Fold-recognition and comparative modeling of human α2,3-sialyltransferases reveal their sequence and structural similarities to CstII from Campylobacter jejuni
© Sujatha and Balaji; licensee BioMed Central Ltd. 2006
Received: 28 November 2005
Accepted: 19 April 2006
Published: 19 April 2006
The 3-D structure of none of the eukaryotic sialyltransferases (SiaTs) has been determined so far. Sequence alignment algorithms such as BLAST and PSI-BLAST could not detect a homolog of these enzymes from the protein databank. SiaTs, thus, belong to the hard/medium target category in the CASP experiments. The objective of the current work is to model the 3-D structures of human SiaTs which transfer the sialic acid in α2,3-linkage viz., ST3Gal I, II, III, IV, V, and VI, using fold-recognition and comparative modeling methods. The pair-wise sequence similarity among these six enzymes ranges from 41 to 63%.
Unlike the sequence similarity servers, fold-recognition servers identified CstII, a α2,3/8 dual-activity SiaT from Campylobacter jejuni as the homolog of all the six ST3Gals; the level of sequence similarity between CstII and ST3Gals is only 15–20% and the similarity is restricted to well-characterized motif regions of ST3Gals. Deriving template-target sequence alignments for the entire ST3Gal sequence was not straightforward: the fold-recognition servers could not find a template for the region preceding the L-motif and that between the L- and S-motifs. Multiple structural templates were identified to model these regions and template identification-modeling-evaluation had to be performed iteratively to choose the most appropriate templates. The modeled structures have acceptable stereochemical properties and are also able to provide qualitative rationalizations for some of the site-directed mutagenesis results reported in literature. Apart from the predicted models, an unexpected but valuable finding from this study is the sequential and structural relatedness of family GT42 and family GT29 SiaTs.
The modeled 3-D structures can be used for docking and other modeling studies and for the rational identification of residues to be mutated to impart desired properties such as altered stability, substrate specificity, etc. Several studies in literature have focused on the development of tools and/or servers for the large-scale/automated modeling of 3-D structures of proteins. In contrast, the present study focuses on modeling the 3-D structure of a specific protein of interest to a biochemist and illustrates the associated difficulties. It is also able to establish a sequence/structure relationship between sialyltransferases of two distinct families.
Sialyltransferases (SiaTs) catalyze the transfer of sialic acid from CMP-Neu5Ac donor substrate to the terminal non-reducing saccharide of glycoproteins or glycolipids [1–4]. They are type II transmembrane proteins with a short, cytoplasmic N-terminal domain followed by a transmembrane domain, a flexible stem region of variable length and a catalytic domain. SiaTs use a variety of glycoconjugates as acceptor substrates in vivo; they can also use mono-, di- or oligo-saccharides as acceptor substrates in vitro. The sialic acid residue can be transferred in α2,3-linkage to Gal, in α2,6-linkage to Gal, GlcNAc or GalNAc and in α2,8-/α2,9-linkage to another sialic acid. SiaTs constitute a superfamily and have been further classified as ST3 (α2,3), ST6Gal (α2,6 to Gal), ST6GalNAc (α2,6 to GalNAc) and ST8 (α2,8/9) families on the basis of the linkage in which sialic acid is transferred . Further classification viz., ST3Gal I, ST3Gal II, etc., is based on acceptor specificity and amino acid sequence.
Eukaryotic SiaTs share four sequence motifs in their catalytic domain; these are L- (large), S- (small), and VS- (very small) motifs  and motif III . The roles of conserved residues found within the sialylmotifs have been investigated by site-specific mutation analyses in ST6Gal I. Residues in the L-motif have been implicated in binding donor substrate  whereas those in the S-motif have been implicated in binding both the donor and acceptor substrates [8, 9]. Mutation of the conserved His residue in the VS-motif to Lys led to loss of activity . Mutating the conserved histidine in VS-motif to alanine gave rise to an enzyme with no activity. Similarly, mutations of histidine and tyrosine residues in motif III to alanine in ST3Gal I also resulted in complete loss of enzyme activity . These motifs are common to all SiaTs and are thus expected to be involved in shared functions such as donor substrate binding, folding and maintaining proper 3-D structure, and catalysis.
