Structure of a lectin from Canavalia gladiata seeds: new structural insights for old molecules
© Delatorre et al; licensee BioMed Central Ltd. 2007
Received: 06 October 2006
Accepted: 02 August 2007
Published: 02 August 2007
Lectins are mainly described as simple carbohydrate-binding proteins. Previous studies have tried to identify other binding sites, which possible recognize plant hormones, secondary metabolites, and isolated amino acid residues. We report the crystal structure of a lectin isolated from Canavalia gladiata seeds (CGL), describing a new binding pocket, which may be related to pathogen resistance activity in ConA-like lectins; a site where a non-protein amino-acid, α-aminobutyric acid (Abu), is bound.
The overall structure of native CGL and complexed with α-methyl-mannoside and Abu have been refined at 2.3 Å and 2.31 Å resolution, respectively. Analysis of the electron density maps of the CGL structure shows clearly the presence of Abu, which was confirmed by mass spectrometry.
The presence of Abu in a plant lectin structure strongly indicates the ability of lectins on carrying secondary metabolites. Comparison of the amino acids composing the site with other legume lectins revealed that this site is conserved, providing an evidence of the biological relevance of this site. This new action of lectins strengthens their role in defense mechanisms in plants.
Most of the biochemical lectin studies have been based on monochromatic view since almost all the properties of these proteins had been commonly reported in terms of lectin-carbohydrate recognition. For several years, the definition of lectins has been improving focused on the carbohydrate-binding properties. The most recent accepted definition establishes lectins as proteins with at least one non-catalytic domain able to recognize and bind reversibly to specific mono and oligosaccharides. They are subdivided into four types: merolectins, hololectins, chimerolectins and superlectins. This classification was conceived in terms of the carbohydrate-binding domain and another unrelated domain . Several studies have tried to find other binding sites that could possibly recognize plant hormones, secondary metabolites and isolated amino acid residues [2–4].
Over 250 non-protein amino acids have been identified in plants . A number of these compounds are intermediates in the synthesis and catabolism of protein amino acids . However, many of these non-protein amino acids may play a role as defensive agents. They show their toxicity in many ways; some of them block the synthesis and the absorption of protein amino acids or can wrongly incorporated into proteins in organisms that feed on these plants. Plants that synthesize non-protein amino acids are not susceptible to the toxicity of these compounds. Seeds from Canavalia ensiformis, which synthesize high quantities of non-protein amino acids, display a biological system capable of discriminating between these amino acids and the others .
Non-protein amino acids are especially abundant in Leguminosae, Liliaceae and in several higher fungi and marine algae. Plant organs rich in these metabolites are seeds (Leguminosae) or rhizomes (Liliaceae). Concentrations in seeds can exceed 10% of dry weight and up to 50% of the nitrogen could be attributed to them. Since non-protein amino acids are often remobilized during germination, they certainly function as N-storage compounds in addition to their role as defense chemicals . If non-protein amino acids are taken up by herbivores, microorganisms or other plants, they may interfere with their metabolism.
Aminobutyric acid (Abu) is a non-protein amino acid that can protect certain plants against pathogens; for instance, when introduced into Arabidopis plants, it has the ability to induce resistance to certain pathogens. Abu protects these plants against pathogens through the activation of natural defense mechanisms of the plant, such as callose deposition, hypersensitive response (HR), and the formation of trailing necroses. Induced resistance is often associated with a process called priming, which is an increased capacity to mobilize cellular defense responses .
Most plant lectins not only play a role in the plant itself (e.g., as a store of nitrogen or as a specific recognition factor) but are also capable of interfering with the functioning of foreign organisms through an interaction with glycoconjugates on the surface or in the digestive tract of these organisms . Although this interference has been reported as a specific event of carbohydrate recognition, it has not been elucidated yet. Stress-regulated pathways for rapid and high gene expression are one of the essential elements in stress acclimation. Salicylic acid, jasmonic acid, systemin, ethylene, and aminobutyric acid have been implicated in the potentiation of gene expression [11, 12], and other signal molecules have been shown to play a similar role .
The content of free protein amino acids in seeds varies among species and increases dramatically after germination. More non-protein amino acids were found in lentil seedlings compared to the seeds . Most plant lectins are probably involved in plant defense . The mechanism of action still remains unclear even though induced response mechanisms are proposed for many pathogen-mediated injuries in plants. The direct interference with viruses and microorganisms is rather exceptional, and the deleterious effects of plant lectins on both predatory invertebrates and animals are well documented .
