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Glycobiology Pages 173-181  

Concanavalin A distorts the [beta]-GlcNAc-(1->2)-Man linkage of [beta]-GlcNAc-(1->2)-[alpha]-Man-(1->3)-[[beta]-GlcNAc-(1->2)-[alpha]-Man-(1->6)]-Man upon binding
Introduction
Results
Discussion
Materials and methods
Acknowledgments
Abbreviations
References

Concanavalin A distorts the [beta]-GlcNAc-(1->2)-Man linkage of [beta]-GlcNAc-(1->2)-[alpha]-Man-(1->3)-[[beta]-GlcNAc-(1->2)-[alpha]-Man-(1->6)]-Man upon binding

Concanavalin A distorts the [beta]-GlcNAc-(1->2)-Man linkage of [beta]-GlcNAc-(1->2)-[alpha]-Man-(1->3)-[[beta]-GlcNAc-(1->2)-[alpha]-Man-(1->6)]-Man upon binding

Davina N.Moothoo, James H.Naismith1

Centre for Biomolecular Sciences, Purdie Building, The University, St. Andrews, Scotland KY16 9ST, United Kingdom

Received on June 26, 1997; revised on August 19, 1997; accepted on August 31, 1997

Carbohydrate recognition by proteins is a key event in many biological processes. Concanavalin A is known to specifically recognize the pentasaccharide core ([beta]-GlcNAc-(1->2)-[alpha]- Man-(1->3)-[[beta]-GlcNAc-(1->2)-[alpha]-Man-(1->6)]-Man) of N-linked oligosaccharides with a Ka of 1.41 × 106 M-1. We have determined the structure of concanavalin A bound to [beta]-GlcNAc-(1->2)-[alpha]-Man-(1->3)-[[beta]-GlcNAc-(1->2)-[alpha]-Man- (1->6)]-Man to 2.7Å. In six of eight subunits there is clear density for all five sugar residues and a well ordered binding site. The pentasaccharide adopts the same conformation in all eight subunits. The binding site is a continuous extended cleft on the surface of the protein. Van der Waals interactions and hydrogen bonds anchor the carbohydrate to the protein. Both GlcNAc residues contact the protein. The GlcNAc on the 1->6 arm of the pentasaccharide makes particularly extensive contacts and including two hydrogen bonds. The binding site of the 1->3 arm GlcNAc is much less extensive. Oligosaccharide recognition by Con A occurs through specific protein carbohydrate interactions and does not require recruitment of adventitious water molecules. The [beta]-GlcNAc-(1->2)-Man glycosidic linkage PSI torsion angle on the 1->6 arm is rotated by over 50° from that observed in solution. This rotation is coupled to disruption of interactions at the monosaccharide site. We suggest destabilization of the monosaccharide site and the conformational strain reduces the free energy liberated by additional interactions at the 1->6 arm GlcNAc site.

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

Introduction

Protein carbohydrate interactions underpin an immense and diverse range of biological processes including immune response, cell differentiation, inflammation, and infection (Ni and Tizard, 1996; Lowe and Ward, 1997). Given the key role of protein carbohydrate recognition in so many processes, it is unsurprising that there is considerable interest in potential therapeutic strategies to control, utilize, or block protein carbohydrate binding. The very ubiquity of carbohydrates that makes them so important as therapeutic targets, presents an enormous problem of selectivity as there is considerable potential for unwanted interactions. Understanding the molecular basis of high affinity oligosaccharide protein interactions is required for the rational design of biologically active oligosaccharide analogues.