The residues that are not conserved across the families are expected to generate differential acceptor specificity, oligomerization, protein-protein interaction, etc. A recent sequence analysis study identified linkage- (family-) specific sequence motifs . Two motifs were found to be unique to the ST3Gal family: 185TTx(4)YPE193 and 209FKxxDxxW216 (human ST3Gal I numbering; accession no. AAA36612). The former motif is contiguous to the L-motif. These motifs, being specific to the ST3 family, are expected to contribute to the characteristic linkage- and acceptor substrate-specificities of the family members .
Knowledge of the 3-D structure of SiaTs is crucial to understand the origin of the substrate specificity and to rationalize the site-specific mutation data on the conserved residues in sialylmotifs. This knowledge will also help in establishing the structure-function relationship in this family of proteins and thereby in generating SiaTs with modified substrate specificity for chemo-enzymatic synthesis of oligosaccharides. However, the 3-D structure of none of the eukaryotic SiaTs is known to date. In view of this, the 3-D structures of six human SiaTs belonging to ST3Gal family have been modeled using fold-recognition and comparative modeling methods. Six different ST3Gals were considered for modeling since their pair-wise sequence similarity ranges from 41 to 66% and they are expected to share the same fold because of their biochemical functional similarities.
Fold-recognition servers identified CstII, a α2,3/8 dual-activity SiaT from Campylobacter jejuni as the homolog of all the six ST3Gals. The generated 3-D models have acceptable stereochemistry. It was also possible to provide a structure-based rationalization for the functional behavior of many of the site-specific mutants. Independent modeling of the six ST3Gals leading to the similar structures enhanced the confidence levels in the generated models. The results also establish that the GT29 and GT42 family SiaTs share sequence and structural similarities.
Results and discussion
Sequence similarity between ST3Gals
Accession numbers and modelled regions of human SiaTs
NCBI accession number
Length (number of residues)
Roles deduced for some of the residues which are conserved in the eukaryotic SiaT superfamily and whose mutations have been experimentally characterized#
Equivalent residue in ST3Gal I
Role deduced for the mutated residue from the modeled 3-D structures‡
Mutations in L motif¶
Structural role: involved in disulphide bridge
Structural role: part of hydrophobic core†
Structural role: part of hydrophobic core†
Structural role: is buried and hydrogen bonds with side chains of N147 and E178 (ST3Gal I numbering). N147 is replaced by Ser in ST3Gal V, but Ser does not form hydrogen bond with Arg
Structural role: part of the hydrophobic core†
Structural role: These are at beginning of strand β4 and are solvent exposed
Mutations in S motif§
Structural role: helix J nucleator
Functional role: close to ribose
Structural role: forms disulphide bridge
Structural role: part of the hydrophobic core
Mutations in motif-III*
loss of activity
Functional role: close to phosphate
Secondary structure prediction
A consensus secondary structure was derived for each SiaT based on the results from eight secondary structure prediction servers (see Additional file 2). The region predicted by the TMHMM server as the transmembrane domain is predicted to be helical in all the ST3Gals. The sequence and length of the region between the transmembrane domain and L-motif in the six ST3Gals are different; this region has only helices but the number of helices varies between 3 and 5. The significance of this variability and its relevance (if any) to differences in acceptor substrate specificity are as yet unknown. The order of occurrence of the secondary structural elements from the L-motif onwards is very nearly the same in all the ST3Gals. The L-motif region is made of coils and strands. The S-motif begins with a helix, immediately followed by a strand. The six-residue-long VS-motif is partly helical. The region between the L- and S-motifs has a mixture of strands and helices. Of the two ST3 family-specific motifs, TTx(4)YPE is part of a strand and FKxxDxxW is in coil conformation.
Overall, 25–32% of residues are in helices and 9–12% residues in strands. The conservation of the nature and order of occurrence of secondary structural elements is strongly suggestive of the conservation of the overall fold in these ST3Gals. It can be inferred from the predicted secondary structures that ST3Gals belong to the α/β class, as defined in the SCOP database . Other glycosyltransferases (GlyTs) whose 3-D structures have been determined so far also belong to the same class. Within this class, there are three fold types designated as nucleotide-diphospho-sugar transferases, UDP-glycosyltransferase/glycogen phosphorylase and α-2,3/8-sialyltransferase CstII.
