2 Centre De Recherches Sur Les Macromolécules Végétales, Cnrs (affiliated with Université Joseph Fourier), 601 rue de la Chimie, BP 53, 38041 Grenoble Cedex 9, France
3 Institute of Chemical Technology, Technická 5, 166 28 Prague, Czech Republic
Received on November 18, 2002; revised on December 9, 2002; accepted on December 17, 2002
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Abstract |
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Key words: blood group antigens / galactosyltransferase / glycosyltransferase / molecular modeling
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Introduction |
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Enzymes belonging to this 3-Gal(NAc)T family share common features. They all use a UDP-nucleotide sugar as donor, retain the configuration of the Gal (or GalNAc) transferred, and their activity is strictly dependent upon the presence of a divalent cation (generally Mn2+). Nevertheless, they differ by their fine specificity:
3GalT, blood group B transferase, and iGb3-S only use UDP-Gal as donor, whereas blood group A transferase and Forss-S use UDP-GalNAc. In addition, the mouse AB glycosyltransferase derived from a cis-AB gene can transfer sugar from both donors (Yamamoto et al., 2001
). The amino acid basis for Gal versus GalNAc specificity has been well defined for the blood group A and B transferases, which differ by only four amino acids (Yamamoto and McNeill, 1996
). On the other hand, very little is known about the basis for the acceptor specificity: N-acetyllactosamine and lactose are the acceptor for
3GalT and iGb3-S, respectively. Blood group enzymes require substitution by a L-fucose on position 2 of the acceptor galactose, whereas Forss-S requires a N-acetyl group at the same location. Details for donor and acceptor specificities are listed in Table I.
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Analysis and comparison of glycosyltransferase crystal structures in the goal of elucidating the basis of substrate recognition and catalysis has been complicated by the occurrence of large movement of one or two loops involved in substrate binding. First evidenced in inverting glycosyltransferases for ß2-GlcNAc transferase (Ünligil et al., 2000) and then ß4-Gal transferase (Ramakrishnan and Qasba, 2001
), this conformational change has also been demonstrated to occur in retaining glycosyltransferases (Boix et al., 2001
). In
3GalT, opening of the acceptor site is dependent on a donor substrate-induced conformational change (Boix et al., 2002
). Among retaining glycosyltransferases, only
3GalT (Boix et al., 2001
, 2002
) and LgtC (Persson et al., 2001
) have been crystallized in this substrate buried state, also called Form II. From the several crystal structures that have presently been obtained in Form I (open) and Form II (closed), it can now be inferred that not only the presence of UDP or UDP-sugar is necessary for obtaining the "locked" conformation but also that this substrate should be added to the crystallization medium. Crystals obtained in the absence of substrate and then soaked with UDP do bind the substrate in the active site, but the ordering of loops does not occur in the solid state. Owing to the difficulties in obtaining cocrystals of glycosyltransferases with their substrates, molecular modeling is an alternative to approaching the role of flexible loops in substrate binding.
In the present article, we propose to use the recent high-resolution structure of 3GalT (Boix et al., 2001
) as a template to model the other related enzymes with
3Gal(NAc) transferase activity listed in Table I. Docking of nucleotide-sugars on one hand and of oligosaccharide acceptor on the other will lead to the comparison of the architecture of the binding sites. Comparison with the recently solved blood group A and B synthases (Patenaude et al., 2002
) validates our modeling method and allows for comparing the two conformational forms of the enzymes. In addition, the different sequence motifs involved in substrate binding have been defined, and their precise role has been investigated.
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Results |
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For two of the enzymes, 3GalT and iGb3-S, a terminal galactose is required for acceptor, and no substitution is tolerated at the C-2 position of this residue. Examination of the models indicated that region LBR-H, and particularly the presence of an additional bulky Trp (Trp356 in
3GalT), is responsible for the fine specificity. The Trp356 in
3GalT makes a barrier for any substitution on the Gal ring, whereas in the other enzymes the smaller Ala or Thr residue together with basic residues 323RK (Forss-S) or 328QL (GTA and GTB) form a pocket accommodating either Fuc or NHAc group on C2 of the Gal ring. The size of the first amino acid of region LBR-H (Trp356 in
3GalT) seems to distinguish whether the first monosaccharide of acceptor could be branched or not.
Tryptophan residues are also involved in defining the acceptor specificity at longer range, that is, with the reducing sugar of the di- or trisaccharide. Again, 3GalT, which uses lactose or N-acetyllactosamine as acceptor, displays a Trp residue stacking with the Glc (or GlcNAc) ring at the reducing end, thus limiting the possibilities of substitution or branching for this residue. This selection of the flat, ribbonlike conformationwhich can be adopted by equatorialequatorial linked carbohydrate (such as cellulose and chitin, but also lactose and N-acetlyllactosamine) by pavement of Trp residueshas previously been observed in polysaccharide-binding protein module. Blood group A and B transferases, which use either
Fuc1-2ßGal1-3GlcNAc (type 1) or
Fuc1-2ßGal1-4GlcNAc (type 2) acceptors, present a PG/S motif in the LBR-D region instead of the AW motif characteristic of
3GalT.
