are seen. In one mode the inter sugar glycosidic linkage is distorted to maximize the methyl group interaction with a hydrophobic pocket. This is the first evidence that a lectin can bind a specifically recognized sugar in two distinct ways.

Results

Overall structure

One tetramer of Con A is present in the asymmetric unit of the Con A Man [alpha]1-2 Man [alpha]-OMe complex, and each monomer binds one disaccharide molecule and 2 metal ions. The overall structure of the protein has been described in detail to a resolution of 0.94 Å and will not be dealt with here (Deacon et al., 1997). The structure has good geometry (Table I) and poorly ordered regions are consistent with previous Con A structures (Naismith and Field, 1996; Deacon et al., 1997; Moothoo and Naismith, 1998). The structure superimposes onto the 1.75 Å Man [alpha]1-2 Man-Con A structure with an average root mean square deviation of 0.19 Å for all C[alpha] (0.12 Å for the C[alpha] of [beta]-sheet residues) and onto the 0.94 Å native structure with an average root mean square deviation of 0.34 Å (0.21 Å for the c[alpha] of [beta]-sheet residues only).

Sugar binding

The structure shows well defined difference density for both sugar rings of the disaccharide in two of the four subunits denoted A and D (Figure 1). In these two subunits the mode of binding is clearly different (Figures 2 and 3). We were unable to unambiguously model a second sugar ring into residual electron density in the remaining two subunits (B and C), only one sugar ring was placed in the model for these two subunits. In subunit B difference density is visible for a second sugar ring sitting in the an extended binding region framed by residues 12-14, 98-100, 207-208, and 227-228 as seen in subunit A. Surprisingly however, electron density is also visible for a second sugar ring in the binding region framed by residues Gly-98, Ser-168, and the loop Thr-226 to Leu-229 as seen in subunit D. Figure 3.


Figure 1. Fo-Fc electron density observed in the binding site for subunits A (top left) and D (top right) (both contoured at 3 [sigma]) and subunit B (below, contoured at 1[sigma]). Maps were calculated after CNS simulated annealing with all sugars omitted.

Subunit A

Figure 2 is a schematic diagram of the hydrogen bonding network between the sugar and the protein. The nonreducing sugar is recognized by the monosaccharide binding site via a combination of hydrogen bonds, a polar contact and van der Waals interactions (Table II, III). The interaction is similar to that observed for methyl [alpha]-d-mannopyranoside. There is a shift in the position of the sugar ring of 0.3 Å out of the binding site with respect to the methyl [alpha]-d-mannopyranoside complex. Such a distortion has been seen in the Con A pentasaccharide complex (Moothoo and Naismith, 1998a). The reducing sugar is recognized via 1 polar contact and 15 van der Waals interactions. The van der Waals interaction is considerably more extensive than that observed for the reducing sugar in the Man [alpha]1-2 Man complex. Table III.

The increase in contacts is possible due to a rotation (~50°) in the psi dihedral angles of the inter-sugar glycosidic linkage of Man [alpha]1-2 Man [alpha]-OMe (Table IV) with respect to the major solution conformation. The major solution conformation of this linkage is observed in subunit D (see below) and was observed in the Man [alpha]1-2 Man complex (Moothoo and Naismith, unpublished observations). The result of the glycoside linkage rotation is that the reducing sugar ring is rotated about a 6-fold axis (Figure 4) placing the anomeric methyl group in a hydrophobic pocket formed by Tyr-12, Leu-99, and Tyr-100. This maximizes its interaction with these residues and also maximizes the interaction of the C6 atom of the sugar with the protein. The conformation we observe is very close to a minor solution conformation (Peters, 1991) and to that observed in the crystal structure of carbohydrate (Srikrishnan, 1991). The conformation we see lies ~1 kcal mol-1 (Perez et al., 1995) above the major solution conformation (Peters, 1991; Dowd et al., 1995). A larger distortion of the glycosidic linkage was observed in the pentasaccharide-Con A complex which placed the glycosidic linkage ~3 kcal mol-1 above the global minimum (Moothoo and Naismith, 1998). In this case a distortion in the interaction between the sugar and the monosaccharide binding site was also observed.

The structurally conserved water molecule observed in all Con A-oligosaccharide structures is observed here making contacts with the protein. This water, however, no longer makes a hydrogen bond with the sugar as seen in the Man [alpha]1-2 Man complex (Moothoo and Naismith, unpublished observations), but it does make van der Waals contact with C6 of the reducing terminal sugar. The hydrogen bond between Arg-228 NH2 and the reducing sugar seen in the Man [alpha]1-2 Man structure is also lost as a result of the rotation of the psi angle.