Template identification by fold-recognition servers
Two approaches were employed to identify the potential templates: (1) Submitting a multiple sequence alignment (MSA) of all the six ST3Gals and (2) Submitting each of the six ST3Gal sequences individually. In the former, MSA for the entire sequence from N- to C-terminus (Figure 1) was submitted to the FUGUE server; the templates that were identified had very low confidence levels (Z-score for the top hit = 2.54; guess). Even the GeneSilico metaserver identifies templates with very low confidence levels (pcons5 score for the top hit = 0.15; unreliable); the α2,3/8 dual-activity sialyltransferase CstII from Campylobacter jejuni (PDB id 1RO7 ; referred to as CstII henceforth) has a pcons5 score of 0.09. However, the alignment with CstII began from only the L-motif onwards of ST3Gals; no template was identified for the region preceding the L-motifs, most likely due to the very low sequence similarity in this region of the ST3Gals. In view of these, MSA starting from the L-motif onwards up to the C-terminus was submitted to these servers. Both the servers identify CstII as the top hit (Z-score = 5.2; likely and pcons5 score = 0.32; unreliable).
The alignments generated by different servers do not agree with each other in some regions. The disagreement was resolved based on secondary structure states of the residues at some regions. For example, the residues 215–254 of ST3Gal I are aligned differently with CstII by the four fold-recognition servers (Figure 2); even the secondary structure states of the aligned residues are different (Figure 2). A similar mismatch was found for the corresponding region of other ST3Gals also. For such regions, other template(s) that would satisfy the predicted secondary structure in that region were identified by submitting only the relevant part of the sequence to the fold-recognition servers and/or PSI-BLAST (see Additional file 4). Thus, the use of pair-wise target-template alignment seems to be more appropriate than deriving templates based on multiple sequence alignment .
Sequence alignment for regions preceding L-motif in ST3Gals
The membrane-association region in CstII is at the C-terminus  unlike the human SiaTs, which have the transmembrane domain at the N-terminus (see Additional file 5). Consequently, the N-terminus of ST3Gals (~150 residues containing the cytoplasmic and transmembrane domains and the stem region) and the C-terminus of CstII (~90 residues; containing the membrane-association region) are left out of alignment generated by the fold-recognition servers. The alignment begins with the N-terminus of CstII and L-motif of ST3Gals; specifically, Lys2 of CstII aligns with Arg140 (ST3Gal I numbering), the second residue of the L-motif (Figure 2). Reversing the directionality of the polypeptide chain in the C-terminus of CstII (i.e., from residue 210 onwards) sets the transmembrane domain of ST3Gal in a position equivalent to the membrane-association region of CstII. The N-terminal region preceding the L-motif of ST3Gals was thus modeled following the Cα-trace of the CstII C-terminus in reverse direction. A considerable amount of similarity in secondary structures was also observed in these regions.
Modeling 3-D structures starting from alignments
The 3-D structure of CstII (PDB id 1RO7; A chain) is the main template for modeling the 3-D structures of all the ST3Gals. Additional templates have been used for regions, which do not have a match in CstII by separately submitting the sequence of these regions to fold-recognition servers and PSI-BLAST (see Additional file 6). Even after this step, suitable templates could not be found for some regions immediately following the transmembrane domain; these regions were not modeled (Table 1). The combined sequence alignments (see Additional file 6) were used to model the 3-D structures of ST3Gals. Only the backbone conformation of the template is taken, and side chains are modeled independently, in regions where the template – target sequences disagree. Modeller uses a loop algorithm to model regions for which no template is specified. Twenty-five models were generated for each ST3Gals. The different structures vary in their backbone conformation, especially in regions that did not have a template, and in side chain conformations.
Stereochemical evaluation of the predicted models
The stereochemical properties and quality of all the models were evaluated by MODELLER, PROCHECK and Verify3D (see Additional file 7). Three to four models were selected for each ST3Gal based on these evaluations. For all the selected models, the value of the objective function, reported as current energy by MODELLER, is in the same range as that if the template is aligned with its own sequence. On an average, 87% of the residues are found in the allowed region of Ramachandran map; PROCHECK considers the model to be very good if it has 90% of the residues in the most favored region. The inter-atomic distances are within acceptable range. Verify3D score is greater than zero for the region from the L-motif onwards but the score drops below 0 for certain regions preceding the L-motif. The models were also evaluated using Colorado3D server, which facilitates the change of amino acid window size when calculating the overall score. Two window sizes, 5 and 21, were used to calculate the average Verify3D and ProsaII score per residue for each of the top models and 25 models generated for the template. The scores calculated using these two window sizes were found to be very similar (see Additional file 7). The template and target models were rendered with the residues color-coded based on ProsaII (see Additional file 8) and verify3D (see Additional file 9) scores. With ProsaII score-based coloring, most of the residues are green and yellow (i.e., average score) in both the target and template proteins (see Additional file 8). With verify3D score-based coloring, even the template proteins has residues in red color (i.e., bad score) although the number of such residues are more in the targets (see Additional file 9).