Con A binds to mannose and glucose with weak affinity (Ka (association constant) 0.82 × 104 M-1) (Mandal et al., 1994b; Williams et al., 1992). The structure of the protein was reported in the 1970s (Hardman and Ainsworth, 1972; Reeke et al., 1975) and has now been extended to 1.0Å (Helliwell and Helliwell, 1996). The 2.9Å [alpha]-Man-OMe Con A complex was reported in 1989 (Derewenda et al., 1989) and provided a clear structural basis for the mannose/glucose selectivity (Goldstein and Poretz, 1986). This structure was extended to 2.0Å resolution (Naismith et al., 1994), and a high resolution study of the glucose complex has been completed (Harrop et al., 1996). The sugar is firmly anchored to the protein by direct hydrogen bonds and van der Waals contacts burying some 75% of its accessible surface. The site of interaction between [alpha]-Man-OMe and Con A is known as the monosaccharide binding site. Such detail was not available at the oligosaccharide level until the trimannoside ([alpha]-Man-(1->3)-[[alpha]-Man-(1->6)]-Man) complex (Ka 3.37 × 105 M-1; Mandal et al., 1994b) of Con A was reported (Naismith and Field, 1996). This structure showed that trisaccharide recognition does not require recruitment of adventitious water molecules to mediate protein sugar hydrogen bonds. It also showed that all three sugar residues were involved in direct contacts (van der Waals and hydrogen bonding) to the protein. The 1->6 terminal mannose binds in the monosaccharide site in a very similar manner as that observed in [alpha]-Man-OMe Con A complex (Derewenda et al., 1989; Naismith et al., 1994) and probably serves as the anchor to bind the other sugar residues. A second report of the trimannoside Con A complex has appeared (Loris et al., 1996) and confirms the first except that in one of the four subunits the trimannoside apparently adopts a different conformation. No functional explanation of this occurrence could be given, and it is unclear whether this is an artefact of crystallization.

Con A belongs to the homologous plant lectin protein family. The function of these lectins in vivo remains unclear. They have, however, proved to be a valuable source of fundamental information on protein carbohydrate recognition. The structures of lectin oligo- and/or monosaccharide complexes have recently been reviewed (Rini, 1995). Plant lectins are not true monosaccharide binding proteins. They are designed to recognize high order carbohydrates, but will bind smaller fragments of the oligosaccharides, such as mannose or galactose. The structures of oligosaccharide complexes of Lathyrus ochrus isolectins I and II (LOLI and LOLII; Bourne et al., 1992, 1994a,b), Griffonia simplicifolia (Delbaere et al., 1993), and Erythina corolladendron (Shaanan et al., 1991) provided experimental evidence that recruitment of water to mediate hydrogen bonds may play an important role in binding oligosaccharides to plant lectins. Mediation of hydrogen bonds by water is a common feature of transport proteins (Mowbray and Cole, 1992; Sharff et al., 1993; Quiocho and Ledvina, 1996), which completely envelop the sugar and form extremely tight complexes (Ka values in the 109 M-1 range). However, oligosaccharide complexes of other proteins (Sixma et al., 1992; Weis et al., 1992; Wright, 1992; Merritt et al., 1994a,b; Stein et al., 1994; Hester et al., 1995; Hester and Wright, 1996; Vandenakker et al., 1996; Wright and Hester, 1996; Wright and Kellogg, 1996) do not appear to require recruitment of additional water molecules for specific protein saccharide interactions. Interestingly structures of a FAB fragment on its own and complexed to carbohydrates from Salmonella typhumurium (Rose et al., 1990; Cygler et al., 1991, 1992; Zdanov et al., 1994) contain a single structurally conserved water molecule which mediates protein oligosaccharide interactions in an analogous manner to the structural water seen in Con A (Naismith and Field, 1996). A wealth of microtitration calorimetry data are available for protein carbohydrate complexes, principally on Con A (Chervenak and Toone, 1995; Mandal et al., 1994a; Mandal and Brewer, 1993; Toone, 1994; Weatherman et al., 1996; Williams et al., 1992) although other lectins and proteins have been studied (Bains et al., 1992; Bundle et al., 1994; Schwarz et al., 1991, 1993; Surolia et al., 1996). These data are complimentary to that obtained by crystallography and quantify the binding affinity. The precise contributions of hydrogen bonding, van der Waals contacts, sugar conformational strain, water rearrangement and surface area occlusion to binding affinity are the subject of intensive investigation as it holds the key to progress in developing modeling methodologies. Combining structural studies with calorimetry data is a powerful way to start delineating the contributions of each of these processes (Gupta et al., 1997).

The pentasaccharide shown in Figure 1 is found in the core of N-linked glycans of the complex type. It binds to Con A with an affinity Ka 1.41 × 106 M-1 and is the tightest carbohydrate Con A complex (Mandal et al., 1994b). We now report the crystal structure of concanavalin A complexed to the pentasaccharide at 2.7Å resolution. All five sugar residues were modeled into electron density, four sugars make direct protein carbohydrate hydrogen bonds and all five make van der Waals contacts with the protein. The structural basis of the specificity of Con A for the pentasaccharide is clear. Compared to other Con A carbohydrate complexes there appear to be subtle changes in the structure of the protein and in the interactions between the sugar residues and the protein. The differences are particularly evident at the monosaccharide site and may partly explain the thermodynamic characteristics of oligosaccharide binding. The conformation of the pentasaccharide is rotated with respect to the observed solution conformation and the predicted energetic minima.