We report here the crystal structure of a native and complexed lectin isolated from Canavalia gladiata seeds, describing a new binding pocket in ConA-like lectins, which may be related to pathogen resistance, a site where a non-protein amino acid, such as α-amino butyric acid, can bind.
Results and discussion
Overall structure of CGL
Statistics of data collection, refinement and quality of the structure
Total number of observations
Total number of unique observations
Resolution limit (Å)
Cell parameters (Å)
a = 100.90
a = 100.91
b = 115.35
b = 115.75
c = 241.08
c = 241.62
Resolution range (Å)
Number amino acid residues in biological assembly
Number of water molecules
RMS deviations from ideal values
Bond lengths (Å)
Bond angles (degrees)
Average B value for whole protein chain (Å 2)
Residues in most favored regions
Residues in additional allowed regions
Residues in generously allowed regions
Among the leguminous lectins isolated from the tribe Diocleinae, only those purified from the seeds of C. ensiformis, C. brasiliensis, C. maritima, Dioclea grandiflora, D. guianensis and Cratylia mollis have three-dimensional structures resolved by X-ray crystallography [14, 15]. These lectins showed respectively 98%, 98%, 99%, 84%, 82% and 81% similarity in their amino acid sequences as compared with CGL.
The oligomeric structures of CGL and CGL-αMM involve two 'canonical' dimers, and these dimers are associated by salt bridges between β-strands. The model was examined by Xfit, and one region around Asp 139 showed a well-defined electron density (fo-fc contoured at 5σ), not elucidated for the model, the atomic coordinates for this structure were deposited without the elucidation of this density with PDB access 1 WUV. After an analysis of the amino acid and secondary metabolite content in leguminous seeds, α-aminobutyric acid (Abu), a non-protein amino acid, was chosen according to electron density. The atomic coordinates for the structure in complex with Abu and α-methyl-mannoside (CGL-αMM) was deposited in PDB with access code 2D7F.
An interesting feature of the presence of the Abu molecule in CGL structures is the great increase in electron density in loops. Previous ConA structures showed some poor density regions at N-terminal residues and in surface loops at residues 118–122, 149–151, 160–163 [16–18]. The CGL structure presents well-defined electron density in these loops and is the first ConA-like structure that shows a stable loop in 118–122 region.
In both CGL crystal structures, the amino acids involved in metal binding are conserved. The calcium metal ion bond drives the trans-to-cis isomerization of the Ala207-Asp208 peptide bond, which is conserved in all known legume lectin crystal structures. This cis-peptide bond contributes to the stabilization of the binding pocket by orienting the positions of residues Asn14 and Arg228.
The sugar-binding site is a highly conserved region in ConA-like lectins. The role of conserved amino acid residues in monosaccharide interactions with lectins has been extensively reported. Commonly, five to eight H-bonds occur between monosaccharides and the Diocleinae lectin domain amino acids Asn14, Leu99, Tyr100, Asp208 and Arg228 ; CGL-αMM displays an alpha-methyl-mannoside recognition involving seven H-bonds with specific amino acid residues and another with a water molecule. The nitrogen side chain of Asn14 forms a single H-bond with αMM O4 oxygen at 2.6 Å; the main chain nitrogens of Leu99 and Tyr100 are connected with αMM O5 and O6 oxygens. O5 is at 3.0 Å from Leu99 and O6 is at 2.9 Å and 2.98 Å from Leu99 and Tyr100, respectively. Oxygens from Asp 208 side chain are the CGL closest atoms from the αMM. They are positioned at 2.5 Å from O7 and 2.7 Å from O6, and the main chain nitrogen of Arg 228 makes a 2.9 Å H-bond with αMM O3 oxygen.
α-aminobutyric acid (Abu) interaction
Amino acids of Abu hydrophobic pocket, showing the conservation in lectins isolated from seeds of leguminous from various tribes.
Abu hydrophobic pocket
The finding of Abu in CGL strongly indicates the capability of lectins to bind secondary metabolites. Some legume lectins possess a hydrophobic binding site with high affinity for adenine and certain adenine-derived plant hormones . The binding of adenine has been described mainly for tetrameric legume lectins, including DBL, PHA-E and SBA [3, 4]. This new property of lectins supports their role in defense mechanisms in plants.
When Abu concentration increases in seedlings, it promotes the synthesis of ethylene. Once lectins are consumed at the end of germination, it's reasonable to assume that there could be a correlation between lectin-binding substances such as Abu and the development of plant response against stress and pathogens [11, 22].
