Crystal structures of Erythrina cristagalli lectin with bound N-linked oligosaccharide and lactose

Kathryn Turton3, Ramanathan Natesh1,3, Nethaji Thiyagarajan3, John A. Chaddock4 and K. Ravi Acharya2,3

3 Department of Biology and Biochemistry, University of Bath, Claverton Down, Bath BA2 7AY, United Kingdom, and 4 The Health Protection Agency, Porton Down, Salisbury SP4 0JG, United Kingdom

Received on April 19, 2004; revised on June 1, 2004; accepted on June 11, 2004


    Abstract
 Top
 Abstract
 Introduction
 Results and discussion
 Materials and methods
 References
 
Erythrina cristagalli lectin (ECL) is a galactose-specific legume lectin. Although its biological function in the legume is unknown, ECL exhibits hemagglutinating activity in vitro and is mitogenic for T lymphocytes. In addition, it has been recently shown that ECL forms a novel conjugate when coupled to a catalytically active derivative of the type A neurotoxin from Clostridium botulinum, thus providing a therapeutic potential. ECL is biologically active as a dimer in which each protomer contains a functional carbohydrate-combining site. The crystal structure of native ECL was recently reported in complex with lactose and 2'-fucosyllactose. ECL protomers adopt the legume lectin fold but form non-canonical dimers via the handshake motif as was previously observed for Erythrina corallodendron lectin. Here we report the crystal structures of native and recombinant forms of the lectin in three new crystal forms, both unliganded and in complex with lactose. For the first time, the detailed structure of the glycosylated hexasaccharide for native ECL has been elucidated. The structure also shows that in the crystal lattice the glycosylation site and the carbohydrate binding site are involved in intermolecular contacts through water-mediated interactions.

Key words: crystal structure / Erythrina cristagalli lectin / Erythrina corallodendron lectin / N-glycosylation / protein–carbohydrate interaction


    Introduction
 Top
 Abstract
 Introduction
 Results and discussion
 Materials and methods
 References
 
Erythrina cristagalli lectin (ECL) is a galactose-specific legume lectin. Although its function in the legume is unknown, in vitro ECL has been shown to have hemagglutinating activity and to be mitogenic for human T lymphocytes (Iglesias et al., 1982Go). Recently, a conjugate comprising a catalytically active derivative of Clostridium botulinum neurotoxin A coupled to ECL has been used to selectively target nociceptive afferents (Duggan et al., 2002Go). The specificity of ECL in retargeting the potent endopeptidase activity of botulinum neurotoxin A to nociceptive afferents in vitro points to the potential therapeutic use of this conjugate in the treatment of chronic pain.

ECL has been well studied in terms of carbohydrate binding. It interacts more strongly with fucosyllactose and fucosyllactosamine (Moreno et al., 1997Go; Surolia et al., 1996Go; Teneberg et al., 1994Go) than with N-acetyllactosamine, lactose, N-acetylgalactosamine and galactose (Iglesias et al., 1982Go). ECL differs subtly from the other Erythrina lectins in that it has a similar affinity for fucosyllactose and fucosyllactosamine, whereas other members of the family exhibit a preference for fucosyllactose (Moreno et al., 1997Go). The crystal structure of native ECL (nECL) has recently been determined in complex with lactose and fucosyllactose, providing insight into its altered carbohydrate specificity (Svensson et al., 2002Go).

The nECL protomer adopts a jelly-roll topology, in common with other legume lectins. Each protomer contains one Ca2+ and one Mn2+ ion, both of which are required for carbohydrate binding activity (Emmerich et al., 1994Go). These ions are situated close to the carbohydrate-binding site (combining site) and help maintain the correct spatial orientation of combining site residues (Derewenda et al., 1989Go). A cis-peptide bond between residues Ala88 and Asn89 holds the side chain of Asn89 in the correct orientation for carbohydrate binding (Svensson et al., 2002Go).

nECL has been shown to be glycosylated at Asn17 and Asn113, with partial occupancy at Asn113 and the nature of the bound heptasaccharide has been characterized (Ashford et al., 1991Go). The major component of the carbohydrates bound to ECL contains two N-acetylglucosamine (GlcNAc) residues, one fucose, one xylose, and three mannose residues. Native ECL exists as a dimer in which two protomers associate back-to-back, forming a handshake motif. This noncanonical mode of dimerization was first observed in Erythrina corallodendron lectin (ECorL) (Shaanan et al., 1991Go), which shares 96% sequence identity with nECL. In ECorL, the ordered heptasaccharide bound to Asn17 is believed to prevent formation of the canonical dimer (Shaanan et al., 1991Go) and it has been suggested that this may explain why nECL dimers also adopt the handshake motif (Svensson et al., 2002Go). However, the reported crystal structure of nECL did not provide the structural details of the bound heptasaccharide.

