2 Molecular Biophysics Unit, Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560012, India; and 3 Bioinformatics Centre, Indian Institute of Science, Bangalore 560012, India
Received on May 12, 2003; accepted on June 19, 2003
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Abstract |
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Key words: affinity enhancement / cross-linking / multiple binding sites / oligosaccharide binding
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Introduction |
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Garlic lectin belongs to a well-conserved family of bulb lectins that are found in monocotyledonous families, such as Amaryllidaceae, Alliaceae, Araceae, Liliaceae, and Orchidaceae (Barre et al., 1996). The structures of several members of this group are now available, all belonging to the ß-prism-II fold comprising three antiparallel four-stranded ß-sheets arranged as a 12-stranded ß-barrel. The bulb lectins, owing to their high mannose specificity, exhibit unique biological properties, such as selective inhibitory activity against HIV and other retroviruses and selective agglutination of rabbit but not human erythrocytes (Balzarini et al., 1991
; Pusztai et al., 1993
). These properties, which may lead to several useful applications, are presumably due to their ability to bind glycoproteins containing oligomannosides (Hester and Wright, 1996
; Kaku et al., 1991
). Although all bulb lectins have exclusive specificity toward mannose, their agglutination specificities and hence their biological roles vary. For example, lectins from Amaryllidaceae and Orchidaceae families (such as snowdrop lectin) are potent inhibitors of HIV due to their ability to bind to glyco protein 120, whereas garlic lectin, which has no detectable antiretroviral activity, can bind to high-mannose glycoproteins, such as invertase and alliinase with very high affinity (Barre et al., 1996
; Dam et al., 1998
).
The structural basis for these differences in binding specificities will provide a framework to study the molecular mechanisms of the larger functional roles of lectins. Complexes of lectins with large oligosaccharides are not easily amenable to X-ray crystallography, due to the inherent difficulties in crystallizing them, thus making it important to study them by computational methods. Here we seek to study the structures of complexes of garlic lectin with di-, tri- penta-, hepta-, octa-, and nonamannosides by a molecular modeling approach to explain its very high affinity to oligosaccharides exhibiting high mannose structures, specificities for the linkages in these complex sugars as well as the role of multiple binding sites in the lectin.
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Results |
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A systematic study was carried out to demonstrate that the inference from the methylated mannose studies holds good for oligosaccharides as well. It was realized that the procedure used for this also helps in determining whether the sugars exhibit differences in affinity among the six binding sites on the garlic lectin dimer. Docking of the dimannosides Man1-2Man, Man
1-3Man, Man
1-4Man, and Man
1-6Man into the six sites was therefore attempted. Starting with the nonreducing end, each of the two mannose residues was separately docked into each site by superposing the ring atoms of the model on to the ring atoms of the crystallographically observed mannose molecule. Interactions with the protein were assessed at each stage both by graphics inspection as well as through measurement of hydrogen bonds and van der Waals interactions between the protein and the sugar. In each case, docking of the second residue into the primary site led to unacceptable steric clashes. The nonreducing residues of the dimannosides could be successfully docked into all the six sites except in the case of Man
1-4Man, which could be docked into only four sites. Docking of even the first residue of this dimannoside into sites E and I led to unacceptable steric clashes. However, energy minimization (see later discussion) led to acceptable docking at these sites as well. Thus 12, 13, 14, and 16 linkages with the nonreducing ends overlaying with the crystallographically identified mannoses, in all the six sites, is possible, at least at the disaccharide level, although the docking of Man
1-4Man at two of the sites was somewhat contrived. Therefore all the terminal mannosyl residues in the nine sugars considered are candidates to be docked at the binding sites. Also, binding at any site appeared to be completely independent of binding at other sites. During the procedure, the conformation of the oligosaccharides and the influence of conformational changes on the binding strengths were also evaluated. A range of 40° around each dihedral angle specified by SWEET for each sugar was searched at intervals of 5°. The systematic conformational search varying the dihedral angles clearly indicated that such changes did not significantly alter binding in any of the cases.