Figure 1 The N-glycan found on the surface of many mammalian cells. The pentasaccharide is boxed, the sugar bound by the monosaccharide site is shown in boldface type. The reducing sugar of the pentasaccharide is shown in italics.

Results

Overall structure of the pentasaccharide protein complex

Two tetramers of Con A are found in the asymmetric unit of the unit cell, giving eight independent 237 residue molecules. Each monomer is a sandwich of two [beta] sheets, the overall fold being identical to the native structure of the protein first reported in the 1970s (Hardman and Ainsworth, 1972; Reeke et al., 1975). The underlying structure changes little on carbohydrate binding. Specific differences in protein structure are dealt with later. The protein main chain is well ordered except for the loop from Asn 118 to Glu 122, this region is consistently disordered or weakly ordered in all structural studies of the protein. The saccharide binding site is remote from the subunit interface.

Differences to native structure

Changes in the positions of Tyr 12, Thr 15, Asp 16, Leu 99, Tyr 100, Ser 204 to Pro 206, Gly 224, and Arg 228 are seen when comparing the structures of native and the pentasaccharide complex of Con A. The changes at Tyr 12, Leu 99, Tyr 100, and Arg 228 are broadly similar to those seen for the [alpha]-Man-OMe complex and have already been discussed in detail by Helliwell and coworkers (Derewenda et al., 1989; Naismith et al., 1994). The main chain at Thr 15 moves approximately 0.3Å to accommodate the 1->3 Man. The situation for Asp 16 is complicated by crystal contacts in the native and pentasaccharide structures and its position is varied in the subunits of the pentasaccharide structure. The change centered on His 205 is quite pronounced (0.3Å on average) and appears to be a result of accommodating the 1->3 arm GlcNAc residue. The main chain at Gly 224 moves substantially (0.5 Å) as result of the carbonyl oxygen forming a polar contact to O4 of the 1->6 arm GlcNAc residue. Displacement of water molecules (and crystal contacts) at the monosaccharide site has already been reported (Derewenda et al., 1989; Naismith et al., 1994); we see a similar pattern for the other four sites. Water molecules or hydrogen bonding crystal contacts are often close to the positions of the hydrogen bonding groups of the sugar. The two 1->3 binding sites are filled by a tight crystal contact in the native structure, involving the loop from Trp 182 to Ser 184. Whether or not this has relevance to the proposed peptide binding site of Con A (Scott et al., 1992) is unknown. In summary, the binding site is preformed in the native structure with water molecules (or crystal contacts) filling the positions of the various sugar residues in the native structure.

Conformation of the bound pentasaccharide

The Con A pentasaccharide complex displays well defined electron density for all five sugar residues in six of the eight monomers. In the other two monomers (both in the same tetramer) which have higher average B-factors; the GlcNAcs on the 1->3 arm are not well defined. As with our trimannoside Con A complex, the glycosidic conformations of the central [alpha]-Man-(1->3)-[[alpha]-Man-(1->6)]-Man of the pentasaccharide (Table I) in all eight subunits are very similar to those reported for conformation of the trimannoside molecule by NMR studies (Homans, 1995). Electron density clearly shows that the anomeric hydroxyl (on the reducing mannose) is found in the alpha configuration (Figure 2) this appears to be a result of the anomeric oxygen's participation in crystal contacts. The N-linked glycan does not have a reducing sugar at this position and the 'reducing" mannose is [beta] 1->4 linked to a GlcNAc sugar (Figure 1). We can see no structural hindrance to Con A binding such a beta linkage. In a second study of the trimannoside Con A complex (Loris et al., 1996), a second minority solution binding conformation of trimannoside was found in one of four subunits, in direct contrast to our result, where the same majority solution conformation of sugar was found in all four subunits (Naismith et al., 1994). There is no evidence for any alternate conformation of the pentasaccharide in this structure in any of the eight subunits. The conformation of the 1->3 arm [beta]-GlcNAc-(1->2)-Man glycosidic linkage is very close to that observed in solution (Homans, 1995). However, the [psi] dihedral angle (Table I) of the 1->6 arm [beta]-GlcNAc-(1->2)-Man glycosidic linkage is dramatically different (~50°) from the solution minimum (Homans, 1995). This distortion is required to avoid penetration of the GlcNAc sugar residue into protein structure; the consequences of this are discussed later.