Despite the importance of oligosaccharide interactions in glycoproteins, only little structural knowledge (using X-ray crystallography) has been gained over the years. This is mainly due to the inherent mobility and chemical heterogeneity of the oligosaccharides that prevent crystallization. However, in a few cases (such as ECorL) this has been achieved (Shaanan et al., 1991Go). Here we report the crystallization and structures of native and recombinant ECL (nECL and recECL, respectively) in three new crystal forms. From the nECL structure, we were able to glean the detailed picture of most of the bound oligosaccharide on Asn113 (a hexasaccharide portion of the heptasaccharide). The protomers of nECL adopt the legume lectin fold and demonstrate structural equivalence of the recombinant form despite its lack of glycosylation. We examined the structural effects of glycosylation on quarternary structure by comparing the dimers formed by native and recombinant ECL. We confirm that dimers of both recombinant and native ECL adopt the handshake motif and suggest that structural factors other than the presence of glycosylation induce the protein to form a noncanonical dimer. Furthermore, we have studied the mode of lactose binding, which is similar to that observed for ECorL.


    Results and discussion
 Top
 Abstract
 Introduction
 Results and discussion
 Materials and methods
 References
 
nECL
nECL cocrystallized with lactose in space group P65, with two molecules per crystallographic asymmetric unit and 67% of the crystal volume occupied by solvent. The final model (dimer) at 2.0 Å resolution (Rcryst 18.97%, Rfree 20.92%) contains 477 amino acids, 2 calcium and manganese ions, 2 lactose molecules, 4 GlcNAc residues, 2 fucose residues, 1 xylose, 2 mannose residues, 4 HEPES molecules, and 456 water molecules (Table I). The estimated Luzzati coordinate error is 0.22 Å. Analysis of the Ramachandran plot revealed that the side chains of more than 99% of the amino acids have allowed or additional allowed conformations. The side chains of Tyr106 in each molecule adopt generously allowed conformations. The first three residues of the heptasaccharide bound to Asn113 were modeled into the electron density in one of the two protein molecules in the asymmetric unit, whereas electron density for six of the seven sugar residues was observed in the other.


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Table I. Crystallographic data processing and refinement statistics

 
recECL
recECL crystallized in triclinic form, with four molecules per unit cell (49% of the crystal volume occupied by the solvent). The final model at 2.13 Å resolution (Rcryst 19.1%, Rfree 24.1%) contains 958 amino acids (molecule A, 1–239; molecule B, 1–239; molecule C, 1–240 and molecule D, 1–240), 4 calcium and manganese ions, 3 glycerol molecules, and 879 water molecules (Table I). The Luzzati coordinate error is 0.23 Å, and the average B factor for protein atoms is 24.52 Å2. |Fo| – |Fc| electron density in the combining site of three protomers was identified as glycerol, which had been included in the cryoprotectant solution used during data collection. A single residue, Tyr106, in only one of the four protomers is located in a generously allowed region of the Ramachandran plot. Possible alternative conformations were observed in the electron density map for the side chains of Ser120, but not in all of the molecules in the unit cell.

recECL was also cocrystallized with lactose in space group P21, with four molecules per asymmetric unit (49% solvent). The final model at 1.7 Å resolution (Rcryst 17.79%, Rfree 20.31%) contains 959 amino acids, 4 lactose molecules, 4 calcium and manganese ions, and 1119 water molecules (Table I). The Luzzati coordinate error is 0.20 Å, and the average B factor for protein atoms is 19.99 Å2. Five amino acid residues are located in generously allowed regions of the Ramachandran plot—Tyr106 from each protomer and Asp221 from one of the four protein molecules. Several side chains were observed to have potential alternative conformations (Met95, Asp161, Leu180, and His234) but not in all of the protomers.