Role of the nine ligand binding regions
The present dissection of the acceptor binding site allows researchers to clarify the role of each binding regions in cation, donor, and acceptor binding. The amino acids that are predicted to directly interact with the substrate have been labeled in Figure 5. The specific role of each of the nine sequence motifs can be summarized as follows.
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Discussion |
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Our docking of N-acetyllactosamine in the acceptor site was also done from the 3GalT/UDP complex. Because the crystal structure of the enzyme in complex with LacNAc has been recently published (Boix et al., 2002
), this gives a direct comparison between the model and the crystal structure and therefore a validation of our modeling approach. Indeed, the location of the acceptor disaccharides is correctly predicted, especially all hydrophobic interaction, with a particular role of the tryptophan residues. Small variations exist between the predicted and observed hydrogen bond networks, mainly due to the orientation of the hydroxyl group at O-6'.
Comparison with the crystal structures of blood group A and B transferases
Crystal structures of both blood group A and B transferases have been very recently elucidated in the native state and as complexes with UDP and acceptor analog (Patenaude et al., 2002). Superimposition of the protein backbone (Asp83Arg176 and Cys196Pro345) of the model structure on the crystal structure yielded a rms of 0.94 Å and 0.87 Å for GTA and GTB enzymes, respectively, thus confirming the quality of the homology modeling. In the crystal structures complexed with UDP and H-type substrate, two peptide regions adjacent to the active site are disordered: one loop (177195) and the C-terminus region (346354) therefore resulting in the open conformation (Form I) of the binding site cleft. On the opposite, the modeled structures correspond to the Form II conformation, which has been proposed to be induced by the binding of the nucleotide sugar (Persson et al., 2001
; Boix et al., 2002
; Ramakrishnan et al., 2002)
. The roles of regions LBR-B, LBR-H, and LBR-I in binding the ligands could then be inferred from the model. Figure 2 displays a comparison of Form I (crystal) and Form II (model) of GTA.
The differences are maximum for the nucleotide-sugar binding site (Figures 2C and 2D): this substrate is completely buried in Form II. Side chains of amino acids from the C-terminal region (Lys346, His348, and Arg252) stack together and join amino acids from the basis of the long 4 helix (Trp181 and Gln182) to form a lid over the pyrophosphate moiety of the nucleotide sugar (Figure 2E). These five amino acids show a high degree of conservation among the family of glycosyltransferases studied. Such closing of the lid by hydrophobic contacts is also observed in LgtC structure (Persson et al., 2001
) where Pro248 in the C-terminal region interacts with the His78Ile79 cluster. When ordering these two regions in our model of GTA, additional contacts are established between the protein and the nucleotide-sugar: Lys346 (LBR-H) and Arg252 (LBR-I) of the C-terminal domain make salt bridges to the phosphate groups, whereas Val184 (LBR-B) interacts with the uracil ring. The GalNAc moiety of the nucleotide-sugar also presents additional contact with the protein. The orientation of Gal/GalNAc proposed from the present modeling study varies slightly from the one that has been deduced (also from modeling) but starting from the Form I crystals structure, where very few contacts were predicted to occur. In our model, GalNAc is stabilized by a larger number of hydrogen bonds (Figure 4), including Ser185, which was disordered in the crystal. Also, the N-acetyl moiety interacted with Gly268, one of the two crucial amino acids in term of AB specificity (see later discussion), therefore explaining why GalNAc is favored over Gal in the larger site of GTA.
In the acceptor site, the differences between Forms I and II are less drastic (Figure 2C and D). Nevertheless, ordering of bulky Lys346 reduces the space available for the fucose residue. In the present model, this results not only in a different orientation of the fucose but also for the Gal moiety. This latter residue, presents a slightly different orientation than in the GTA or GTB crystals, resulting in a better stacking with Trp300, as previously observed in the crystal structure of 3GalT/lactose complex (Boix et al., 2002
).
Comparison with biochemical data on GTA and GTB
The differences in amino acids between GTA and GTB are limited to four residues: Arg176Gly, Gly235Ser, Leu266Met, and Gly268Ala (Yamamoto et al., 1990). Among these four differences, three are located in regions that we identify as LBRs. As discussed, Leu266Met and Gly268Ala substitutions (region LBR-E) have a crucial effect on the shape of the nucleotide binding pocket and directly affect the specificity for the nucleotide sugar. Indeed, mutagenesis studies demonstrated that when only one of the two positions is substituted by its analog counterpart in the other enzyme, the resulting enzyme displays both A and B activity (Yamamoto et al., 1996
; Seto et al., 1999
). The mouse glycosyltransferase that naturally displays this dual specificity also has only one difference (Met to Gly) with human BGT (Yamamoto et al., 2001
). Our modeling study rationalizes the concerted action of the two amino acids involved. As shown in Figure 3A, they form together a bottleneck that size controls the possibility to accept an N-acetyl group in a subsite.
