2 Bioinformatics Centre, Indian Institute of Science, Bangalore 560 012, India, and 3 Molecular Biophysics Unit, Indian Institute of Science, Bangalore-560 012, India
Received on March 10, 2004; revised on August 19, 2004; accepted on August 20, 2004
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Abstract |
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Key words: ß-prism-I fold / carbohydrate recognition / jacalin-related lectin / plant lectin / sequence analysis
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Introduction |
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Plant lectins are broadly divided into five classes based on their subunit folds (Bettler et al., online data available at www.cermav.cnrs.fr/lectines). These are legume lectins, ß-prism-I lectins, monocot mannose-binding lectins, trefoil lectins, and hevein domain lectins. The best characterized of these are the legume lectins, the analyses of which has led to great insights into the structural basis for proteincarbohydrate recognition and has also provided a basis to explore many potential applications including disease diagnosis (Lis and Sharon, 1998; Sharma and Surolia, 1997
). In recent years there has been good progress in the understanding of other lectin families as well. The ß-prism-I lectins, which are also commonly known as jacalin-related lectins (JRLs), are a classic example. Although only recently discovered, these lectins have become an important subject in plant biochemistry, physiology, and medicine (Aucouturier et al., 1989
; Peumans et al., 2001
; Tamma et al., 2003
). It is of primary interest to carry out a systematic analysis of the available members of this family in an effort to elucidate their molecular mechanisms of action as well as to exploit their potential applications. Here we report a bioinformatics study of JRLs and present a description of various features leading to sequencestructurefunction correlations among them.
JRLs derive their name from jacalin, the first member to be identified from the seeds of jackfruit (Bunn-Moreno and Campos-Neto, 1981). It has since triggered a lot of research in the area, which has resulted in the identification of many related lectins from different plants. Jacalin also happens to be the first member of the family to be studied by X-ray crystallography (Sankaranarayanan et al., 1996
). The structure revealed a novel lectin fold, named the ß-prism-I fold. Lectins in this family have been found to exhibit a repertoire of functions, such as specific binding to the tumor associated T-antigen (Jeyaprakash et al., 2002
), binding with specific regions of HIV and hence its inhibition (Tamma et al., 1996
), potent and selective stimulation of distinct T cell functions (Lafont et al., 1997
), and a unique ability to specifically recognize immunoglobulin A1 from human serum (Hashim et al., 2001
). Many biomedical applications automatically spring out of these functions, some examples of which are detection of specific tumors and hence diagnosis of cancer (Sujathan et al., 1996
), affinity chromatography of IgA1 (Booth et al., 1995
), and selective immunostimulation (Lafont et al., 1996
).
Each of the four subunits in jacalin is made of a major -chain of 133 amino acids and a minor ß-chain of 20 amino acids. The crystal structure of jacalin indicated that each of its subunits exhibited a type I ß-prism fold, comprised of three Greek keys (four-stranded ß-sheets) contributed by both the chains (Sankaranarayanan et al., 1996
). The crystal structures of other lectins in this family, artocarpin from Artocarpus integrifolia (Pratap et al., 2002
), Helianthus tuberosus lectin (heltuba; Bourne et al., 1999
), Maclura pomifera agglutinin (MPA; Lee et al., 1998
), and Calystegia sepium lectin (calsepa; Bourne et al., 2004
) determined subsequently, confirm this fold to be characteristic of the family, although significant differences in quaternary associations were observed. These crystal structures also indicate one carbohydrate-binding site per subunit. Residues forming the binding site emerge from different loops at one end of the prism. Based on the known sugar specificities, lectins in this family can be broadly divided into two classes: (1) the galactose-specific lectins and (2) the mannose/glucose-specific lectins (Peumans et al., 2001
). Some characteristics important for carbohydrate specificity have also been discerned from these structures.