Overall structure
Protomers of ECL adopt the conserved jelly-roll fold that is characteristic of legume lectins (Figure 1). This structural motif comprises a six-stranded back ß-sheet, a curved seven-stranded front ß-sheet, a short five-membered ß-sheet, and a set of loops connecting the three sheets. Both the six- and seven-stranded ß-sheets are entirely antiparallel. The N- and C-termini associate together, forming the first two strands of the six-stranded ß-sheet.



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Fig. 1. The structure of ECL dimer. Protomers of ECL associate together back-to-back to form noncanonical dimers via the handshake motif as was first observed for ECorL. The dimer structure is stabilized by two hydrogen bonds and a series of contacts between side chains in four strands of the flat, six-stranded ß-sheet. The protomers are tilted with respect to one another such that the N- and C-termini play no part in the dimer interface. Each protomer adopts the conserved jelly-roll fold characteristic of legume lectins. The N-linked carbohydrate (on Asn113), lactose bound at the combining site and HEPES molecules (from the crystallization medium) are shown. The manganese and calcium ions bound in the vicinity of the combined site are shown as small spheres.

 
The crystal structures of nECL and recECL at 2.0 Å and 2.13 Å resolution, respectively, superimpose with an overall root mean squared deviation of 0.26 Å (C{alpha} atoms). Comparison of the two structural models indicated that there are no significant differences between the crystal structures of nECL and recECL, thus confirming that recECL adopts native-like structure. The main difference between the native and recombinant forms is the absence of N-linked carbohydrate in recECL.

Each ECL protomer contains one Ca2+ and one Mn2+ ion, both located close to the carbohydrate combining site. The metal ions are approximately 4.2 Å apart, and each coordinate two water molecules and make contacts with four amino acids in the vicinity of the combining site. One of these structural waters also stabilizes the conserved cis-peptide bond (Ala88–Asp89) that correctly orients the side chain of Asp89 for carbohydrate binding. Mn2+ makes contacts with the side chains of Glu127, Asp129, Asp136, and His142, and Ca2+ makes contacts with Asp129, Phe131, Asn133, and Asp136 (Table II). In addition to binding the calcium ion, the side chains of Phe131 and Asn133 are also implicated in lactose binding (see following discussion).


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Table II. Contacts between ECL and metal ions

 
ECL exists as a biologically active dimer and has not been isolated in monomeric form. Both nECL and recECL form noncanonical back-to-back dimers via the handshake motif in which the two protomers are tilted with respect to each other (Figure 1). This mode of dimerization has previously been reported for ECorL and the basic winged bean lectin (Prabu et al., 1998Go; Shaanan et al., 1991Go). The dimer interface exists between the six-stranded sheets of each protomer and is stabilized by two hydrogen bonds between the side chain of Lys171 in one protomer and Thr193 in the other and a number of van der Waals contacts between residues in four strands of the six-stranded ß-sheet—the strands formed by the N- and C-termini do not take part in stabilizing the quarternary structure. Residues Arg73, Glu79, Gln80, Pro81, Tyr82, Thr83, Arg84, Lys116, Gln117, Asp118, Asn119, Asn148, Asp161, Asn162, Gln164, Lys171, Ile191, Thr193, Gln202, Val203, and Asp221 mediate these contacts at the dimer interface.

Binding of N-linked glycosylated saccharide
The N-linked oligosaccharide bound to nECL is covalently bound to Asn113, which is part of the loop structure located between strands ß5 and ß6. This residue is the only possible site of attachment for N-linked glycosylation in the 241-amino-acid sequence of ECL. In one of the two protein molecules (molecule B) in the asymmetric unit, there was enough electron density to model six of the seven sugar residues bound to Asn113 (Figure 2). The modeled hexasaccharide has the profile: {alpha}D-Man-(1 -> 3)-[ß-D-Xyl-(1 -> 2)]-ß-D-Man-ß-D-GlcNAc-(1 -> 4)-[{alpha}-L-Fuc (1 -> 3)]-D-GlcNAc and does not make contacts with any other parts of the lectin protomer to which it is bound, although it might communicate with the lactose moiety bound in the combining site of a symmetry-related molecule through water mediated interactions.



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Fig. 2. Structure of bound N-linked hexasaccharide to nECL. Details of the surrounding residues for the hexasaccharide at the glycosylation site (Asn113). In the crystal lattice, the N-linked carbohydrate makes indirect interaction with the combining site through water-mediated interaction.