Man [alpha]1-2 Man [alpha]-OMe buries a total of 61Å2 of additional surface area over Man [alpha]1-2 Man upon binding to Con A. Significantly, this increase is mainly due to burying of apolar surface accessible area; the anomeric methyl group and C6 of the sugar and Tyr-12 and Asp-16 of the protein. Burying apolar surface area is energetically favorable and therefore contributes favorably to the binding affinity.

Table II. Hydrogen bonding and polar contact distances between Man [alpha]1-2 Man [alpha]-OMe and the protein
Sugar Protein Distancea
Monosaccharide binding sugarb
O3 Arg-228 N 3.1
O4 Asn-14 ND2 3.0
O4 Asp-208 OD1 2.7
O4 Arg-228 N 3.2c
O5 Leu-99 N 3.2
O6 Gly-98 N 3.3 c
O6 Leu-99 N 3.0 c
O6 Tyr-100 N 3.1
O6 Asp-208 OD2 3.0
Second sugar
O6 (A subunit) Tyr-12 OH 3.0 c
O3 (D subunit) Thr-226 OG1 3.3
O4 (D subunit) Ser-168 OG 3.3 c
aAveraged across the four subunits, typically ± 0.1 Å.
bIn subunit A this is the non-reducing sugar, in subunit D this is the reducing sugar.
cDistance is less than 3.5 Å, but the donor-acceptor angle deviates substantially from the linearity expected for a hydrogen bond and is thus classified as a polar contact.

Table III. Van der Waals interaction (<4.0 Å) between the carbohydrate and protein
Sugar Contacts Residue
Primary binding sugara,b 42 Tyr-12, Asn-14, Gly-98, Leu-99, Gly-207, Asp-208, Gly-277, Arg-228
Second sugar ring (A subunit) 15 Tyr-12, Leu-99, Tyr-100
Second sugar ring(D subunit) 10 Gly-98, Ser-168, Thr-226
aAveraged across the four subunits.
bIn subunit A this is the nonreducing sugar ring, in subunit D this is the reducing sugar ring.

Table IV. Dihedral angles around the inter-sugar glycosidic linkage of Man [alpha]1-2 Man [alpha]-OMe and Man [alpha]1-2 Man
  [phiv]a [psi]a
Man [alpha]1-2 Man [alpha]-OMe: A subunit 78.3 -91.9
Man [alpha]1-2 Man [alpha]-OMe: D subunit 69.6 -146.6
Man [alpha]1-2 Manb 64.6 -140.8
Man [alpha]1-2 Man[alpha]-OMec (major solution conformation) 60 -150
Man [alpha]1-2 Man[alpha]-OMec (minor solution conformation) 70 -80
Man [alpha]1-2 Man[alpha]-OMec (minor solution conformation)   -90
Man [alpha]1-2 Man[alpha]-OMe d (solid state conformation) 64.2 -105.3
a[phiv] is O-5 C-1 O-X C-X, [psi] is C-1 O-X C-X C-(X-1) (IUPAC convention).
bFrom 1.75 Å Con A-Man [alpha]1-2 Man structure (Naismith and Moothoo, unpublished observations).
cFrom Peters, 1991.
dFrom Srikrishnan, 1989.

Subunit D

Figure 2. Schematic diagram of the hydrogen bonds made between the disaccharide and the protein in subunits A (left) and D (middle). The monosaccharide binding site is shown boxed. The GlcNAc [beta]1-2 Man derived from the pentasaccharide (Moothoo and Naismith, 1998a) structure is shown for comparison on the right.

In this subunit the disaccharide is recognized by an alternative binding site to that seen in the A subunit (Figure 2). It is the reducing terminal mannose residue which is recognized by the monosaccharide binding site via hydrogen bonds, van der Waals interactions, and a polar contact. The non-reducing terminal sugar is recognized by the site occupied by the 1-6 arm GlcNAc in the pentasaccharide bound structure (Moothoo and Naismith, 1998) and interacts with Gly-98, Ser-168, and Thr-226 (Table II, III).