Other non-protein amino acids, such as the Abu isomers β-aminobutyric acid (BABA) and γ-aminobutyric acid (GABA), have been reported to be physiological inducers in many processes related to response-induced resistance and stress . Actually, non-protein amino acids seem to be involved in ethylene-dependent induced systemic resistance . If our hypothesis is true, lectins may have an important role in this mechanism, which would define a lectin-dependent ISR pathway.
It should be highlighted that this discussion above is simply a hypothesis and that we don't really have any certainties regarding the real relevance of this site and the binding of Abu to the lectin. But the fact that the amino acids present in this site are conserved in legume lectins as mentioned before is undoubtedly an evidence that an important biological activity is mediated by this site.
The common carbohydrate-binding site of lectins has been reported as the main target in the study of lectins, but evidence of new biological sites leads to questions about the physiological function of these proteins, first considered storage proteins and later found to play a role in plant defense mechanisms.
The increase in Abu concentration in the seedling corresponds to the increasing induction of hypersensitive response in adult plants considering the high concentrations of HR inducers at this time of plant ontogeny as described by Boege and Marquis . In agreement, lectin concentration in seeds declines drastically by the end of germination and, therefore, is lower in the seedling (Figure 6).
Our study supports other investigations proposing a function for lectins beyond simple carbohydrate recognition. Plant lectins are proteins which may also interact with secondary metabolites, displaying cell recognition, and defense functions in plant physiology. Here, we described a new interaction site not related to the sugar-binding site in a ConA-like lectin that may be related to plant defense.
Crystallization and Data Collection
The purification of Canavalia gladiata lectin (CGL) was performed as discribed by Cecatto et al.  and Moreno et al. . Crystals of the Canavalia gladiata lectin were grown by the hanging drop vapor diffusion method, drops contained 3 μL of protein solution and 3 μL of 0.1 M Tris-HCl, pH 8.5, with 2.0 M ammonium sulfate. Cryoprotected native CGL crystals diffracted to a resolution of 2.3 Å using a synchrotron radiation source and indexed, integrated and scaled at the same resolution. Low resolution data sets have been collected in capillar without cryoprotection at 2.9, 3.0 and 3.1 Å resolution in room temperature to confirm the presence of Abu. Native crystals were also soaked with a solution containing 1 mM α-methyl-mannoside. The complexed crystals diffracted to 2.3 Å. The data were indexed, integrated and scaled at 2.3 Å using MOSFLM  and SCALA . Crystals belong to the orthorhombic space group C2221. The calculated Matthews coefficient indicated a tetramer in the asymmetric unit, and this protein assumes the same oligomerization in biological conditions.
Molecular Replacement and Refinement
The crystal structures CGL and CGL-αMM were determined by molecular replacement using the MolRep program . Canavalia ensiformis lectin (ConA) structure without the complexed sugar and water molecules (PDB code 5CNA) was used as the search model . The Abu coordinates were obtained by PRODRG program . The initial structures were refined using REFMAC5  and water molecules were added to the models using XtalView . The crystal structures were deposited in the PDB  with accession code 1 WUV and 2D7F. The crystallographic statistics data are shown in Table 1. An omit map contoured at 3 σ to the aminobutyric acid was generated using CCP4 Omit program .
Mass Spectrometry Analysis
The presence of Abu in the molecule of native CGL was analyzed in an ESI-Q-ToF/MS/MS experiment. An amount of 1 mg of CGL was dissolved in 1 mL of a solution containing 50% acetonitrile and 5% TFA. The protein solution was diluted 100 × and injected in a nano-electrospray ionization source and analyzed in a Micromass™ quadrupole time-of-flight instrument with a resolution of 8,000 and accuracy of 10 ppm. The spectral data were processed using a Biolynx 4.0 program.
Canavalia gladiata lectin
Canavalia gladiata lectin in complex with α-methyl-mannoside
Canavalia ensiformis lectin
Electrospray ionization source
Quadrupole-time of flight analyzer
systemic acquired resistance
induced systemic resistance.
This work was partly financed by Fundação Cearense de Apoio ao Desenvolvimento Científico e Tecnológico (FUNCAP), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Universidade Regional do Cariri (URCA), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Laboratório Nacional de Luz Síncrotron (LNLS), Laboratório de Espectrometria de Massas/LNLS, Campinas – Brazil, and FAPESP (SMOLBNet, 01/07532-0). Benildo Sousa Cavada and Walter Filgueira de Azevedo Jr. are senior investigators of CNPq. BSC, AHS and WFA are senior investigators of CNPq. We also thank Dr. A. Leyva for English language editing of the manuscript.
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