BACKGROUND
sialyltransferases  catalyze the transfer of sialic acid from cmp-neu5ac donor substrate to the terminal non-reducing saccharide of glycoproteins or glycolipids  <cit> .
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 α <dig> -linkage to gal, in α <dig> -linkage to gal, glcnac or galnac and in α <dig> -/α <dig> -linkage to another sialic acid.
siats constitute a superfamily and have been further classified as st <dig> , st6gal , st6galnac  and st <dig>  families on the basis of the linkage in which sialic acid is transferred  <cit> .
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- , s- , and vs-  motifs  <cit>  and motif iii  <cit> .
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  <cit>  whereas those in the s-motif have been implicated in binding both the donor and acceptor substrates  <cit> .
mutation of the conserved his residue in the vs-motif to lys led to loss of activity  <cit> .
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  <cit> .
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-  specific sequence motifs  <cit> .
two motifs were found to be unique to the st3gal family: 185ttxype <dig> and 209fkxxdxxw <dig> .
the former motif is contiguous to the l-motif.
these motifs, being specific to the st <dig> family, are expected to contribute to the characteristic linkage- and acceptor substrate-specificities of the family members  <cit> .
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  <dig> to 66% and they are expected to share the same fold because of their biochemical functional similarities.
fold-recognition servers identified cstii, a α <dig> / <dig> 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 gt <dig> and gt <dig> family siats share sequence and structural similarities.
RESULTS
sequence similarity between st3gals
pairwise sequence similarity between st3gal i, ii, iii, iv, v and vi ranges from  <dig> to 66% .
the similarity is higher  in the region from the l-motif up to the c-terminus.
st3gal i and ii are more similar to each other than they are to other four st3s as has been noted previously  <cit> .
the six st3gal sequences were also multiply aligned using the tcoffee server .
the level of confidence in the alignment is quite high, as judged by the confidence scores generated by the tcoffee algorithm, except in regions encompassing the stem, transmembrane and n-terminal cytoplasmic domains .
the motifs of the siat superfamily  and linkage-specific motifs of the st <dig> family align with each other.
a cysteine residue in the stem region is conserved in all the six st3gals .
secondary structure prediction
a consensus secondary structure was derived for each siat based on the results from eight secondary structure prediction servers .
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  <dig> and  <dig>  the significance of this variability and its relevance  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 st <dig> family-specific motifs, ttxype 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  <cit> .
other glycosyltransferases  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- <dig> /8-sialyltransferase cstii.
template identification by fold-recognition servers
two approaches were employed to identify the potential templates:  submitting a multiple sequence alignment  of all the six st3gals and  submitting each of the six st3gal sequences individually.
in the former, msa for the entire sequence from n- to c-terminus  was submitted to the fugue server; the templates that were identified had very low confidence levels .
even the genesilico metaserver identifies templates with very low confidence levels ; the α <dig> / <dig> dual-activity sialyltransferase cstii from campylobacter jejuni  has a pcons <dig> score of  <dig> .
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 .
in the second approach, complete sequence from n- to c-terminus of the six st3gals was used separately as query to search for homologs in the pdb database using blast and psi-blast.
no significant hits were obtained.
among the fold-recognition servers, only ffas <dig> and the genesilico metaserver identified cstii as a hit and the alignment began from the l-motif region of st3gals.
however, if only the sequence from l-motif onwards is used as query, then even fugue and sam-t <dig> servers identify cstii as the possible template with a high level of confidence .
the template-target alignments generated for motif regions  when st3gal sequences were submitted individually were same as that obtained by submitting the multiple sequence alignment.
in all the cases, the secondary structures of the target and template residues in the alignment regions 250– <dig>  were entirely different .
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– <dig> of st3gal i are aligned differently with cstii by the four fold-recognition servers ; even the secondary structure states of the aligned residues are different .
a similar mismatch was found for the corresponding region of other st3gals also.
for such regions, other template 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 .
thus, the use of pair-wise target-template alignment seems to be more appropriate than deriving templates based on multiple sequence alignment  <cit> .
sequence alignment for regions preceding l-motif in st3gals
the membrane-association region in cstii is at the c-terminus  <cit>  unlike the human siats, which have the transmembrane domain at the n-terminus .
consequently, the n-terminus of st3gals  and the c-terminus of cstii  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, lys <dig> of cstii aligns with arg <dig> , the second residue of the l-motif .
reversing the directionality of the polypeptide chain in the c-terminus of cstii  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  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 .
even after this step, suitable templates could not be found for some regions immediately following the transmembrane domain; these regions were not modeled .
the combined sequence alignments  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 .