 
Influence of glycosylation on the quarternary structure
ECL shares 96% sequence identity with ECorL, which was thought to dimerize in a noncanonical fashion because of the glycosylation attached to Asn17. Based on their high sequence identity, one would expect ECL to dimerize in the same way as ECorL, but the form of ECL studied in this investigation lacks the N-linked glycosylation site at position 17 (which is located at the opposite end of the molecule compared with Asn113 in ECL). This raises an interesting issue regarding its mode of dimerization: If there is no heptasaccharide bound to residue 17, what might force ECL to form a noncanonical dimer? Several studies have been undertaken to rationalize the various oligomerization states of legume lectins in terms of their amino acid sequences, shape complementarity of protomers, interaction energy between protomers, and hydrophobic surface area buried on oligomerisation (Elgavish and Shaanan, 2001Go; Manoj and Suguna, 2001Go; Prabu et al., 1999Go; Srinivas et al., 2001Go). The results of these studies indicate that the observed modes of oligomerization are energetically more favorable than any alternative quarternary structures.

We confirm that both native and recombinant forms of ECL associate into dimers back-to-back via the handshake motif. Because recECL is unglycosylated, this demonstrates that the presence of N-linked glycosylation does not influence the mode of dimerization in ECL and suggests that factors intrinsic to the primary structure of the lectin dictate its quarternary structure. Examination of legume lectin sequences, focusing on the regions forming interfaces between protomers and dimers, has identified amino acid residues that might influence the mode of oligomerization (Manoj and Suguna, 2001Go). Extrapolation of these results to ECL reveals that the primary structure of this lectin contains features (Glu2, Glu12, Lys55, Arg73, and Lys171) that indicate it could be expected to form ECorL-type dimers. Lectins that do not form canonical dimers have an acidic residue at the position corresponding to Lys55 in ECL and a charged residue equivalent to Glu12 that comes into close contact with a charged residue (Glu2) that would form unfavorable interactions if a concanavalin A–type dimer were formed (Manoj and Suguna, 2001Go). Arginine and lysine residues at positions equivalent to 73 and 171 in ECL are conserved among lectins forming ECorL-type dimers.

Lactose binding
The combining site is located in a shallow cleft on the surface of ECL and accommodates the galactose moiety of bound carbohydrates. The crystal structures of the nECL-lactose and recECL-lactose complexes superimpose with an overall root mean squared deviation of 0.27 Å. The spatial arrangement of residues in the combining sites of both forms of the lectin are identical, confirming that recECL binds lactose in the same way as nECL. Both nECL and recECL bind lactose through a set of structural water molecules. These water molecules mediate indirect hydrogen bonds between Gly107, Asn133, Ala218, and Gln219 and the O2, O3, and O6 of galactose and O2 of glucose. Lactose also makes contacts with more water molecules in the combining site (Figure 3). Hydrophobic stacking interactions were observed between the aromatic ring of Phe131, the galactose ring of the bound lactose and the side chain of Tyr106, forming a sandwich with the galactose ring between the aromatic side chains. The side chain of Tyr106 adopts a generously allowed configuration in the lactose-bound structures, as determined by the Ramachandran plot. In the unliganded recECL structure, Tyr106 in only one of the four protein molecules was located in an additional allowed region of the plot. Thus it appears that the conformation of Tyr106 is affected by carbohydrate binding.



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Fig. 3. Binding of lactose to ECL. Hydrophobic interactions were observed between the side chains of Tyr106 and Phe131 and the galactose ring of the lactose molecule. This sandwiches the sugar ring between the aromatic side chains of the two amino acids. Lactose makes a number of contacts with side chains of combining site residues, but only the galactose moiety is accommodated in the shallow cleft of the combining site. There are no direct hydrogen bonds between the lectin and disaccharide; instead, interactions are mediated by a set of structural water molecules. Key residues involved in carbohydrate binding are Leu86, Asp89, Gly107, Asn133, Ala218, and Gln219.