With this background, we carried out a systematic database analysis extending to several jacalin-like molecules to derive common minimum principles characterizing (1) the jacalin fold and (2) the features generating carbohydrate-recognition capability as well as the determinants of specificity. The vast number of sequences, a significant amount of biochemical data, as well as a few crystal structures reported enable a simultaneous analysis of all known members of the family to develop a broader perspective of the functionalities as well as potential uses of these lectins.
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Results |
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Multiple alignment and phylogenetic analysis
A multiple alignment carried out with the sequences of the domains identified reflects the overall similarities (Figure 2). The multiple alignment indicates that the second half of the sequence is more conserved in the different members of the family than the first half (Figure 2). A consensus profile derived for the alignment, with the help of the PRATT profile creation tool, indeed confirms this. The profile listed using the PROSITE syntax thus reads, F-x(2,3)-N-x(3,5)-G-x-[FHY]-G-x(3,4)[AGNQST]-x(3,4)-L-x(3,8)-G-F-x(0,1)-G-x(2)-G-x(2)-[ILV]-x(2)-[FILV]-[DGS], ranging from residues 86 to 128 of the jacalin -chain. Mapping the profile onto the structure reveals that the conserved residues lie predominantly in the first and the third sheet regions of the ß-prism (Figure 2, also Figure 3).
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Analysis of quaternary associations
Examination of the quaternary associations exhibited by different crystal structures indicate that there are three different types known so far, although the five crystal structures exhibit very high similarities in their individual subunits. The three different quaternary types can be attributed to interactions involving three different interface sets: (1) interface set A involving residues 102109, 131133 from the A chain and 1115 from the B chain (numbers correspond to 1JAC) as seen in the tetramers of jacalin, MPA, and artocarpin; (2) interface set B involving residues 1925 and 5160 (residue numbers from 1OUW) as seen in the dimer of calsepa; and (3) interface set C involving residues 711, 119124, and 2026 (residue numbers from 1C3K), as seen in the octamer of heltuba, the first two segments significantly overlapping with the interface set A and the third segment partly overlapping with the interface set B (Figure 5). Thus jacalin, artocarpin, and MPA, which can be described as dimers of dimers (Sankaranarayanan et al., 1996), have their first dimerization interface on the back of the first sheet, forming extensive interactions between the first sheets in the two subunits (Figure 5). Calsepa shows an interaction surface on a face opposite to that in jacalin (equivalent to residues 38 of jacalin), leading to a dimer achieved through a completely different mode. Heltuba, on the other hand, has two interaction surfaces distributed on opposite faces of the prism, which enable it to associate with two distinct modes of the dimer of the jacalin type as well as the dimer of the calsepa type (albeit with altered intersubunit orientations), thus leading to a closed octameric arrangement.
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Furthermore, the surfaces generated by the homology models were analyzed for the presence of exposed hydrophobic residues on their surfaces. Such residues are listed in Table II, and those corresponding to either the A or the B (extended to encompass the remaining part of interface set C) interface sets are differently highlighted. A large number of JRLs contained some exposed residues corresponding to interface set A, indicating the possibility of associations through this segment as in jacalin. Some JRLs, such as those in banana and mulberry, contained exposed hydrophobic residues predominantly in this segment. Some others, such as those from wheat and Convolvulous species, contained a clearly identifiable cluster (as determined by visual inspection to identify their presence on the same face of the prism) corresponding to interface set B, indicating the possibility of associations, as in calsepa. Some other JRLs, such as those from rice and Brassica species, showed such residues on both surfaces, corresponding to interface set C, indicating the possibility of interactions as in heltuba. Yet some other JRLs show exposed clusters distributed on other segments, suggesting other modes of quaternary association.
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Structural features of carbohydrate recognition
Posttranslational modification
Apart from the known cases of posttranslational cleavage leading to a major chain and a minor ß chain in galactose-specific JRLs (Houles-Astoul et al., 2002
), such as jacalin (Sankaranarayanan et al., 1996
), MPA (Lee et al., 1998
), and the galactose variety from Morus nigra (Rouge et al., 2003
), the multiple alignment carried out here indicates that some lectins of B. napus also contain a free amino-terminal group, aligning in structure with the N-terminal glycine of jacalin and other galactose-specific lectins (Figure 2). The sugar specificity of the domains in this case is not individually known, but they are generally annotated as mannose-specific and myrosinase-binding lectins.