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  <dig> 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,  <dig> and  <dig>  were used to calculate the average verify3d and prosaii score per residue for each of the top models and  <dig> models generated for the template.
the scores calculated using these two window sizes were found to be very similar .
the template and target models were rendered with the residues color-coded based on prosaii  and verify3d  scores.
with prosaii score-based coloring, most of the residues are green and yellow  in both the target and template proteins .
with verify3d score-based coloring, even the template proteins has residues in red color  although the number of such residues are more in the targets .
characterization and comparison of modeled st3gal structures
the st3gal fold is characterized by a six-stranded  parallel β-sheet flanked on the two sides by strands β <dig> and β5' in an antiparallel orientation; strand β <dig> is present in only some st3gals .
helices e, f and i share a common interface and are in spatial proximity of strands β <dig>  β <dig>  β <dig> and β <dig> .
helices a and b are very small i.e.,  <dig> to  <dig> residue long.
helices b and k' are found in only some st3gals.
the 3-d structures of st3gals compare well with each other to a large extent.
strands β <dig>  β <dig>  β <dig>  β <dig>  β <dig> andβ <dig> and helices e, f and i in various st3gals superpose well on each other .
the length of the loop region between helices e and f is variable : it is shorter in st3gal i and ii compared to that in the other four st3gals.
it has been reported that st3gal i and ii do not bind substrates that contain glcnac attached to terminal galactose whereas the other four do bind such substrates, albeit with varying affinities  <cit> .
the relationship between the size of h6-h <dig> loop and the observed differences in the acceptor substrate specificities needs experimental validation.
the conformation of the region from helix c to strand β <dig> also varies in different st3gals.
this difference is due to differences in the amino acid sequences, which, in turn, required the use of different templates for modeling these regions.
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 .
the similarity is to be expected since cstii was the main template for deriving the models.
helix b is 8– <dig> residues long in cstii; in st3gals, it is only a helical loop formed by a few residues in the alignment region 226– <dig> .
helix j is not as prominent in cstii as it is in the modeled st3gals.
the average rms deviation between the target  and template  structures is calculated to be  <dig>  Å by the ssm server and  <dig>  Å by the dali server .
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
cstii and st3gals are both sialyltransferases and use the same donor substrate, cmp-neu5ac.
the crystal structure of cstii has been determined in complex with the donor substrate analog, cmp-3-fluoro-neunac   <cit> .
the modeled st3gal structures were superposed on the structure of cstii; for this purpose, the backbone atoms of the residues constituting the l-, s- and vs-motifs were used as reference atoms.
this enabled the identification of residues that are likely to interact with cmp-neu5ac in st3gals.
the residues that are found within  <dig> Å from cmp-neu5ac were found to be part of the l-, s- and vs-motifs, motif iii and one of the st3gal family-specific motifs viz., ttxype .
the second family-specific motif fkxxdxxw is in spatial proximity of ttxype and seems to have a role in binding the acceptor substrate .
in this putative binding mode, the loop between β <dig> and helix i is near cytosine, beginning of l-motif is near ribose, tyr <dig>  is close to phosphate, middle of l-motif is close to phosphate and sialic acid, and tyr <dig> , beginning of s-motif, his of vs-motif are close to sialic acid .
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  <cit> .
quantitative analysis of rat st6gal i indicated the presence of only one disulphide bond although the enzyme has seven cysteine residues  <cit> .
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 .
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 .
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 .
relationship between family gt <dig> and family gt <dig> siats
eukaryotic  <cit>  and prokaryotic  <cit>  siats have been classified into four families based on sequence similarity in the cazy database  <cit> :  family gt <dig> contains viral and eukaryotic siats; these enzymes have α <dig> -, α <dig> -, and α <dig> -activities;  family gt <dig> contains bacterial polysiats mainly from escherichia coli and neisseria meningitides;  family gt <dig> contains siats from campylobacter jejuni and haemophilus influenzae and  family gt <dig> contains α <dig> -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 gt <dig> whereas human st3gals belong to family gt <dig>  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.
the l-, s- and vs-motifs characteristic of mammalian siats are thought to be absent in prokaryotic siats  <cit> .
the residues in cstii which correspond to these motif regions were identified by the structure-based sequence alignment generated by fold-recognition servers.
a multiple sequence alignment of  <dig> experimentally characterized st3gal sequences  was submitted to the fugue server, which aligned these to cstii .
using this alignment, multiple sequence alignments of experimentally characterized st3gals and family gt <dig> siats were merged  and sequence logos were generated .
several residues in the l-, s- and vs-motif regions were found to be either strictly conserved or have conservative replacements in gt <dig> family siats.
this suggests that family gt <dig> siats also have the l-, s- and vs-motifs .
conserved residues are found in other regions also .
one such is the proline residue immediately after the l-motif ; this residue is conserved in st <dig> family also  <cit> .
family gt <dig> is actually a superfamily consisting of st3gal, st6gal, st6galnac and st8sia families  <cit> .
cstii was identified as the top hit by the fold-recognition server ffas <dig> 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 gt <dig> family also share the cstii fold and thereby establish the structural similarities between gt <dig> and gt <dig> family members.
on the contrary, cstii was not identified as a potential template when representative members of gt <dig> and gt <dig> families were submitted to ffas <dig> server.
this indicates the absence of any detectable structural similarities of gt <dig> and gt <dig> families with gt <dig> and gt <dig> family siats.