The substitution Gly235Ser is located in the acceptor site (region LBR-D) and may be involved in small differences in acceptor recognition that have been defined by chemical mapping of the acceptors (see later discussion). Arg176Gly mutation does not affect the A-specificity but gives an 11-fold increase in kcat (Seto et al., 1997). This amino acid is not involved in the substrate binding but is located at the surface of the enzyme. Analysis of the surface indicates that it is located about 8 Å from LBR-A, which closes the site above the nucleotide sugar. Depending on the orientation of the Arg176 side chain, it can participate to a basic cluster with K123 and K124 of LBR-A, two basic amino acids of A and B transferases at the surface of the binding site. Modifying this basic cluster could affect the turnover of the nucleotide-sugar and the catalysis products. Progress in the understanding of the catalytic mechanism is needed to explain fully the role of these residues.
Chemical mapping studies with modified synthetic acceptors demonstrated a special role for O-4 of the galactose residue (Lowary and Hindsgaul, 1993, 1994
). Indeed, in the present models, this hydroxyl group is involved in a strong hydrogen bond with His233 for both GTA and GTB enzymes. Mapping studies on the fucose residue indicated some differences in specificity because only the B enzyme requires the methyl group at C6-Fuc (Mukherjee et al., 2000
). In our model, this methyl group interacts with Leu329 in a region (LBR-G) where there is no difference between A and B transferases. Nevertheless, in our prediction, the methyl group of the fucose is also spatially close to the region LBR-D, where GTA and GTB differs by a Gly235Ser substitution.
Conclusion
The elucidation of the catalytic mechanism of glycosyltransferases remains one of the most challenging problems in structural glycobiology (Ly et al., 2002). Particularly, the catalytic event that allows transfer of a monosaccharide with retention of configuration has not yet been elucidated: the double displacement reaction via formation of a glycosyl-enzyme intermediate that has been proposed by analogy with glycosylhydrolases (Gastinel et al., 2001
) has been revised recently and is now almost abandoned (Boix et al., 2002
; Ly et al., 2002
), although no clear alternative mechanism could be proposed. In this context, the aim of the present study was to clarify the role of conformational changes as well as the basis of substrate and acceptor binding in one family of glycosyltransferases and to rationalize their specificities. The sequence motifs playing a role in ligand binding have been identified: the sugar donor specificity (UDP-Gal versus UDP-GalNAc) is due to the relative size of two crucial amino acids, whereas in the acceptor site, tryptophan residues play a key role in defining the fine specificity. Because glycosyltransferases are widely used for oligosaccharide synthesis in biotechnological approaches, such a modeling study could provide some structural basis for rational engineering of specificities and design of new transferase activities.
The present article has been limited to enzymes belonging to one family of homologous enzymes; it may nevertheless be extended to other glycosyltransferases. Among the crystal structures that have been determined recently, all of the enzymes for which Mn2+ is required for activity share the same fold, despite a lack of sequence similarities. In such cases, fold recognition methods are a powerful tool for identifying family of enzymes that can share the same fold (Breton et al., 2002). Combination of fold recognition study and molecular modeling may help in predicting the acceptor specificities of putative glycosyltransferase sequences from newly determined genomes. This approach may be helpful in the emerging area of glycogenomics.
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Materials and methods |
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UDP-Gal/UDP-GalNAc was docked into the binding site in a conformation and location similar to what has been observed for UDP-Gal complexed with 3GalT (Boix et al., 2001
) and for UDP-2F-Gal complexed with LgtC (Persson et al., 2001
). A Mn2+ ion was located between the pyrophosphate group and aspartate groups of the DXD motif of protein. Atom types and energy parameters available for carbohydrates (Imberty et al., 1999
) were used together with parameters developed for the sugarpyrophosphate linkage (Petrova et al., 1999
). For disaccharide acceptors, the conformation at the glycosidic linkage was selected according to crystal structures, when available (Pérez et al., 2000
), and to previously calculated energy maps (Imberty et al., 1995
). In all cases, the monosaccharides on the nonreducing side have been located in the binding site as observed for lactose and LacNAc acceptor in complex with
3GalT (Boix et al., 2002
).
Four models were therefore generated: GTA/UDP-GalNAc/Fuc1-2ßGal1-4ßGlcNAc/Mn2+, GTB/UDP-Gal/
Fuc1-2ßGal1-4ßGlcNAc/Mn2+, Forss-S/UDP-GalNAc/ßGalNAc1-3
Gal/Mn2+, and iGb3-S/UDP-Gal/ßGal1-4ßGlc/Mn2+. The complex
3GalT/UDP-Gal/ßGal1-4ßGlcNAc/Mn2+ was also generated for comparison. In all complexes, several cycles of energy minimization were performed to optimize the geometry of all ligands and also of protein side chains in the binding site and its vicinity. Energy calculations were performed using the TRIPOS force field (Clark et al., 1989
) in the Sybyl package with addition of energy parameters developed for carbohydrates (Imberty et al., 1999
).
Validation of the models was performed by superimposition with the crystal structure of GTA and GTB enzymes complexed with UDP and disaccharidic acceptor analog (code 1LZI and 1LZJ; Patenaude et al., 2002).
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Acknowledgements |
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1 To whom correspondence should be addressed; e-mail: imberty{at}cermav.cnrs.fr
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Abbreviations |
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References |
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