A majority of JRLs do not exhibit such cleavage and function as single protomers. Even in these cases, the region corresponding to the cleavage site in jacalin aligns with a glycine containing loop connecting two ß-strands. Structural superpositions carried out here indicate that the glycine(s) in this loop are oriented toward the sugar and interact by forming hydrogen bonds through their peptidyl nitrogens. The architecture of the binding site in the crystal structures suggests that either a break in the loop resulting in a free amino group as in jacalin or a glycine containing loop as in artocarpin (Figure 6) are key features in creating the appropriate contours to form the binding pocket, irrespective of the specificity. A visual inspection of the homology models built for other lectins also reflect this property (the coordinates for the models are available online at http://144.16.93.115/lectins/jacalin/models). Replacing this glycine with a larger residue would lead to steric clashes with the sugar atoms in all the JRLs and hence abolish binding to a significant extent.
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BL3 and the conserved GXXXD motif
Simultaneous analysis of the various crystal structures (jacalin, artocarpin, heltuba, MPA, and calsepa), facilitated by structural superpositions, reveal that the conformation of BL1 and BL2 vary significantly in these structures, whereas the conformation of BL3 remains constant (Figure 7). The same trend was observed in the homology models of the other JRL domains as well. Apart from the conserved conformation, there is also a much higher degree of sequence conservation in this region (Table III). A glycine and an aspartic acid (corresponding to residues 121 and 125 in jacalin) separated by a three-residue spacer, are seen to be predominantly conserved in the known JRL domains, as seen in Figure 2. The spatial position of this loop suggests that it is actually involved in forming the binding pocket. The interactions of the conserved glycine and aspartic acid with the sugar are also invariant, irrespective of the carbohydrate specificity, thus pointing to the conclusion that this feature is a key determinant of generating carbohydrate-recognition capability in general, which, in conjunction with the features from other loops, brings about the structural basis of specificity. A tryptophan in this loop is well conserved but not crucial for activity, because it is compensated either by other hydrophobic residues in the same loop or by hydrophobic residues from other loops. For example, Leu89 in BL2 and Leu139 in BL3 in artocarpin can play the same role as Trp123 in jacalin.
Deviations from the conserved GXXXD motif are observed in a few JRLs (Figure 2, Table III), such as some domains of B. napus, Hordeum vlugare, Oryza sativa, and Triticum aestivum, where a conservative substitution such as an asparagine or a glutamic acid is seen in the place of aspartic acid in most of them. Some of these also show a glycine and an aspartic acid, not directly aligned with the expected position but in the immediate neighborhood, which may be important for lectin activity. A histidine and an arginine are also found in some domains (from H. tuberosus, Hordeum vulgare). Two JRLs from T. aestivum and B. napus show extreme divergence with a hydrophobic residue in this position, although a glutamine is present within the loop in both. A substitution of the aspartic acid is also largely coupled with a substitution of the glycine, perhaps indicating directed evolution to alter primary sugar specificities.