CONCLUSIONS
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  <cit> .
this latter aspect is especially beneficial for the chemoenzymatic synthesis of carbohydrates and in turn, for glycomics .
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.

 sialyltransferases catalyze the transfer of sialic acid from cmp-neu5ac donor substrate to the terminal non-reducing saccharide of glycoproteins or glycolipids. 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 α -linkage to gal, in α -linkage to gal, glcnac or galnac and in α -/α -linkage to another sialic acid. siats constitute a superfamily and have been further classified as st, st6gal, st6galnac and st 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-, s-, and vs- 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. 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- specific sequence motifs. two motifs were found to be unique to the st3gal family: 185ttxype and 209fkxxdxxw. the former motif is contiguous to the l-motif. these motifs, being specific to the st 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 to 66% and they are expected to share the same fold because of their biochemical functional similarities. fold-recognition servers identified cstii, a α / 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 gt and gt family siats share sequence and structural similarities.  sequence similarity between st3gals pairwise sequence similarity between st3gal i, ii, iii, iv, v and vi ranges from to 66%. the similarity is higher in the region from the l-motif up to the c-terminus. st3gal i and ii are more similar to each other than they are to other four st3s as has been noted previously. the six st3gal sequences were also multiply aligned using the tcoffee server. the level of confidence in the alignment is quite high, as judged by the confidence scores generated by the tcoffee algorithm, except in regions encompassing the stem, transmembrane and n-terminal cytoplasmic domains. the motifs of the siat superfamily and linkage-specific motifs of the st family align with each other. a cysteine residue in the stem region is conserved in all the six st3gals. secondary structure prediction a consensus secondary structure was derived for each siat based on the results from eight secondary structure prediction servers. 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 and the significance of this variability and its relevance 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 st family-specific motifs, ttxype 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 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- /8-sialyltransferase cstii. template identification by fold-recognition servers two approaches were employed to identify the potential templates: submitting a multiple sequence alignment of all the six st3gals and submitting each of the six st3gal sequences individually. in the former, msa for the entire sequence from n- to c-terminus was submitted to the fugue server; the templates that were identified had very low confidence levels. even the genesilico metaserver identifies templates with very low confidence levels; the α / dual-activity sialyltransferase cstii from campylobacter jejuni has a pcons score of. 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. in the second approach, complete sequence from n- to c-terminus of the six st3gals was used separately as query to search for homologs in the pdb database using blast and psi-blast. no significant hits were obtained. among the fold-recognition servers, only ffas and the genesilico metaserver identified cstii as a hit and the alignment began from the l-motif region of st3gals. however, if only the sequence from l-motif onwards is used as query, then even fugue and sam-t servers identify cstii as the possible template with a high level of confidence. the template-target alignments generated for motif regions when st3gal sequences were submitted individually were same as that obtained by submitting the multiple sequence alignment. in all the cases, the secondary structures of the target and template residues in the alignment regions 250– were entirely different. 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– of st3gal i are aligned differently with cstii by the four fold-recognition servers; even the secondary structure states of the aligned residues are different. a similar mismatch was found for the corresponding region of other st3gals also. for such regions, other template 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. 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. consequently, the n-terminus of st3gals and the c-terminus of cstii 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, lys of cstii aligns with arg, the second residue of the l-motif. reversing the directionality of the polypeptide chain in the c-terminus of cstii 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 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. even after this step, suitable templates could not be found for some regions immediately following the transmembrane domain; these regions were not modeled. the combined sequence alignments 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. 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 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, and were used to calculate the average verify3d and prosaii score per residue for each of the top models and models generated for the template. the scores calculated using these two window sizes were found to be very similar. the template and target models were rendered with the residues color-coded based on prosaii and verify3d scores. with prosaii score-based coloring, most of the residues are green and yellow in both the target and template proteins. with verify3d score-based coloring, even the template proteins has residues in red color although the number of such residues are more in the targets. characterization and comparison of modeled st3gal structures the st3gal fold is characterized by a six-stranded parallel β-sheet flanked on the two sides by strands β and β5' in an antiparallel orientation; strand β is present in only some st3gals. helices e, f and i share a common interface and are in spatial proximity of strands β β β and β. helices a and b