Lectincarbohydrate complexes
Next the construction of complexes with all the nine oligosaccharides was attempted. Altogether 18 models were constructed with the same sugar docked in the same way in all the six binding sites. In view of the calculations outlined, only the nonreducing ends were used for docking at the primary sites. Thus the first model involved Man1-2Man attached to all the six binding sites with the first residue superposing on the crystallographically observed mannose molecule in each case. Three more lectindisaccharide complexes were similarly constructed. Two models could be constructed using the trisaccharide, one with one of the terminal mannosyl residue at the six primary sites and the other with the other terminal mannosyl residue at the sites. Likewise, three models each could be constructed with the penta-, hepta-, octa-, and the nona-oligosaccharides, because each of these sugars has three nonreducing mannosyl residues.
Energy minimization was carried out on all the 18 models. The root mean square (RMS) deviations in C positions between the original and the minimized models varied between 0.36 Å in the case of a complex with a disaccharide and 0.99 Å in the case of a complex with an oligosaccharide involving nine mannose residues. These values compare well with the RMS deviations in C
positions between the two monomers in the original crystallographic model (0.85 Å). The RMS deviations when the monomers in the refined models of the dimers were superposed were between 0.86 to 0.90 Å in most cases. The values exceeded 1 Å (1.031.41 Å) in the case of three complexes with oligosaccharides containing eight or nine mannose residues. Similar RMS deviations were exhibited by the atoms in binding site residues and the sugar, when the original and the minimized models were compared. RMS deviations involving these atoms in the initial models indicated sites E and I to be most symmetric (0.610.72 Å) and sites G and K the least symmetric (1.341.77 Å), with respect to the noncrystallographic dyad that relates the two monomers in the lectin dimer. The same trend, though with somewhat higher RMS deviations, is maintained in the energy minimized models as well. The values cited indicate that the minimization had proceeded sensibly.
The interactions at each of the six sites were separately examined in all the models. Energy was calculated individually for the interaction of each carbohydrate molecule. Also calculated in each case were the surface area buried on complexation and shape complementarity of their surfaces. Unacceptable steric clashes in a few instances expressed themselves as high interaction energies, and such instances could be easily identified. The interaction energy, hydrophobic surface area buried on complexation and shape complementarity for the complexation of each carbohydrate molecule with the different sites on the lectin are listed in Table I.
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Linked lectin molecules: exploratory effort
Singly linked pairs of lectin molecules
The next obvious step was to explore the possibility of a given oligosaccharide simultaneously binding to and hence linking two or more lectin molecules. Considering that each dimer has six binding sites and each carbohydrate molecule has one to three terminal mannosyl residues capable of binding to these sites, the number of possible distinct pairs or groups of linked lectin molecules becomes large. The possibility, even in principle, of linking three molecules exists only in the case of the pentasaccharide and the higher oligomers. Careful visual examination showed that the docking of any of the oligosaccharides simultaneously to three lectin molecules in any manner would lead to severe steric clashes, which could not be expected to be relieved through energy minimization. Therefore only linked lectin pairs were considered.
The disaccharides have only one nonreducing end each. Therefore they cannot link two lectin molecules. In the trisaccharidelectin complex, in which a trisaccharide is bound to each of the binding sites, there are six free terminal residues, one belonging to each of the six trisaccharides bound to the lectin, which are capable of docking into one of the six binding sites in a neighboring lectin dimer. In each of the complexes involving higher oligosaccharides, there are 12 such free terminal residues. Each of them can in principle dock into any one of the six sites in another dimer. In practice, all the possibilities are not realized owing to steric clashes. All the possible linked dimers were generated by systematically superposing, one by one, each of the terminal residues in each energy-minimized complex onto the bound mannose in each of the six binding sites in another lectin dimer. Of the 72 (or 36 in the case of the trisaccharide) linked dimersthose involving more than 20 steric clashes with an interatomic distance of 2.3 Å or lesswere rejected as sterically unacceptable. This criterion is arbitrary, but appeared reasonable. This resulted in the singly linked dimers listed in Table II. In Table II, each pair is identified in terms of the terminal residues involved and the binding sites used. For example, among the pairs linked by the pentasaccharide, M1FM3G refers to the linked pair in which the terminal mannose M1 occupies the binding site F in one dimer and the terminal mannose M3 occupies binding site G in other.
