Overall structure of CGL
The crystal structure of native CGL and in complex with α-methyl-mannoside (CGL-αMM) provides a new source of information for the understanding of lectins and reveals a new binding site for a non protein amino acid at the monomers contact interface of each canonical dimer of leguminous lectins. The overall structure of native CGL and CGL-αMM has been refined at the 2.3 Å and 2.31 Å resolutions, respectively (Figure 1). The lectin CGL model was refined in the absence of ligand to produce the Fo-Fc map. This map is unbiased insofar as the atomic coordinates used for phase calculations have never been refined together with the ligand. The ligand was finally inserted to produce the final coordinates after some further refinement. The models present good stereochemistry, and the Ramachandran plot is consistent with a geometrically well-defined structure (Table 1).
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
Abu was found at the monomers contact interface of CGL-αMM (Figure 2). The Fo-Fc omit map in figure 3a is contoured at 3σ cutoff and confirms that the map is unbiased and the electron density clearly has Abu fitted in the map. Abu ligand is accessible to the solvent surface by the carboxyl group and interacts with one chain through hydrogen bond with Asp139 and an interstitial water molecule, which interacts with Asn 124 and with the other chain through hydrogen bonds with Ala 125 and with another interstitial water interacting with Gln 137, and through hydrophobic interaction with Leu126 and Val 179 (Figure 2). The presence of Abu at the monomers interface increased the intermolecular contacts and strongly stabilized the canonical dimers. This interaction decreased the vibrational spectrum and increased the X-ray scattering in loop regions. In order to obtain further evidence for the presence of ABU in the structure, we collected data in the absence of glycerol at room temperature. Analysis of these low resolution CGL structures (solved at approximately 3.0 Å resolution), also indicates presence of the electron density for the Abu. At room temperature, protein crystals were mounted in sealed thin-walled glass capillary tubes for X-ray data collection, these conditions lead to a loss in the resolution power. The best data set diffracted to 3.0 Å and shows ABU in a non-conclusive electron density map, but the electron density calculated at 2.3 Å shows clearly that the map corresponding to the ABU density. Due to the distance of atoms is really difficult to consider that this density refers to a set of ordered waters in both structures. The structure solved in the absence of glycerol and showing appropriate density for ABU is shown in figure 3b.
Hydrophilic interactions occur between Abu and Asp139 by H-bonds. The OD2 hydroxyl oxygen from Asp139 and Abu oxygen display at 2.4 Å. The same Abu molecule oxygen builds another H-bond with a water molecule at 2.6 Å and the Abu nitrogen also interacts with a water molecule at 2.5 Å (Figure 4).
The residues that compose this hydrophobic pocket are highly conserved in other ConA-like lectins from Phaseoleae tribe, such as, lectins from Canavalia brasiliensis seeds (ConBr), Canavalia maritima seeds (ConM) and Dioclea grandiflora seeds (DGL). These residues were also found to be highly conserved in various lectins from Phaseoleae, Glycineae and Sophoreae tribes from Fabales order. Lectins isolated from seeds of Bowringia mildbraedii (BMA), Dolichos lablab (Dlab) and Glicine max (GML and SBA) presented in their amino acid sequences the same residues that were found in CGL Abu site or, in some cases, an amino acid conservative change (Table 2). All the residues listed form the monomer-monomer interface of the dimmers of these legume lectins. The conservative changes in Abu site, specifically in Ala125, do not seem to be a problem to perform the interaction with molecules like Abu because the hydrogen bond occurs between the Abu and the main chain of this residue. In addition, residues corresponding to Leu 126 and Val 179, which are effective in hydrophobic anchoring of Abu, and the ones corresponding to Asp 139 and Gln 137, responsible by hydrophilic contacts, are highly conserved in spite of the relative phylogenetic distance of these leguminous tribes. It is important to consider that the origin of the genes that codify plant lectin rose from a unique ancestral and the little differences found between them were originated by divergence evolutionary processes . But structures are more conserved than sequences during evolution. Therefore some differences found in the amino acids of Abu site not necessarily mean an absence of functional correlations between the CGL and other lectins .
The mass spectrometry analysis of CGL reveals a very well purified spectrum showing the peaks of ions with charge from +18 (m/z = 1419.8669) to + 28 (m/z = 913.1152) (Figure 5a). The deconvolution of this spectrum reveals two peaks to CGL exact mass; the first peak (m/z = 12770.0010) is the double-charged ion and the second one (m/z = 25541.0020) represents the mono-charged ion and the exact mass of the protein (Figure 5b). The presence of Abu in the CGL structure was confirmed by mass spectrometry. The mass spectra of Abu displayed m/z (+H) = 104.1131 ± 0.1 Min the low mass spectrum in the CGL ESI-Q-ToF MS analysis. The fragmentation of this ion in an MS/MS experiment revealed an ion-fragment with m/z = 86.05 ± 0.1, this mass referred to Abu molecular mass with a common loss of a water group (~18 Da). (Figure 5c).
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.
CGL reveals an interaction site for a non-protein amino acid, which may be involved in defense mechanisms and the induction of response against pathogens in synergy with jasmonate, salicylic acid and abscisic acid pathways. During the final step of the germination of leguminous seeds, Abu concentration increases. In lentil experiments shown by Rozan and collaborators , the total content of non-protein amino acids in non-germinating seeds was very low. This content increased after four days of germination and then even more when the stage of seedling was reached . There's a possibility that at least a part of the increase in Abu concentration may be explained by the release of this molecule when the canonical dimmer is disrupted, when the lectins start to be consumed during final steps of germination . Consequently free Abu increases in the seedlings. This hypothesis is schematically depicted in Figure 6.
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).