 
The mode of lactose binding in ECL is similar to that observed for ECorL (Shaanan et al., 1991Go), although there are subtle differences in their carbohydrate specificities (Moreno et al., 1997Go; Teneberg et al., 1994Go). The altered carbohydrate specificity of ECL compared to ECorL is postulated to be due to differences in their amino acid sequences at positions 111 and 125 (Svensson et al., 2002Go)—substitution of residues at these positions causes rotation of the side chain of Val92, which is thought to induce structural changes in the combining site. Analysis of the crystal structure of nECL revealed that although there are no contacts between these three residues, Val92 makes contacts with Val126, which is located close to the combining site. It is possible that substitutions causing movement of Val92 might indirectly affect carbohydrate binding. However, the real effect of Val92 on carbohydrate binding remains to be fully analyzed.

The structures of recECL unliganded and in complex with lactose (at 2.13 Å and 1.70 Å resolution, respectively) superimpose with an overall root mean squared deviation of 0.21 Å. A direct comparison of side chain positions in the combining sites of the two structures revealed that there are no structural rearrangements on lactose binding. This indicates that the amino acid residues involved in carbohydrate recognition are optimally oriented in the ECL protomer.

Comparison with previously reported structure of nECL in the presence of lactose
As already mentioned, the crystal structures of nECL in complex with lactose and 2'-fucosyllactose were previously reported (Svensson et al., 2002Go). Comparison of the amino acid sequence of nECL used in this investigation with that reported for native ECL (hereafter referred to as SvenECL) revealed 10 differences: Asn16Asp, Asp17Asn, Ile25Leu, Ile59Met, Met62Ser, Ser175Pro, Leu180His, Ala181Val, Glu206Asp, and His234Gln (where the first residue is that in nECL and the second is that in SvenECL). The differences at positions 25, 181, and 206 represent conservative substitutions. The residues at positions 16 and 17 appear to be swapped in nECL compared with SvenECL, but because the side chains are of the same size and shape, these amino acids cannot be distinguished from one another on the basis of their electron density. The sequence of recECL was determined by DNA sequencing of multiple clones from two independent sources of E. cristagalli (Stancombe et al., 2002) and analysis of the electron density maps confirmed this sequence at all 10 locations where it differs from SvenECL (obtained by mass spectrometric peptide mapping, tandem mass spectrometry, and X-ray structure; Svensson et al., 2002Go). However, it is important to note that none of the sequence differences affect amino acids that are involved in carbohydrate binding or stabilizing the dimer interface. The main difference is the absence of a potential N-linked glycosylation site at residue 17 from nECL. The electron density maps for nECL were carefully inspected to ensure that there was no evidence of residue 17 being asparagine instead of aspartic acid, but no density was observed that might represent bound carbohydrate. Therefore we are confident that the sequence of nECL matches that of recECL.

Conclusions
In this investigation, nECL was crystallized in a new crystal form in complex with lactose and recECL was crystallized for the first time in two different crystal forms, both unliganded and in complex with lactose. We confirm that the tertiary structure of both forms of ECL is homologous to the known structures of other legume lectins, with protomers adopting the jelly-roll (legume lectin) fold. We have confirmed that nECL protomers associate back-to-back via the handshake motif, and we have shown that recECL protomers dimerize in the same way. Following comparison of native and recombinant forms of the lectin, it can be concluded that the presence of bound oligosaccharide does not influence the tertiary or quarternary structure of ECL. Furthermore, comparison of the structures of nECL and recECL in complex with lactose confirms that the presence of N-linked glycosylation on nECL has no major effect on the structure of the combining site or carbohydrate binding. Thus we confirm that recECL is native-like in terms of both its structure and biological activity.


    Materials and methods
 Top
 Abstract
 Introduction
 Results and discussion
 Materials and methods
 References
 
Protein purification and crystallization
nECL was purchased from Sigma (Dorset, UK). recECL was expressed and purified as previously reported (Stancombe et al., 2003Go). Briefly, E. cristagalli seeds (Sandeman Seeds, Oxford Botanical Gardens, UK) were germinated and genomic DNA, obtained from leaf material, was amplified by polymerase chain reaction and cloned into vector pMTL1015. For expression, this clone was transformed into Escherichia coli BL21 (DE3) cells and cultured at 8-L fermentation scale at 37°C until static growth (OD600 = 30). The protein was solubilized from inclusion bodies and subsequently refolded and purified on an immobilized lactose matrix. After extensive washing, recECL was eluted by the addition of 0.3 M lactose.