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The intersite distances (Sij), measured as the distance between the centers of the bound mannose rings, were calculated. Only those pairs (Figure 3) of terminal residues in the complex in which the distance between centers of the two rings (Tij) lies within ± 3 Å of any of the Sij's were retained for further calculations. This distance filter reduced the number of pairs of terminal residues to be considered for double linkages. For example, in the case of the nonasaccharide, the number came down from 1980 to 188. To each possible pair, a lectin dimer was docked such that the least squares deviation between the 12 ring atoms in the pair of terminal residues and those in the bound mannoses in the 2 concerned sites in the incoming lectin dimer is a minimum. Among the double-linked dimers thus constructed, those with more than 40 unacceptable contacts (interatomic distance 2.3 Å or less) were rejected. The remaining possible double linked dimers are listed in Table II.
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Further calculations on linked lectin molecules
One anomaly in the calculations using a single, energy-minimized conformation for each oligosaccharde was immediately obvious. Several possible singly linked lectin pairs involving the heptasaccharide could be identified (Table II). The same was true with those involving the nonasaccharide, too. However, none of them could be identified when the octasaccharide was used as the link. This appeared strange as the heptasaccharide could be obtained by the deletion of a mannosyl residue at one end of the octasaccharide, whereas the nonasaccharide resulted when a mannosyl residue was added at another end of the octasaccharide. Careful examination of interactions involving the three oligosaccharides in their minimum energy conformations showed that this anomaly was due to a steric effect. Several linkages involving M1 and M4 of the heptasaccharides occur. However, when a mannosyl residue is added to M7, the added residue, in the minimum energy conformation, prevents the interaction of M4 with the second lectin in the pair. The nonasaccharide results from the addition of a mannosyl residue (M5) to M4. Now, an M1M5 linkage is possible in spite of the presence of M9. It could also be seen that an M1M4 linkage involving the octasaccharide becomes possible when the conformation of the M6-M7-M8 arm is varied.
This observation indicated that the conformational flexibility, although it did not matter much at the disaccharide level, needs to be taken into account to avoid misleading results on the higher oligosaccharides. It is reasonable to assign three possible values, one close to that in the energy-minimized conformation and two at 20° on either side of it, for each torsion angle. Two torsion angles are involved in defining the conformation about a 12 or 13 linkage. Three are involved when the linkage is 16. Then the number of conformers to be considered for the hepta-, the octa-, and the nonasaccharides work out to be 314 (5 million), 316 (
43 million), and 318 (
387 million), respectively. These are formidable numbers, and it is nearly impossible to explore all the conformers. A comparison of the results involving the trimer, the pentamer, and the heptamer indicated them to be apparently on expected lines. It was therefore decided to keep the conformation of the pentamer fixed as corresponding to the energy-minimized structure. The number of torsion angles to be varied in the heptamer, octamer, and nonamer then reduced to 4, 6, and 8, respectively, and the number of different conformers to be dealt with to 81, 729, and 6561, respectively.
The same method used in calculations with energy minimized oligosaccharide structures, was used for searching pairs of lectins linked by the different conformers. The single linkages found possible with the heptamer, octamer, and nonamer are given in Table III. Understandably many more linkages than those listed in Table II could be identified when the conformational flexibility of the oligosaccharides was also partially taken into account. The same is true about double linkages (Table III). In general, several pairs of conformers can lead to a given double linkage.
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Discussion |
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As indicated earlier, the first attempt was to dock all the nine sugars individually to each of the six binding sites on the lectin. This led to 18 possible lectinsugar complexes in which all the sites are occupied by the same sugar. These calculations showed that oligosaccharides bind to the protein through their nonreducing ends only. Energy minimization indicated docking at all the sites to be possible in most instances. The interaction energies and surface area buried on complexation were not dramatically higher in the case of higher oligomers. In fact these indices could not explain even the higher affinities of the lectin toward dimannosides compared to that for mannose. It may, however, be noted that not unexpectedly, the surface area buried on complexation (including the nonpolar component) generally increased with increased size of the oligosaccharide. As already mentioned, it was also ascertained that no sugar can simultaneously bind to two or more sites, as the distances between binding sites are higher than those between the terminal sugars in the oligosaccharides. It is in this context that the possibility of each sugar binding to more than one lectin molecule was systematically explored.