Discussion

The interactions between Con A and the pentasaccharide, the structural basis of specificity

The pentasaccharide sits in an extended continuous groove on the surface of the protein (Figure 3) and buries over 1000 Å2 of surface area. In terms of buried surface area it is one of the largest continuous protein carbohydrate interfaces characterized. For discussion we have split the continuous carbohydrate binding site into five components; in reality these overlap and share several protein residues. The five sites are the 1->3 arm GlcNAc site, the 1->3 Man site, the reducing Man site, the monosaccharide site, and the 1->6 arm GlcNAc site. These sites correspond to the cognate sugar residues shown in Figure 1. This is in contrast to the oligosaccharide structures of LOLI and LOLII that have effectively two discrete binding sites on the protein surface (Bourne et al., 1992, 1994a,b).


Figure 2 The difference electron density map contoured at 3[sigma] was calculated by removing the sugar from the model, then refining with simulated annealing (2000K) followed by Powell minimization and B-factor protocols. All five sugars are in good electron density (figure produced using O (Jones et al., 1991).

Table I . Torsion angles of the glycosidic linkages

  [phiv]a (0) [psi]a (0) [omega]a (0)
[alpha]-Man-OMeb,c 63 ± 5 ND ND
Trimannosideb
[alpha]-Man-(1->6)-Man 66 ± 3 -171 ± 3 73 ± 5
[alpha]-Man-(1->3)-Manb 67 ± 2 -112 ± 5 ND
Pentasaccharidec
1-6 arm [beta]-GlcNAc-(1->2)-Manb -75 ± 3 -129 ± 3 ND
1-3 arm [beta]-GlcNAc-(1->2)-Manb -85 ± 6 -78 ± 5 ND
[alpha]-Man-(1->6)-Man 71 ± 3 179 ± 2 72 ± 3
[alpha]-Man-(1->3)-Manb 65 ± 2 -102 ± 2 ND
Solution valuesd
[beta]-GlcNAc-(1->2)-Manb -92 ± 16 -83 ± 14 ND
[alpha]-Man-(1->6) Manb 70 ± 20 -170 ± 20 60 ± 20
[alpha]-Man-(1->3) Manb 80 ± 15 -116 ± 25 ND
aThe torsion angles [phiv], [psi] and [omega] are: [phiv] is O5 - C1 - OX - CX, [psi] is C1 - OX - CX - C(X-1), and [omega] is OX - CX - C(X-1) - C(X-2). Standard deviations are given. The sugar at the monosaccharide site is shown in boldface type.
bOne or more angles are not defined (ND) for these sugars.
cAveraged over all ordered fully ordered sugars.
dHomans, 1995.


Figure 3 (a) The pentasaccharide Con A complex. The binding site is an extended groove on the surface of the protein. (Figure generated by RASTER3D; Merritt and Murphy, 1994).

Fig. 3. (b) A schematic representation of the hydrogen bonds between the protein and the pentasaccharide.

The monosaccharide site was first identified by Helliwell and coworkers (Derewenda et al., 1989); the reducing Man and 1->3 Man sites were defined by the determination of the trimannoside complex (Naismith and Field, 1996). The residues forming these sites in the pentasaccharide complex are unchanged from these earlier descriptions. The interactions at these sites are listed in Tables II and 2. There are a number of subtle changes in the interactions at the monosaccharide site compared to previous descriptions that we deal with more fully later.

The 1->6 arm GlcNAc site is formed by Gly 98, Ser 168 and the loop Thr 226 to Leu 229. One face of the sugar residue is effectively parallel to the protein surface. The loop from Thr 226 to Leu 229 interacts extensively with the sugar residue and forms one side of the binding site. Gly 98 and Ser 168 sit either side of the N-acetyl group, their contacts are listed in Tables II and III. Gly 98 is a key residue, mutation to any other amino acid would abolish binding; as the side chain would intrude into the binding site clashing with the 1->2 linked sugar. Ser 168 hydrogen bonds to the sugar; presumably loss of this H-bond would diminish the interaction and any increase in the size of the residue would inhibit binding of the sugar conformation we observe. The 1->6 arm GlcNAc sugar makes a fairly extensive interaction with the protein (Tables II, II). The 1->3 arm GlcNAc site is much less well defined, the side chain of His 205 caps O4 of the sugar (Figure 3). The ring of the sugar sits on a shallow cleft defined by Pro 13 and the side chain of Tyr 12. There are no hydrogen bonds between this sugar residue and the protein and only 7 van der Waals contacts.