Characterization and comparison of modeled ST3Gal structures
Comparison of the modeled structures with CstII structure
The modeled 3-D structures of ST3Gals are similar to, but not exactly same as, that of CstII (Figure 3). The similarity is to be expected since CstII was the main template for deriving the models. Helix B is 8–10 residues long in CstII; in ST3Gals, it is only a helical loop formed by a few residues in the alignment region 226–231 (Figure 1). Helix J is not as prominent in CstII as it is in the modeled ST3Gals. The average RMS deviation between the target (ST3Gals) and template (CstII) structures is calculated to be 1.9 Å by the SSM server and 2.4 Å by the DALI server (see Additional file 10). The 3-D structure of no other protein was found to be similar to that of ST3Gals by the SSM and DALI servers.
Residues involved in binding to CMP-Neu5Ac, the donor substrate
Location of residues whose functional importance has been studied by site-specific mutations
Site-directed mutagenesis has been used to investigate the role of several residues conserved in SiaT superfamily [7–10, 21]. Quantitative analysis of rat ST6Gal I indicated the presence of only one disulphide bond although the enzyme has seven cysteine residues . All the modeled ST3Gals have one disulphide bond between two conserved cysteine residues, one present at the beginning of the L-motif and the other in the middle of the S-motif (Figure 6C). These two cysteine residues come in spatial proximity of each other when no specific constraints were used for the purpose of bringing them together. This disulfide bridge holds the β-strand of L-motif and the helix of S-motif together and is away from the putative CMP-Neu5Ac binding site (Figure 6C). Hence, mutation of either of these two residues is expected to destabilize the enzyme and consequently, lead to loss of activity. Structural/functional roles have also been deduced for other residues that are conserved in the SiaT superfamily based on the modeled 3-D structures; these deductions are in consonance with the results of experimental site-specific mutation studies (Table 2; Figure 6D).
Relationship between family GT29 and family GT42 SiaTs
Eukaryotic [3–5] and prokaryotic [22–27] SiaTs have been classified into four families based on sequence similarity in the CAZy database : (a) family GT29 contains viral and eukaryotic SiaTs; these enzymes have α2,3-, α2,6-, and α2,8-activities; (b) family GT38 contains bacterial polySiaTs mainly from Escherichia coli and Neisseria meningitides; (c) family GT42 contains SiaTs from Campylobacter jejuni and Haemophilus influenzae and (d) family GT52 contains α2,3-SiaT from Neisseria gonorrhoeae, Neisseria meningitides and few hypothetical SiaTs from Haemophilus influenzae. No sequence-based evolutionary relationship among these SiaT families has been established till date. Surprisingly, CstII was identified as the template for modeling the 3-D structures of human ST3Gals by fold-recognition servers; CstII belongs to family GT42 whereas human ST3Gals belong to family GT29. The modeled 3-D structures were found to be stereochemically acceptable and also were able to provide qualitative explanations for some of the site-specific mutagenesis data.
Family GT29 is actually a superfamily consisting of ST3Gal, ST6Gal, ST6GalNAc and ST8Sia families . CstII was identified as the top hit by the fold-recognition server FFAS03 even for the human ST6Gal, ST6GalNAc and ST8Sia family members; the E-value in these cases is comparable to that obtained for ST3Gals. This suggests that other members of the GT29 family also share the CstII fold and thereby establish the structural similarities between GT29 and GT42 family members. On the contrary, CstII was not identified as a potential template when representative members of GT38 and GT52 families were submitted to FFAS03 server. This indicates the absence of any detectable structural similarities of GT38 and GT52 families with GT29 and GT42 family SiaTs.