The binding mode seen in subunit A is unavailable in this subunit due to crystal contacts and likewise this mode would be prohibited by crystal contacts in subunit A. The phi and psi inter-glycosidic linkage angles are close to those observed in the Man [alpha]1-2 Man structure which lies in an energetic minimum. Although the nonreducing terminal sugar of Man [alpha]1-2 Man [alpha]-OMe occupies the binding site of the 1-6 arm GlcNAc of the pentasaccharide, the two sugar rings are in different positions. The number of interactions made by the non-reducing terminal sugar with the protein are more than halved in this complex compared with the GlcNAc sugar due to this different position of the sugar rings, and the absence of the N-acetyl group. There is 11 Å2 less polar buried surface area buried overall upon binding in this subunit than subunit A but 23 Å2 more sugar apolar surface area buried upon binding compared to subunit A. In this subunit, Tyr-12, which is not involved in sugar binding, is moved 1.3 Å towards the monosaccharide sugar. The side chain of Asp-16 is markedly different than the other subunits with its chi1 angle rotated about 90° and there is a 10° rotation in the chi-1 angle of Thr-226 which is involved in carbohydrate binding in this subunit.

Subunit B

Once a single sugar ring had been modeled into the monosaccharide binding site, there remained additional difference electron density for a second sugar ring in two different places, analogous to the binding modes seen in subunits A and D (Figure 1). As a crystal structure is a static average of many molecules, some proportion of molecules apparently bind the saccharide in a manner analogous to the seen in subunit A the remainder analogous to subunit D. Unlike subunits A and D no crystal contacts interfere with the two sites. The resolution of the data and resulting map quality prevent accurate modeling of this disorder and our model contains only a single sugar bound at the monosaccharide site.

In subunit C once the monosaccharide was built into the model, we were unable to model in a second sugar.

Discussion

The structure of the Man [alpha]1-2 Man [alpha]-OMe complex presented here represents the first demonstration that a carbohydrate can be bound specifically in two distinct environments by the same lectin. The obvious question arises as to why this phenomenon was not observed in the Man [alpha]1-2 Man complex? The crystal packing arrangement in that complex would preclude the binding arrangement seen in subunit D in this report. Thus, either methyl glycoside formation results in equalization of the energy of interaction at both sites, or crystallization kinetically selects one mode in preference to the other. Modeling protocols are not currently advanced enough to distinguish between these two possibilities.


Figure 3. Man [alpha]1-2 Man [alpha]-OMe complex, both binding modes are shown (figure generated by RASTER3D; Merrit and Murphy, 1994).

Statistical thermodynamics gives the relative occupancy for the freely two inter-converting but mutually exclusive states as 1:e-[Delta][Delta]G°/RT, where [Delta][Delta]G° is the difference in free energy between the states. In our case we define [Delta][Delta]G° = [Delta]G°weak - [Delta]G°strong where [Delta]G°strong is the intrinsic free energy of binding of the higher affinity mode and [Delta]G°weak is the intrinsic free energy of binding of the lower affinity mode. This results in the ration of binding modes ([weak mode] / [strong mode] ) as given in Equation 1.

[weak mode] / [strong monde] = e-([Delta]G°weak-[Delta]G°strong)/RT (1)

When two binding modes are exclusive, the measured equilibrium constant (Kmeas) is

Kmeas = [complex] / [protein][sugar] (2)
[complex] = [strong mode] + [weak mode] (3)

By standard thermodynamic treatment it follows that:

[Delta]G°meas = [Delta]G°strong - RT1n(1+e-[Delta][Delta]G°/RT) (4)


Figure 4. The disaccharide observed in the Man [alpha]1-2 Man complex (solid line) superimposed onto the Man [alpha]1-2 Man [alpha]-OMe complex observed in subunit A (broken line). The rotation about the inter-sugar glycosidic linkage is obvious.

In the simplest case where the sites are equivalent [Delta][Delta]G° = 0, [Delta]G°meas = [Delta]G°strong + RTln2, the measured [Delta]G° contains a statistical enhancement of 0.41 kcal mol-1 to the intrinsic binding free energy of the site. At the other extreme where [Delta][Delta]G° is very large, [Delta]G°meas = [Delta]G°strong, effectively collapsing to the simple single site case. The electron density maps we observe are of insufficient quality to accurately experimentally calculate the ratio between the binding modes. However in subunit B, the residual electron density for a second sugar appears by inspection to be approximately as strong in both locations. Accepting the error inherent in this imprecise approach we suggest an upper limit of 2:1 for the ratio of occupancy between the two modes. Using this value as a limit, Equation 1 yields a value of [Delta][Delta]G° (maximum difference in free energy of binding between the two modes at 25°C) of 0.46 kcal mol-1.