LOLI and II also bind sugars 1->2 linked to the monosaccharide site and both have a Gly in an analogous position to Gly 98. However, in the octasaccharide structures (Bourne et al., 1992, 1994b) which contain a [beta]-GlcNAc-(1->2)-Man disaccharide, the PHI torsion angle of GlcNAc sugar is rotated almost 180° with respect to that in our structure. This is because in LOL I and II Ser 168 is replaced by an Asn that would clash with the N-acetyl group. The interactions at this site are much less extensive in LOLI and II than in Con A, having less than half the number of van der Waals interactions. The lectin from Dioclea grandiflora is homologous to Con A and binds the trimannoside with similar affinity (Chervenak and Toone, 1995). It has a Gly at position 98 but does not recognize the pentasaccharide (Gupta and Brewer, 1996). This is due to changes at Ser 168 (to Asn), which creates a steric clash with the N-acetyl group, and at Thr 226 (to Gly), which removes a hydrogen bond and van der Waals contacts. Interestingly Dioclea grandiflora and Con A both bind the high mannose type structures shown in Figure 4 with similar affinity (Mandal et al., 1994b; Gupta and Brewer, 1996) approximately Ka 9 × 105 M-1. These high mannose sugars precipitate both Con A and Dioclea grandiflora from solution (Gupta and Brewer, 1996), suggesting they are able to bind two Con A molecules. The mode of binding of these sugars is not yet clear but we suggest in Figure 4 how high mannose sugars may bind to Con A. The [alpha] 1->2 linked terminal mannose residues would sit in the 1->3 and 1->6 GlcNAc sites identified by this study and unlike GlcNAc would not clash with the Asn 168 residue in Dioclea grandiflora.

Table II . Hydrogen bonding and polar contact (<3.5 Å) distances between sugar and protein in the six fully ordered sites
Protein Sugar Average distance
(typically ±0.1Å)
  GlcNAc, 1->6 arm  
Thr-226 OG1 O3 2.6
Gly-224 Oa O4 2.8
Thr-226 Oa O6 3.4
Ser-168 OG O7 2.6
  1->6 Man  
Arg-228 N O3 3.0
Asn-14 ND2 O4 2.9
Arg-228 Na O4 3.3
Asp-208 OD1 O4 2.8
Leu-99 N O5 3.0
Leu-99Na O6 2.9
Tyr-100 N O6 3.0
Asp-208 OD2 O6 3.0
  Reducing Man  
OWb O2 2.7
Asp-16 OD2 a,c O2 3.3
Tyr-12 OH O4 2.8
  1->3 Man  
Pro-13 Oa O3 2.9
Thr-15 N O3 2.9
Thr-15 OG1 O3 3.0
Thr-15 OG1 O4 2.6
Asp-16 Na O4 3.0
aThese contacts are within hydrogen bonding distance; however, the donor-H-acceptor geometry differs substantially from the linearity expected for a hydrogen bond. The sugar at the monosaccharide site is shown in boldface type.
bOW is the structurally conserved water molecule. This is hydrogen bonded to Asn-14, Asp-16, and Arg-228.
cThis contact is absent in some units due to crystal contacts.

Table III . Van der Waals contacts (<4.0Å) between Con A and the pentasaccharidea
Sugar Residue
1->6 arm GlcNAc Gly-98 (6), Leu-99 (1), Ser-168 (5), Gly-224 (2), Thr-226 (7), Arg-228 (2), Leu-229 (2)
1->6 Man Tyr-12 (1), Asn-14 (2), Gly-98 (3), Leu-99 (9), Tyr-100 (5),
Ala-207 (2), Asp-208 (9), Gly-227 (4), Arg-228 (9)
Reducing Man Tyr-12 (3), Leu-99 (2), Tyr-100 (1)
1->3 Man Tyr-12 (2), Pro-13 (3), Asp-14 (4), Thr-15 (10), Asp-16 (4)
1->3 arm GlcNAc Tyr-12 (2), Pro-13 (2), His-205 (3)
aAveraged over the six well ordered binding sites. The sugar at the monosaccharide site is shown in boldface type.