are very small i.e., to residue long. helices b and k' are found in only some st3gals. the 3-d structures of st3gals compare well with each other to a large extent. strands β β β β β andβ and helices e, f and i in various st3gals superpose well on each other. the length of the loop region between helices e and f is variable: it is shorter in st3gal i and ii compared to that in the other four st3gals. it has been reported that st3gal i and ii do not bind substrates that contain glcnac attached to terminal galactose whereas the other four do bind such substrates, albeit with varying affinities. the relationship between the size of h6-h loop and the observed differences in the acceptor substrate specificities needs experimental validation. the conformation of the region from helix c to strand β also varies in different st3gals. this difference is due to differences in the amino acid sequences, which, in turn, required the use of different templates for modeling these regions. 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. the similarity is to be expected since cstii was the main template for deriving the models. helix b is 8– residues long in cstii; in st3gals, it is only a helical loop formed by a few residues in the alignment region 226–. helix j is not as prominent in cstii as it is in the modeled st3gals. the average rms deviation between the target and template structures is calculated to be Å by the ssm server and Å by the dali server. 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 cstii and st3gals are both sialyltransferases and use the same donor substrate, cmp-neu5ac. the crystal structure of cstii has been determined in complex with the donor substrate analog, cmp-3-fluoro-neunac. the modeled st3gal structures were superposed on the structure of cstii; for this purpose, the backbone atoms of the residues constituting the l-, s- and vs-motifs were used as reference atoms. this enabled the identification of residues that are likely to interact with cmp-neu5ac in st3gals. the residues that are found within Å from cmp-neu5ac were found to be part of the l-, s- and vs-motifs, motif iii and one of the st3gal family-specific motifs viz., ttxype. the second family-specific motif fkxxdxxw is in spatial proximity of ttxype and seems to have a role in binding the acceptor substrate. in this putative binding mode, the loop between β and helix i is near cytosine, beginning of l-motif is near ribose, tyr is close to phosphate, middle of l-motif is close to phosphate and sialic acid, and tyr, beginning of s-motif, his of vs-motif are close to sialic acid. 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. 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. 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. 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. relationship between family gt and family gt siats eukaryotic and prokaryotic siats have been classified into four families based on sequence similarity in the cazy database: family gt contains viral and eukaryotic siats; these enzymes have α -, α -, and α -activities; family gt contains bacterial polysiats mainly from escherichia coli and neisseria meningitides; family gt contains siats from campylobacter jejuni and haemophilus influenzae and family gt contains α -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 gt whereas human st3gals belong to family gt 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. the l-, s- and vs-motifs characteristic of mammalian siats are thought to be absent in prokaryotic siats. the residues in cstii which correspond to these motif regions were identified by the structure-based sequence alignment generated by fold-recognition servers. a multiple sequence alignment of experimentally characterized st3gal sequences was submitted to the fugue server, which aligned these to cstii. using this alignment, multiple sequence alignments of experimentally characterized st3gals and family gt siats were merged and sequence logos were generated. several residues in the l-, s- and vs-motif regions were found to be either strictly conserved or have conservative replacements in gt family siats. this suggests that family gt siats also have the l-, s- and vs-motifs. conserved residues are found in other regions also. one such is the proline residue immediately after the l-motif; this residue is conserved in st family also. family gt is actually a superfamily consisting of st3gal, st6gal, st6galnac and st8sia families. cstii was identified as the top hit by the fold-recognition server ffas 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 gt family also share the cstii fold and thereby establish the structural similarities between gt and gt family members. on the contrary, cstii was not identified as a potential template when representative members of gt and gt families were submitted to ffas server. this indicates the absence of any detectable structural similarities of gt and gt families with gt and gt 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. this latter aspect is especially beneficial for the chemoenzymatic synthesis of carbohydrates and in turn, for glycomics. 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 objective of the current work is to model the 3-d structures of human siats which transfer the sialic acid in α -linkage viz., st3gal i, ii, iii, iv, v, and vi, using fold-recognition and comparative modeling methods. 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. the 3-d structure of none of the eukaryotic sialyltransferases has been determined so far. 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. unlike the sequence similarity servers, fold-recognition servers identified cstii, a α / 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. 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. the pair-wise sequence similarity among these six enzymes ranges from to 63%.
 the 3-d structure of none of the eukaryotic sialyltransferases 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 α -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 to 63%.  unlike the sequence similarity servers, fold-recognition servers identified cstii, a α / 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 gt and family gt 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. 