The pentasaccharide and higher oligomers have three terminal residues each, which can interact with the sugar binding sites in the lectin. M1 (Figure 2), which is involved in almost all cross-links, may be referred as the primary anchor in all cases, including the trisaccharide. M3 in the pentamer, M4 in the heptamer and the octamer, and M5 in the nonamer could be appropriately called the middle anchor. Geometrically, M3 in the trisaccharide corresponds to the middle anchor in the pentamer. M5 in the pentamer, M7 in the heptamer, M8 in the octamer, and M9 in the nonamer may be called the terminal anchors. This nomenclature will be used in the following discussion wherever appropriate.
The M1-M2-M3 fragment is common to the tri- and pentasaccharides. The torsion angles that define the conformation of this fragment are also very similar in the two sugars; the difference between the corresponding angles in the two cases is in the range of 223°. No single linkages involving M5 of the pentamer exist. Therefore, not surprisingly, the trimer and the pentamer are involved in exactly the same type of single linkages (Table II). Because the calculations used to explore double linkages do not require rigorous superposition of the anchor residues and the mannose molecules bound to the lectin, M5 also figure in such linkages. Two of the three double linkages generated by the trimer are produced by the pentamer also. In the case of the third double linkage, the distance between M1 and M3 in the pentasaccharide just falls outside the appropriate range on account of the slight difference in the conformation of M1-M2-M3 fragment between the trimer and the pentamer. Both tri- and pentasaccharides led to similar type of single cross-linked aggregates, consistent with similarities in their binding affinities.
Interestingly, all the linkages produced by the heptamer and the octamer involve M1. In the case of pairs connected by the nonamer, linkages involving the middle anchor and the terminal anchor also occur. These are the linkages that primarily contribute to the additional numbers in the pairs linked by the nonamer. The numbers directly indicate the potential for the formation of linked lectin molecules. The higher potential of the high-mannose oligosaccharides to form linked molecules as compared to the tri- and the pentamanno-oligosaccharides is reflected in the number of different types of aggregations found possible with it, again correlating with the orders of magnitude higher affinity for the lectin. Admittedly more linkages involving the trimer and the pentamer would have resulted if their conformational flexibility was taken into consideration. However, for reasons already mentioned, it was computationally impossible to carry out calculations for all the possible conformers. Thus, as far as the heptamer and the higher oligomers are concerned, the attempt is to identify linkages additional to those made by the pentamer, as the object of the exercise is comparison of the potential of different oligosaccharides to form single and double linkages. Table III clearly shows that the linkages formed by the heptamer and the higher oligomers would be very substantially higher than those formed by the pentamer for a given conformation of the pentasaccharide. In general, on account of the presence of multiple binding sites on the lectin, linked pairs can be successively linked to other pairs to produce large aggregates. Although no rigorous proof exists for a direct relation between ability to form linkages and affinity, it appears reasonable to expect the ability to form more linkages to result in larger aggregates. The differences in affinity appears to correlate, admittedly qualitatively, with the ability to form single and double linkages.
Typical singly and doubly linked pairs of lectins are illustrated in Figures 4 and 5. Of particular interest are symmetrically doubly linked dimers (Figure 5d), which appear as tetramers with 222 symmetry. "Tetramers" of this type are generated by hepta-, octa-, and nonasaccharides. Indeed modeling involving lower oligomers did not lead to such tetramers. In this type of aggregation, in addition to cross-linking of the two dimers through their F and J sites, the hepta-, octa-, and nona-oligosaccharides are also capable of forming single cross-linked aggregates through their G and K sites with two other protein dimers, which in turn are free to repeat the cross-linking pattern through their F and J sites to more number of protein dimers, thus resulting in the formation of a lattice. It is interesting to note that the second of the three binding sites (F and J sites, see Figure 1) is involved in each subunit in this type of arrangement (Figure 5d) leaving the third site (G and K sites) free for forming an additional single linkage with yet another lectin molecule per subunit. The symmetry in the interactions in the aggregate lends itself to the formation of homogenous aggregates or lattices (Figure 6). As a result, such lattices can be expected to have more intrinsic stability as compared to a heterogeneous aggregate formed by a combination of other linkages demonstrated here.