Contribution of the other sites to affinity


Figure 4 The high mannose N-linked glycan. We predict that the five boxed sugars mimic the pentasaccharide of this study; the sugar we predict that is bound by the monosaccharide site is shown in boldface type. The sugars which we propose bind and cross-link to another Con A molecule are underlined.

The pentasaccharide and trimannoside Con A complexes suggest that there are in principle two additional mannose binding sites (reducing and 1->3); however, calorimetry (Mandal et al., 1994b) and structural analysis (Derewenda et al., 1989) of the [alpha]-Man-OMe Con A complex show that only one sugar is bound. Tabulation of the van der Waals contacts, buried surface area and hydrogen bonds shows that these two Man sites have considerably fewer interactions than the monosaccharide site (Table II, III). From these tables we suggest that the reducing mannose site contributes little to the protein carbohydrate interaction but that the 1->3 Man will contribute to an increase in affinity. The 1->3 arm GlcNAc residue makes very few contacts with the protein, buries little surface area and its binding site is the least well defined. This sugar residue does induce a clear change in the main chain position centered on His 205 when compared to other Con A structures. Given the insubstantial nature of its interaction and distortion in protein main chain we would argue that the 1->3 arm GlcNAc residue does not make a large additional contribution to binding. The 1->6 arm GlcNAc site would appear able to make a substantial contribution to binding as it makes an array of van der Waals contacts with the protein, buries a substantial surface area, and makes specific hydrogen bonds with the protein.

The 1->6 arm GlcNAc site

The disaccharide [beta]-GlcNAc-(1->2)-Man that we predict will bind analogously to the 1->6 arm of the pentasaccharide only liberates 5.2 kcalmol-1 (Mandal et al., 1994b). This is identical to Man which liberates approximately 5.2 kcalmol-1 (E.J.Toone, personal communication). It appears that [beta] 1->2 addition of GlcNAc to the monosaccharide mannose is energetically neutral, despite the large increase in the number of protein carbohydrate contacts. Jencks has pointed out that the free energy of binding a single compound AB to a protein is not the algebraic sum of free energy of binding of the isolated components A and B (Jencks, 1981). The difference between the summation of the free energy of binding A and B and the composite molecule AB, is the interaction energy. The interaction energy has several components, some of which contribute favorably to binding, some of which contribute unfavorably. The most well known of these is the chelate effect, that is, the entropy penalty paid by binding AB (loss of translational and rotational degrees of freedom) is less than binding A and B (Page and Jencks, 1971). The chelate effect always contributes favorably to the binding energy. The chelating disaccharide ([beta]-GlcNAc-(1->2)-Man) fails to increase the affinity for the protein over the simple monosaccharide. We believe we have identified structural changes in the pentasaccharide and to the monosaccharide site that contribute to the interaction energy, decreasing the affinity of binding of the pentasaccharide.

Rotation of the 1->6 arm [beta]-GlcNAc-(1->2)-Man glycosidic linkage

We have identified a large rotation (~50°) in the glycosidic linkage PSI torsion angle from that seen in free solution. The dynamics observed for this linkage in free solution suggest it is a relatively rigid conformation (Homans, 1995). The energy surface for two glycosidic angles in the simple disaccharide (PHI and PSI) [beta]-GlcNAc-(1->2)-Man has been calculated (Imberty et al., 1991). While the energetics of the simple disaccharide will be different from the pentasaccharide, it provides a reasonable approximation to the 1->6 arm [beta]-GlcNAc-(1->2)-Man linkage. Bourne et al., saw an alternative conformation for the [beta]-GlcNAc-(1->2)-Man linkage in their LOL I octasaccharide structure (Bourne et al., 1992). However, their observed glycosidic linkages were within ±30° of a second energetic minimum for this linkage. Plotting the glycosidic torsion angles we have observed on the energy surface suggests that the conformation we see is ~3 kcalmol-1 above the global minimum and is remote from any other minimum. The crystal structure of the Se155-4 antibody Fab fragment in complex with [alpha]-Gal-(1->2)- [[alpha]-Abe-(1->3)]-Man, the [alpha]-Gal-(1->2)-Man [phiv] torsion angle is rotated by about 40° from the theoretical global energy minimum, about 3 kcalmol-1 above the minimum (Cygler et al., 1992). However, Cygler et al. (1992) suggested that this conformation is favored by a hydrogen bond within the sugar molecule which was not predicted by theoretical calculations. We find no such 'compensating" internal hydrogen bond to explain the rotation we observe. We have not found any other examples of the sugar conformation in the bound state being so energetically distant from the conformation seen or predicted for the uncomplexed sugar.