This value of [Delta][Delta]G° gives an enhancement to the intrinsic binding energy of the strong site (Equation 4) of 0.24 kcal mol-1. As [Delta]G°meas is -7.0 kcal mol-1, we estimate that [Delta]G°strong lies between -6.59 kcal mol-1 (both sites equal) and -6.76 kcal mol-1 (maximal suggested free energy difference between the sites), and [Delta]G°weak lies between -6.59kcal mol-1 (equal sites) and -6.3 kcal mol-1. Although our study does not identify which is the strong site and which is the weak site, our work shows that both modes bind more tightly than methyl [alpha]-d-mannopyranoside by at least 1.0 kcal mol-1.

In one binding mode the secondary sugar makes extensive contacts that are particularly "hydrophobic" in nature. However in order to maximize these interactions the minor solution or solid state conformation is accessed. The precise conformation we see is ~1 kcal mol-1 above the global minimum. Thus, a trade-off between van der Waals interactions, conformational strain, and destabilization of the primary site of attachment occurs. In the other mode, the second sugar by adopting the major solution glycosidic conformation avoids the distortion penalty but seems not to harness the full binding potential in terms of hydrogen bonds and van der Waals contacts with the protein.

Materials and methods

Man [alpha]1-2 Man [alpha]-OMe was prepared using standard methodology (Ogawa et al., 1981).

Structure determination

Data were collected to a resolution of 2.75 Å from crystals of the Con A-Man [alpha]1-2 Man [alpha]-OMe complex. Crystallization and data collection have been reported in detail previously (Moothoo and Naismith, 1999).

The structure was determined by the molecular replacement method as implemented in the CCP4 (CCP4, 1994) program AMORE (Navaza, 1994) using all data from 10 Å to 3.8 Å. The tetramer of the methyl [alpha]-d-mannopyranoside-Con A complex (PDB code 5CNA) was used as the search model with all metal ions, sugar molecules, and water molecules removed. The solution obtained was imported into CNS (Brunger and Adams, 1998) and refinement carried out using the free R-factor (Rfree) as a guide. Rfree was calculated on 10% of data which were excluded from all refinement calculations. Rigid body refinement gave an Rfree of 28.0%. Fo-Fc and 2Fo-Fc electron density maps were calculated from this model. Strong density was observed for a single sugar in the monosaccharide binding site of the four subunits. However, only weak density was observed for the second sugar in some subunits and only methyl [alpha]-d-mannopyranoside was included in the model at this stage. A number of changes in protein structure were made at this stage using "O" (Jones et al., 1991). The metal ions were included in the model with zero electrostatic charge. Further refinement proceeded smoothly by alternating cycles of automated CNS refinement (restrained positional and B-factor) and manual intervention using "O." The protein was refined with the Engh and Huber stereochemical parameter dictionary (Engh and Huber, 1991) and included the CNS bulk solvent correction (Brunger and Adams, 1998). Noncrystallographic restraints were applied throughout for both positional and B-factor refinement. Apart from the 10% of measured data excluded to monitor refinement no cut-offs were applied to the data. Sigma electron density maps were calculated using CNS with all data to 2.75Å and included the CNS bulk solvent correction. Water molecules were added to the model in batches if they satisfied four criteria: they corresponded to a peak > 3.5[sigma] in the Fo-Fc map; they made hydrogen bonds with reasonable stereochemistry; they reappeared in at least 1[sigma] in subsequently calculated 2Fo-Fc maps and that a drop in the free R factor was observed. A second sugar molecule was included into subunits A and D when the Fo-Fc map showed density stronger than 3[sigma] for all atoms in the sugar residue. Statistics on the final model are shown in Table I. The coordinates have been deposited with the Protein Data Bank (1BXH) for immediate release.

Acknowledgments

We are grateful to Steve Homans, John Helliwell, and Patrick Wright for discussions, encouragement and advice. We thanks the referees for helpful comments. The research is supported by B.B.S.R.C. Project Grant B08307 (J.H.N.), Glaxo-Wellcome and a B.B.S.R.C. ROPA award (R.A.F.).

Abbreviations

Con A, concanavalin A; GlcNAc, glucosamine; r.m.s., root mean square; Man, mannose; Man [alpha]1-2 Man [alpha]-OMe, methyl 2-O-([alpha]-d-mannopyranosyl)-[alpha]-d-mannopyranoside; Man [alpha]1-2 Man, 2-O-([alpha]-d-mannopyranosyl)-[alpha]-d-mannopyranose.

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