Crystallization was achieved using the vapor diffusion method (hanging drops) at 16°C, and crystals were observed within 3–4 weeks. For crystallization of nECL in space group P65, drops made up of 2 ml protein solution, 2 ml mother liquor (70% 2-methyl-2,4-pentanediol and 0.1 M HEPES, pH 7.0) and 0.4 ml 100 mM lactose solution and were equilibrated over sealed wells containing 800 ml mother liquor. recECL crystallized in space group P1 from drops containing 2 ml protein and 2 ml mother liquor (17% polyethylene glycol [PEG] 3350, 0.3 M sodium chloride, and 0.02 M imidazole). recECL was also cocrystallized with lactose using mother liquor made up of 20% PEG 3350 and 0.2 M imidazole.

X-ray data collection and structure determination
X-ray diffraction data were collected at 100 K using the Synchrotron radiation Source at Daresbury, UK, on station PX14.1 (wavelength 1.483 Å). Raw data images for nECL were indexed and integrated using DENZO (Otwinowski and Minor, 1997Go) and then scaled with SCALEPACK (Otwinowski and Minor 1997Go). X-ray images collected for the recECL crystals were processed and scaled using HKL2000 (Otwinowski and Minor, 1997Go) (Table I).

Initial phases were obtained by the molecular replacement method using the program AMoRe (Navaza, 1994Go). For nECL, the crystal structure of ECorL at 1.95 Å resolution (PDB code 1AX1) (Elgavish and Shaanan, 1998Go) was used as an initial model with the nine nonconserved residues mutated to alanine, and all waters, ligands, and metal ions removed. Refinement was performed using the CNS suite of programs (Brunger et al., 1998Go). After one round of refinement, the crystallographic R factor (Rcryst) dropped to 29.59% (Rfree 32.38%, based on 5% of reflections omitted from the refinement). Calculated phases from the refined structure were used to determine |Fo| – |Fc| and 2|Fo| – |Fc| electron density maps. Careful examination of the |Fo| – |Fc| map allowed mutation of the nonconserved residues back to their native side chains and the addition of calcium and manganese ions to the model. HEPES and lactose were built into electron density of each protomer, with parameter and topology files from the HIC-Up server (Kleywegt and Jones, 1998Go). Similarly glycosylated sugars (three in molecule A and six in molecule B, part of the heptasaccharide bound to Asn113) were modeled into the |Fo| – |Fc| electron density map, riding on the Asn113 residue.

Repeated rounds of refinement and model building were carried out to improve the model. After several rounds of refinement, water molecules were added to the structure if there were peaks in the |Fo| – |Fc| electron density maps with heights greater than 3{Sigma} at hydrogen bond forming distances from the appropriate atoms. 2|Fo| – |Fc| maps were also used to check the consistency in peaks. Water molecules with a temperature factor of >60 Å2 were excluded from subsequent refinement steps.

Subsequent solution of recECL structures was achieved using the refined coordinates of nECL (at 2.0 Å resolution) as a search model. Model building and refinement procedures were undertaken as described for nECL. For recECL in space group P1, apart from the lactose molecule, glycerol was modeled into the carbohydrate-combining site.

The program PROCHECK (Laskowski et al., 1993Go) was used to assess the quality of each structure after the final round of refinement. Analysis of the Ramachandran plot for each structure revealed that over 99% of residues were located in allowed regions of the plot. The refinement statistics for all structures are listed in Table I.


    Acknowledgements
 
We thank Frances Alexander for supply of purified recECL; the staff at the Synchrotron Radiation Source, Daresbury; and Gayatri Chavali and Shalini Iyer for help during X-ray data collection. This work was supported by postgraduate studentships to K.T. and N.T. through a joint agreement between the University of Bath and Health Protection Agency, Salisbury, United Kingdom. The atomic coordinates for nECL (P65), recECL (P1), and recECL (P21) with codes 1UZY, 1UZZ, and 1V00, respectively, have been deposited with the RCSB Protein Data Bank.


    Footnotes
 
2 To whom correspondence should be addressed; e-mail: k.r.acharya{at}bath.ac.uk

1 Present address: ICGEB, Aruna Asaf Ali Marg, New Delhi 110 067, India Back


    Abbreviations
 
ECL, Erythrina cristagalli lectin; ECorL, Erythrina corallodendron lectin


    References
 Top
 Abstract
 Introduction
 Results and discussion
 Materials and methods
 References
 
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