Table IV . Differences among the Con A complexes at the contacts at the carbohydrate binding sites
  H-bonds Polar contactsa Van der Waals contacts
Pentasaccharide complexb
1->6 arm GlcNAc 2 2 25
1->6 Man 6 2 44
Reducing Man 1 1 6
1->3 Man 3 2 23
1->3 arm GlcNAc 0 0 7
Trisaccharide complex
1-> Man 6 2 46
Reducing Man 1 1 8
1->3 Man 3 2 23
[alpha]-Man-OMe complexc
[alpha]-Man-OMe 6 2 48
aPolar contacts are those within hydrogen bonding distance (< 3.5Å) which have geometry inconsistent with a hydrogen bond. The sugar at the monosaccharide site is shown in boldface type.
bAveraged over the six well ordered sites in the pentasaccharide complex.
cThe C and D subunits in this complex are distorted by protein sugar crystal contacts are not included in this comparison (Naismith et al., 1994).

Distortion of the monosaccharide site

A comparison of the monosaccharide site in the pentasaccharide complex and the mannose complex is summarized in Table IV. Superimposing the 237 c[alpha] protein atoms of the pentasaccharide and [alpha]-Man-OMe complexes give an r.m.s. deviation for the 13 sugar atoms of 0.33Å. This distortion in the sugar position relative to the protein is centered around the O2 of the mannose residue which is [beta] 1->2 linked to a GlcNAc residue in the pentasaccharide. Without this movement in the O2 position the GlcNAc residue would sterically clash (approaches < 2.0Å) with residues Thr 222, Ser 168, and Gly 98. The presence of the [beta] 1->2 linkage alters the main chain trace of the loop from Gly 98 to Tyr 100, centered at Leu 99 the average shift is 0.2Å. This region is one of the four loops that form the monosaccharide site. We note that while there is a reduction in the number of van der Waals contacts at the monosaccharide site, the number of hydrogen bonds remains the same (Table IV).

We hypothesize that the [alpha]-Man-OMe complex represents the energetic minimum of the monosaccharide site. We believe the combination of the distortions (discussed above) decrease the free energy liberated by the monosaccharide site lowering the overall affinity of the oligosaccharide for Con A. In short, we suggest these are structural manifestations of the interaction energy. We predict that the free energy liberated by the disaccharide [beta]-GlcNAc-(1->2)-Man (5.2 kcalmol-1) is considerably less than algebraic sum of the free energy liberated separately by GlcNAc and Man at their cognate sites. The [Delta]G0 value for Man is 5.2 kcalmol-1, and although the value GlcNAc is known (5.2 kcalmol-1) it probably reflects binding at the monosaccharide site. Consequently, we have no reliable estimate for the energy liberated by GlcNAc at the 1->6 arm GlcNAc site. However, the extent of the contacts between the protein and sugar suggests this is not zero. Quantitation of the components of the interaction energy that are likely to include solvent reorganization is beyond our reach at present. However, the combination of calorimetry and structural work continues to generate a significant resource for theoretical approaches to oligosaccharide recognition