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The proteincarbohydrate interactions are relatively weak on the biological scale, with dissociation constants usually in the milli to micromolar range (Lee and Lee, 1995). To overcome the problem of weak affinity, nature appears to have evolved a mechanism of multivalency, in which multiple copies of both the oligosachhardies and the interacting segments of lectins are displayed on cell surfaces such that many weak interactions reinforce one another in a cumulative manner. Multivalency not only enhances the affinity or strength of binding but also amplifies binding selectivity or specificity, thus allowing nature to use subtle changes in oligosaccharide structures to create an impressive array of ligands for highly specific biological processes.
3D structures have been determined for more than 200 different lectins, which have provided detailed information about the structure, binding site and the mode of action of individual molecules of these lectins. Yet in most cases, they have not been sufficient by themselves to explain multivalency or generation of specificity in recognizing a wide variety of oligosaccharides. They do, however, provide excellent frameworks to integrate biochemical information through computational approaches and help in addressing some of the crucial issues, as illustrated in this article. Modeling of proteincarbohydrate cross-linked aggregates of garlic lectin reported here, clearly reveal the role of multivalency in increasing specificity to higher oligosaccharides. Formation of large aggregates through cross-linking has been studied earlier using physicochemical, electron microscopic, and X-ray techniques (Bhattacharyya et al., 1990; Lee et al., 1984
, Lee and Lee, 2000
; Brewer, 2002
). The X-ray studies on soybean agglutinin complexed with bivalent pentasaccharides are particularly noteworthy (Olsen et al., 1997
). The garlic lectinoligosaccharide interactions, considered here, present a more complex case because the lectin molecule has six binding sites and the higher oligosaccharides are trivalent. It is perhaps for the first time such a complex set of lectincarbohydrate interactions are sought to be studied through detailed rigorous modeling. Multivalency is a common and in fact characteristic feature of lectins. In addition to providing a qualitative explanation for the dramatic increase in the affinity of the lectin for higher oligosaccharides compared to that for lower ones, the study provides insights into the general problem of the multivalency of lectins.
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Materials and methods |
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The carbohydrates were docked to the desired binding sites on the lectin molecule by superposing the ring of the appropriate residue on the ring of the mannose molecule bound to that site. A locally developed program using Kearsley's (1989) procedure based on Mackay's algorithm was used for this purpose. The model of methylated mannose was constructed using INSIGHT II (version 98) in calculations involving methylated mannose. The same program suite was used for search in dihedral space for visual examination or for ascertaining steric contacts.
Structures of lectin-oligosaccharide complexes were energy-minimized using X-PLOR (Brünger, 1992) with a distance-dependent dielectric constant. In each case 500 steps of minimization were carried out. The main chain atoms of the lectins were restrained with a force constant of 200 kCal/mol/Å2. One lectincarbohydrate hydrogen bond each from the hydroxyl oxygens O2, O3, and O4 from the mannose residue at the primary site were restrained to lie between 2.4 and 3.6 Å with a force constant of 100 kCal/mol/Å2. When calculating interaction energies, amino acid residues with one or more atoms within a distance of 6 Å from any atom in the oligosaccharide were included. Buried surface areas were estimated using Lee and Richard's (1971)
algorithm implemented in the NACCESS package (Hubbard and Thornton, 1993
) with 1.4 Å as the radius of water molecule. The shape complementarity coefficient (Lawrence and Colman, 1993
) was calculated using CCP4 (1994)
with interface of 6Å. The coefficient can vary between 0 and 1, where 1 indicates ideal compatibility between two surfaces. Steric contacts were found using CCP4 (1994)
.
Different sugar linkages that would be accepted by the lectin for noncovalent binding, the residues within a oligosaccharide that would bind to the lectin, as well as aggregations involving single and double linkages between lectins and oligosaccharides, were explored using locally developed programs in a manner described at appropriate places.
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Acknowledgements |
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Footnotes |
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References |
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