The influencing loop
On a detailed analysis of proteinsugar contacts and the interaction networks of the participating residues (tables listing these are available online at http://144.16.93.115/lectins/jacalin/contacts), we observe that a fourth loop, an influencing loop (IL) can also influence the conformation of some of these residues and hence the specificity. This loop corresponding to residues 2023 in jacalin and is found to be longer in the galactose-binding varieties than in the mannose ones (Table III). The residues in this loop interact with residues in BL1, BL2, and BL3 in jacalin (Asn 20 ND2 [IL]-Gly 50 O [BL1], Glu 22 O [IL]Arg 82 NH2 [segment adjacent to BL2], Thr 23 O [IL]Arg 82 NE [segment adjacent to BL2], Ala 24 [IL]Val 75 [BL2; favorable van der Waals interaction], Ile 25 O [residue just outside IL]Leu 124 [BL3] apart from many more hydrophobic interactions) and hence influence the orientation and conformation of these loop regions, thus indirectly influencing carbohydrate binding. In artocarpin and many other mannose-specific lectins, there is a deletion in this region leading to a small loop, resulting in eliminating the indirect influence on carbohydrate binding. In calsepa, a mannose-specific lectin, a longer loop corresponds to this region but has a conformation very different from that of jacalin, and its position does not allow interactions with the binding site loops, as judged from the distances between the corresponding residue sets.
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Discussion |
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Sequencestructure correlations
The overall sequence of the JRLs appears to be well conserved across different plant species in which it is found, in fact leading to their recognition as JRLs, whose subunits bear the ß-prism-I fold, made of three ß-sheets arranged into a prism, with an three fold, running parallel to the sheets. The three fold internal symmetry observed in the fold, however, is not apparent at the sequence level within each subunit, unlike in the case of mannose-specific bulb lectins, where both sequences and structures show the three fold repeats (Hester and Wright, 1996
).
Sequencestructure mapping of members of this family indicate that the strand regions are highly conserved, especially in the first and the third sheets. On the other hand, the loop segments that connect the strands are highly variable in their lengths and sequences, consistent with their role in generating functional diversity. Furthermore, mapping the profile onto the structure reveals that the conserved residues lie predominantly in the first and the third sheet regions of the ß-prism (Figure 2, also Figure 3B). A twofold corollary stems out of this observation. The first is that the conserved sequence forming the profile in the second half of the sequence (made of the first and the third sheets in the structure) plus any sequence segment located appropriately to form the second ß-sheet can be regarded as a fingerprint of the ß-prism-I fold. The second is that the variable regions, which are predominantly in the first half of the sequence, are to a major extent responsible for the differences in detail and hence form causal factors in generating major diversity, both for carbohydrate specificity as well as for quaternary structure. The fact that these residues (from the loops and second ß-sheet forming the first half of the sequence) encompass IL, BL1, as well as the quaternary interface set B, is totally consistent with this argument.
While the members belonging to the Moraceae, Convolvulaceae, and the Musaceae families contain only a single copy of the domain, the members of Brassicaceae exhibit significant diversity with lectins ranging from a single copy to those containing six copies of the same JRL domain within a single sequence (Table I). On the other hand, some other sequences, such as those in O. sativa, contain one to few repeats of the JRL domain but also contain other domains. Sequence analysis indicates that one sequence of rice lectin (CAD40624) containing three copies of the JRL domain also contains a serine/threonine protein kinase domain (55% sequence similarity, e value: 9e52), whereas yet another rice protein contains three copies of the JRL domain and a Lis-H containing cytoskeleton protein domain (50% sequence similarity, e-value: 2e37). There have been earlier examples of coexistence of legume lectin domains with other domains, one of them with a jacalin-like fold in the insecticidal toxin (Chandra et al., 2001). These observations indicate the high propensity of association of this domain either with more of its own kind or with other domains, providing a framework to analyze their larger functional roles.
Available structures of JRLs indicate that the members are capable of adopting a wide range of quaternary associations. The crystal structure solutions of jacalin and artocarpin show tetrameric arrangements, whereas heltuba exhibits a remarkably different octameric association; the recently determined structure of calsepa shows yet another arrangement, that of a stable dimer (Figure 5). The presence of exposed hydrophobic residues spread over different regions in all the modeled JRLs, shown in Table II, indicate the remarkable diversity in quaternary associations that these structures may exhibit. Significant variations in quaternary associations have been observed earlier in the families of legume lectins (Prabu et al., 1999) and bulb lectins (Chandra et al., 1999
) and have also been associated with differences in larger oligosaccharide binding, perhaps a strategy for generating diversity in function over a common subunit scaffold.