Materials and methods

Con A was obtained from Sigma (Poole, United Kingdom) and [beta]-GlcNAc-(1->2)-[alpha]-Man-(1->3)-[[beta]-GlcNAc-(1->2)-[alpha]-Man-(1->6)]-Man was obtained from Dextra Laboratories (Reading, United Kingdom). Crystals of the protein carbohydrate complex were obtained from a sitting drop using a solution containing a 30-fold excess of sugar over Con A (protein concentration 6 mg ml-1), 1 mM MnCl2, and 1mM CaCl2. This was equilibrated against a reservoir buffered at pH 6.0 containing 15% polyethylene glycol 6K as the precipitant Data were recorded as 200 nonoverlapping 20 min 1° oscillations. The data were processed and merged with DENZO and SCALEPACK (Otwinowski, 1993). The space group was assigned as C2 with unit cell parameters a = 176.5Å, b = 122.8Å, c = 124.6Å, [beta] = 134.2°. The asymmetric unit contains two tetramers; Matthew's number 2.5 Da Å-3, ~46% solvent. Data collection and reduction statistics are shown in Table V. The structure was determined by the molecular replacement method as implemented in the CCP4 (CCP4, 1994) program AMORE (Navaza, 1994) using all data from 12Å to 3.4Å. The trimannoside Con A complex of Con A (PDB code 1CVN) (Naismith and Field, 1996) was used as the search model (with all metal ions, sugar molecules, and water molecules removed. Strong difference electron density was observed for all metal ions and 32 of the 40 possible sugars, with weak density for 6 but ambiguous density for 2. A number of changes in protein structure were made manually at this stage using 'O" (Jones et al., 1991). The metal ions were included in the model with zero electrostatic charge and the sugar molecules included when the Fo-Fc map showed density stronger than 3[sigma] for all atoms in the sugar residue. Further refinement proceeded smoothly by alternating cycles of automated X-PLOR (Brunger, 1992) refinement and manual intervention using 'O". The protein was refined with the Engh and Huber stereochemical parameter dictionary. Noncrystallographic restraints were applied throughout for both positional and B-factor refinement. Despite the limited resolution we did carry out restrained B-factor refinement because we had 8-fold non-crystallographic symmetry and saw a clear (>4%) improvement in Rfree. All individual B-factors were reset to the overall average temperature factor after each manual intervention. Apart from the 10% of measured data excluded to monitor refinement no cut-offs were applied to the data. A bulk solvent correction was included in the X-PLOR refinement with parameters for solvent density 0.34e-Å-3, solvent radius 0.25 Å and B factor 50 Å2. Electron density maps were calculated using SIGMAA (Read, 1986) modified coefficients with all data to 2.7 Å and included the X-PLOR 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. Statistics on the final model are shown in Table V. The coordinates and structure factors have been deposited with Protein Data Bank (Bernstein et al., 1977) (entry codes 1TEI and 1TEISF).

Table V . Crystallographic data collection statistics and refinement statistics
Unique reflections 53516
Completeness of data (%) (25.0 - 2.7Å/2.8-2.7Å) 97.1/95.8
Rmerge (I) (%) (25.0 - 2.7Å/2.8-2.7Å)a 9.4/33.7
Average data redundancy (25.0 - 2.7Å/2.8-2.7Å) 4.2/4.1
% of data > 1[sigma] (25.0 - 2.7Å/2.8-2.7Å) 93/83
Refinement
Resolution range (Å) 25-2.7
Rfree (%)b 21.9
R factor (%) 17.9
Bond r.m.s. deviation (Å)c 0.01
Angle r.m.s. deviation (0)c 1.746
Non-crystallographic symmetry r.m.s. deviation (c[alpha] atoms) (Å) 0.12
B-factor bonded atoms r.m.s. deviation (Å2)e 3.26
Ramachandran core/additional (%)d 85.3/14.7
Protein mean B-factor (Å2) 29.1
Sugar mean B-factor (Å2) 35.9
Solvent mean B (Å2) 22.5
Number of protein atoms 14472
Number of sugar atoms 496
Number of solvent atoms 74
Number of metal ions 16
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).
dCore and additionally allowed regions as defined by PROCHECK (Laskowski et al., 1993). No residues are in the generously allowed or disallowed regions. All B-factor calculations exclude the 370 protein atoms (2.6%) and 21 sugar atoms (4.2%) (1->3 arm GlcNAc in F and H subunits) which were stereochemically modeled.
eCalculated 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.

Acknowledgments

We thank the Wellcome Trust (043586/Z/95/Z/MP/RF/PK) for equipment support. We are grateful to Rob Field, John Helliwell, Steve Homans, Charlie Weller, and Trevor Rutherford for discussions, encouragement, and advice. The research is supported by the B.B.S.R.C. (B08307).

Abbreviations

Con A, concanavalin A; GlcNAc, glucosamine; LOLI, Lathyrus ochrus isolectin I; LOLII, Lathyrus ochrus isolectin II; Man, mannose; r.m.s., root mean square.

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