The knowledge of the 3-D structures of glycosyltransferases is important to better understand their biological function and to delineate structure-function relationships, as borne out, for example, in the case of galactosyltransferases [29–31]. This latter aspect is especially beneficial for the chemoenzymatic synthesis of carbohydrates and in turn, for glycomics (see, for example, ). SiaTs are another equally important class of glycosyltransferases but the 3-D structure for none of the human SiaTs is available till date. In light of these, the 3-D structure models of ST3Gals obtained in this study can be used to identify mutations that are likely to alter the donor and/or acceptor substrate specificities, thereby facilitating their use in the chemoenzymatic synthesis of complex carbohydrates and also to refine the predicted structures in the present study. This study has also provided another example of sequentially divergent proteins sharing a common fold to perform the same biochemical function.
The amino acid sequences of the experimentally characterized, human SiaTs belonging to the ST3Gal family (Table 1) were retrieved from the protein sequence database at NCBI http://www.ncbi.nlm.nih.gov. The 3-D structures of proteins were obtained from the protein data bank . The fold classification of proteins is from the SCOP database [12, 34].
Protein sequence databases were searched using BLAST  or PSI-BLAST  servers at NCBI. FFAS03 , FUGUE , PHYRE (successor of 3D-PSSM, ), SAM-T02  and GeneSilico Metaserver  were used for fold-recognition. Multiple sequence alignments were obtained using the TCoffee server [42, 43]. Transmembrane helices were predicted using the TMHMM server v. 2.0 . Secondary structures were predicted using the APSSP , JPRED , NNPREDICT , PROF , PSIPRED , SAM-T99 , SOPMA  and SSPRO  servers. Verify3D [53, 54] and Colorado3D  were used to evaluate the models. DALI  and SSM  servers were used for 3-D structure comparisons. Sequence logos were created using WebLogo (version 2.8.1) . All the servers were used with default values for the various parameters, except where mentioned otherwise.
Software and hardware
BioEdit  was used for display and manipulation of sequences. SwissPDBviewer , Rasmol  and PyMol  were used to visualization and/or rendering. Modeller6v1, a homology modeling software, was used for modeling the 3-D structures [63, 64]. The stereochemical quality of the generated model was assessed using PROCHECK [65, 66]. All the software were run on an Intel Pentium IV desktop personal computer, except for modeller6v1, which was run on a SGI octane workstation. Default values were used for all the parameters, unless specified otherwise.
Secondary structure prediction
The secondary structures of each of the six ST3Gals were predicted separately using eight prediction servers mentioned earlier. The secondary structures were predicted as three states, helix (H), strand (E) and coil (C). A consensus secondary structure was obtained by comparing the predictions of the eight servers. If different secondary structure states are predicted for a residue by the servers, the state that has been predicted by at least five of eight servers was taken as the consensus state; in other cases, it was marked as U (uncertain).
Template-target sequence alignment
The ST3Gal sequences were submitted to fold-recognition servers separately. All the servers provide alignment of the submitted ST3Gal sequence (target) with the sequence of the potential hits (templates). Inspection of the template-target alignments generated by these fold-recognition servers revealed that certain regions of ST3Gals either did not have a template or the template-target secondary structures did not match. Such regions of ST3Gals were separately submitted to PSI-BLAST and fold-recognition servers. The best hits identified from these were then used as additional templates to model the target sequences.
Validation of predicted 3-D structures
The stereochemical properties of predicted 3-D structures were assessed by PROCHECK and the residue environments by Verify3D and Colorado3D. Regions that are found by these servers as poorly modeled were improved by iterative manual adjustment of alignments and re-modeling. In the second stage of structure validation, the ability of the predicted structures to rationalize the results from the site-specific mutagenesis experiments reported in literature was investigated.
BLAST server: http://www.ncbi.nlm.nih.gov/BLAST/
CAZy database: http://afmb.cnrs-mrs.fr/CAZY/
GeneSilico Metaserver: http://genesilico.pl/meta
SCOP database: http://scop.mrc-lmb.cam.ac.uk/scop/
Authors thank Professor Andrej Sali for providing Modeller6v1. Authors also thank Mr. Ronak Y Patel for sharing his experimental database of ST3Gals and the anonymous referee for his/her useful comments. MSS is grateful to Indian Institute of Technology Bombay for teaching assistantship. This work was supported by a grant from the Council for Scientific and Industrial Research, India to PVB (Grant No. 37(1110)/02/EMR-II).
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