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.
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 |
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.
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
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
Table II.
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 |
Table III.
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 |
Table IV.
[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 |
Subunit D
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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
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
In subunit C once the monosaccharide was built into the model, we were unable to model in a second sugar.
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.
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.
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.).
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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