It turns out that all the currently available structures in the family belong to the sequences containing only a single copy of the jacalin-like domain in a protomer. The sequences coupled with the experimentally observed molecular weights for these lectins have provided clues about the nature and mode of possible protomer assemblies for some of the JRLs studied here. These predictions provide a framework to further probe the larger functions of the lectins. It must also be mentioned that the crystal structure of heltuba revealed the presence of an octamer, which appears to be capable of cross-linking N-glycans from different glycoproteins, as against a tetramer suggested by the biochemical studies (Bourne et al., 1999). It would therefore be of great interest to determine the structures of these proteins to get a clearer understanding of the precise nature of their higher-order structures. Despite the lack of thorough knowledge of these aspects, it is clear from the data described here that the lectins of this family exhibit a truly spectacular range of quaternary associations, despite high sequence and subunit structure conservation.
Carbohydrate binding: recognizing patterns that determine sugar specificity
Even though all known JRLs exhibit high sequence conservation in their overall sequences and hence the same subunit structure, marked variations are observed in their abilities to recognize various carbohydrates. The data available so far have led to their broad classification into galactose-specific and mannose-specific classes. Some of them, however, are not exclusive to galactose or mannose, but their primary specificities are consistent with the classification. For example, jacalin, a galactose-specific lectin, has been shown to bind to mannose as well, albeit with a lower affinity (Bourne et al., 2002).
The observation of three distinct clusters in the phylogenetic trees indicate that the sequences can be subclassified into different groups (Figure 4). Most of the plants that appear in cluster 1 are known to be mannose-specific from the various biochemical studies described in the literature. Lectins from B. napus present in cluster 3 are known to be specific to N-acetyl glucosamine and p-aminophenyl -D-mannopyranoside and also capable of binding myrosinase (Taipalensuu et al., 1997
). Thus cluster 1 seems to be predominantly mannose specific, and cluster 3 is specific to sugars containing glucose-mannose plus a high affinity to myrosinase. Cluster 2 seems to have subclassified lectins with galactose and mannose specificity into separate groups. The galactose-specific lectins are all grouped together into a subcluster (2A1) of the Moraceae lectins (2A). It appears from the tree as well as the detailed sequence analysis carried out that lectins from taxonomically related plants tend to have sequences closely related to each other in their overall compositions as compared with those from the taxonomically divergent ones. Yet within the taxonomical cluster (cluster 2), the known galactose-specific lectins in Moraceae group out into a subcluster from the mannose-specific ones from the same plants, indicating the ability of the phylogenetic tree to discriminate between the galactose- and the mannose-binding varieties.
Although lectins from rice are generally known to be mannose-specific, weak binding to galactose containing sugars is also reported (Poola et al., 1986). The phylogenetic tree suggests that some lectin domains of rice as also those from the chosen sequence of A. thaliana are closer to the Moraceae lectins than the cereal or the Brassicaceae lectin domains, respectively. It is possible that differences in carbohydrate specificity occur among the different rice lectins or even among different domains of a single lectin, which could perhaps get reflected as polyspecificity, observed in many biochemical experiments. The lectins from B. napus are also all simply automatically annotated as myrosinase-binding based on their overall sequence similarities (Geshi and Brandt, 1998
). Such annotations, though useful in overall classifications, will not necessarily have the resolution of information required for understanding the finer functional features, such as carbohydrate specificities in this case, which can only be obtained by detailed sequence and structural studies.
A closer look at the multiple alignment in Figure 2 indicates that despite the immediately detectable overall similarities, fine differences in many regions of the polypeptide chain exist, particularly in the loop regions that form the binding sites. When viewed at the level of these individual segments, sequence variation patterns responsible for functional divergence stand out from those responsible for species divergence. The binding site loops of different lectins display variability not only in sequence but also in size. The fact that these regions house residues that are crucial for carbohydrate binding immediately suggests that sequence variability may be a strategy to generate carbohydrate recognition diversity. In legume lectins, a similar strategy has been identified, where the lengths of the last of the four loops dictate binding specificity (Sharma and Surolia, 1997). Furthermore, the density of insertions and deletions in these loop regions is high in JRLs, similar to that in legume lectins, as evident from the number of gaps in the multiple alignment in Figure 2.
The multiple alignment also indicates that the residues involved in various quaternary interactions are also among the variable segments. Although members of this family exhibit high variation in quaternary association, it is not likely to influence monosaccharide specificities. This is because the known crystal structures indicate that the residues involved in quaternary interactions do not make any direct contact with the bound monosaccharide. Differences in quaternary associations, however, could have major implications to the recognition of higher sugar structures, as seen in the case of bulb lectins (Chandra et al., 1999).
Mapping the variations observed in the loop segments from the multiple alignment onto the previously characterized structural features, required for carbohydrate binding, has led to the determination of conservation of each feature. Furthermore, two more features have been identified from this study to be important for defining specificity. From the available crystal structures, it is known that two main differences exist in the binding site residues between the galactose-specific and the mannose-specific JRLs. These relate to (1) posttranslational cleavage and (2) aromatic stacking with the sugar. In addition, the new sequence and structural features identified here are (3) the hydrophobic lid formed by BL1 and (4) presence of an IL that can be correlated with sugar-binding properties in these lectins. The study of all known JRL domains, analyzes each individual feature and provides insight about which of them determine specificities, versus which of them augment specificities in individual cases, as discussed next.
BL1
The crystal structures and the models suggest that residues in BL1 (encompassing the Phe47 in jacalin) in galactose-binding lectins form a hydrophobic lid to the binding pocket, the absence of which, as in the mannose-binding lectins, will allow binding of an oligosaccharide, extending at that end of the prism. The length and sequence of this loop is therefore important for monosaccharide specificity, and perhaps even more so for oligosaccharide specificities.
BL2
The presence of aromatic residues at this position in both galactose- and mannose-specific varieties suggest that the role of the aromatic residue in the second loop BL2, and hence the loop is mainly in enhancing binding in individual cases but is not crucial for determining monosaccharide specificity. The conformation of the loop in different proteins vary significantly, as do the loop sizes, and do not seem to suggest a correlation of the loop size with monosaccharide specificities. For example, the size and orientation of the loop in banana lectin and CCA resemble that of jacalin but not artocarpin. Although not critical for monosaccharide specificity, the spatial position and orientation of the loop suggests that it will strongly influence oligosaccharide binding. A recent crystallographic study involving artocarpin complexes in fact illustrates the influence of the variability of the BL2 loop region in generating diversity in oligosaccharide recognition (Jeyaprakash et al., 2004).
BL3
The loop BL3 appears to play a different role, that of determining lectin activity, by generating carbohydrate recognition capability, judged by (1) its invariant conformation and (2) housing the invariant aspartic acid involved in interactions with both the galactose as well as the mannose. The high sequence conservation in this loop leads to the derivation of a sequence motif GXXXD, where X is any amino acid. However, because the binding site in JRLs is formed by multiple segments not contiguous in the sequence, the signature, although a determinant of carbohydrate binding, does not reflect the carbohydrate specificity, unlike in bulb lectins, where a nine-residue sequence signature is related to their mannose-binding abilities (Ramachandraiah and Chandra, 2000).
IL
The length and conformation of the IL is also seen to discriminate between the two varieties, the longer one interacting with the three loops BL1BL3 in galactose-binding JRLs,
Posttranslational cleavage
Posttranslational cleavage leading to a free N-terminus interacting with the sugar is observed in all galactose-specific JRLs and is not found in the known mannose-specific varieties. The first domains of some lectins of B. napus also contain a free amino-terminal group, aligning in position with the N-terminal glycine of jacalin and other galactose-specific lectins. It is also interesting to observe that the first domains of the three lectins of B. napus appear closer to the galactose-specific ones from Moraceae in the phylogenetic tree, away from the other lectins in the Brassicaceae cluster, suggesting that they could exhibit different specificities either at the mono- or at the oligosaccharide level. The sugar specificity of the domains in this case is not individually known, but they are generally annotated as mannose-specific and myrosinase-binding lectins. Although a free amino terminus will be accessible to the sugar in these domains, an aromatic residue analogous to Tyr78 in jacalin is not present to provide a possible stacking interaction. Moreover there is a remarkable variation in loop lengths in these domains as compared to those in the galactose-specific Moraceae lectins. These domains therefore may not exhibit the preference for galactose, despite having a free amino terminus at this site. The analysis presented provides a useful framework for the design of further experimental work to probe if a posttranslational modification is or is not the key in defining galactose specificity in these lectins.
The cleavage site loop
Despite the presence or absence of cleavage, the glycine-rich loop in the region is seen to provide contours to form a carbohydrate-recognition site and hence, in comibation with BL3, can be said to be the determinants of lectin activity in JRLs.
Thus the analysis indicates that it is a combination of factors that determine specificity, rather than a single discriminatory factor. Although the third loop BL3 and the free N-terminal glycine/glycine-rich loop form the contours of the binding site, the conformation of BL1 seems to determine specificity, whereas the interactions from BL2 enhance binding capabilities in individual cases and will heavily influence oligosaccharide binding. Thus features that are crucial versus features that merely enhance binding in individual cases can be understood. This knowledge also helps in predicting sugar specificity for lectins, for which experimental information is not yet available. Given the pace at which whole genome sequences are determined, the ability to predict primary ligand binding will be a valuable tool for understanding function on a large scale as well as to provide focus to design relevant experiments.
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Materials and methods |
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The individual alignments are available online at http://144.16.93.115/lectin/jacalin/alignments. All alignmentspairwise or multiplehave been carried out with full domain sequences, corresponding to that of the -chain of jacalin. Structural alignments and superpositions have also been carried out with full domains.
Distribution across various life forms was analyzed using the Blink module along with the TaxBrowser tool available online at NCBI (www.ncbi.nlm.nih.gov/sutils/static//blinkhelp.html). The phylogenetic analyses were carried out using the MEGA suite of programs (Kumar et al., 2001). Sequence profiles were derived and analysed using PRATT and PROSITE (Bairoch and Boeckmann, 1992
) tools.
The relevant protein structures were obtained from the Protein databank (Berman et al., 2000). Secondary structure prediction was carried out by using the PHD algorithm (Rost, 1996
), through the NPSA server (http://npsa-pbil.ibcp.fr). Molecular models were built using Modeller 6.0 (Sali and Blundell, 1993
) using homology modeling techniques. Structural equivalences were obtained using the Dali algorithm (Holm and Sander, 1995
). Solvent-accessible surface areas were computed using the Lee and Richards (1971)
algorithm, and residues Ala, Phe, Ile, Leu, Met, Pro, Trp, and Val were considered exposed if their surface areas were greater than 30 Å2. Intersubunit and proteinsugar interactions were computed using the CCP4 Contacts program (CCP4, 1994
). The smaller subunits (of 1820 residues) making up each subunits in the known galactose-specific lectins were appended to the major subunits and treated as one unit for this calculation, so as to ignore the intrasubunit interface.
Insight II software suite (Accelrys, www.accelrys.com) was used to visualize, overlay, and analyze various structures. Appropriate Perl scripts and C programs were written for various needs of file handling and extracting required information, as well as for appropriate representation. Structure-based sequence alignments were also carried out using home-grown Perl scripts that parsed the Dali outputs and produced appropriate sequence equivalences. Carbohydrate specificities were obtained from the literature and have been made available online at http://144.16.93.115/lectins/jacalin.
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Acknowledgements |
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Footnotes |
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Abbreviations |
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References |
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