Plant lectins, especially those purified from species of the Leguminosae family, represent the best studied group of carbohydrate-binding proteins (Van Damme et al., 1998 ). Legume lectins have been used for decades as a model system for studying protein–sugar interactions. Fundamental insights obtained from investigating these protein model systems can often be readily applied to lectins outside this family, such as the pharmaceutically important C-type lectins and galectins (Hamelryck et al., 1998 ).

The legume lectins from the Diocleinae subtribe are highly similar proteins that present significant differences in the potency/efficacy of their biological activities (Delatorre et al., 2006 ). For instance, even though they only differ by three amino-acid residues, Canavalia gladiata lectin (CGL) and C. maritima lectin (CML) present different patterns in several activities, such as in aorta-relaxation experiments (to be published). In spite of these activities being very well documented, little is known about the receptors with which they interact and how this happens. The crystal structures of many plant lectins have revealed how they specifically recognize their carbohydrate ligands (Lis & Sharon, 1998 ) and this may give us a direction for elucidating their mechanism of action.

An outstanding feature of Diocleinae lectins is that although all the monomers have similar tertiary structures, they show different modes of quaternary association. Minor differences in the ratio between the dimeric and tetrameric forms, together with changes in the relative orientations of the carbohydrate-binding sites in the quaternary structures, have been hypothesized to contribute to the differing biological activities possessed by these lectins (Brinda et al., 2004 ).

Native CGL and CML have been crystallized and their structures have been solved in space groups C222 and P2₁2₁2, respectively (Moreno et al., 2004 ; Gadelha et al., 2005 ). Both have tetramers composed of 237 amino-acid monomers in the asymmetric unit. Crystals of CML in complex with trehalose and maltose were subsequently obtained under the same conditions as described for the native protein and presented the same space group P2₁2₁2 (Delatorre et al., 2006 ).

Although the crystallization conditions are different for the crystal complexes and the native proteins, the main cause of the change in the symmetry seems to be the presence of the dimannosides. CGL and CML complexed with Man(α1-2)Man(α1)OMe crystallized in space group I222 with one molecule in the asymmetric unit after 72 h, while the crystals of CGL and CML complexed with Man(α1-3)Man(α1)OMe and Man(α1-4)Man(α1)OMe belonged to space group P3₂ with a tetramer in the asymmetric unit, crystallizing after 48 h.

The importance of the dimannosides in the crystallization process is noteworthy. Native CGL and CML crystallize under different conditions and in different space groups: 0.1 M Tris–HCl pH 8.5, 2.0 M ammonium sulfate (C222) and 0.1 M Na HEPES pH 8.48, 4% PEG 400, 2.0 M ammonium sulfate (P2₁2₁2), respectively. In the presence of the carbohydrates, the crystallization conditions become the same for each sugar: for CML and CGL with Man(α1-2)Man(α1)OMe the condition was 0.1 M Tris–HCl pH 8.0–9.0, 1.8–2.6 M ammonium sulfate, while crystals of CML and CGL complexed with Man(α1-3)Man(α1)OMe and Man(α1-4)Man(α1)OMe only grew in the presence of 4.5–6.5 M sodium formate.

It is well established that legume lectins possess three types of hydrophobic sites based on different ligand affinities (Sharon & Lis, 1990 ). One of these sites is adjacent to the monosaccharide-binding site and participates in interactions involving several hydrophobic sugars. Bouckaert and coworkers described that the O3-linked mannose of Man(α1-3)Man(α1)OMe and the O6-linked mannose of Man(α1-6)Man(α1)OMe bind to the hydrophobic subsite formed by Tyr12, Tyr100 and Leu99 (Bouckaert et al., 1999 ).

Investigations of the binding of Man(α1-2)Man(α1)OMe, Man(α1-3)Man(α1)OMe and Man(α1-6)Man(α1)OMe to ConA in this same hydrophobic subsite revealed significant differences in their affinity (Moothoo et al., 1999 ). Based on this, we have crystallized CML and CGL complexed with the dimannosides Man(α1-2)Man(α1)OMe and Man(α1-3)Man(α1)OMe in order to compare them with the previously reported complexes from ConA and correlate their structure and affinity. The crystal complexes of CGL and CML in the presence of Man(α1-4)Man(α1)OMe represent the first ConA-like structure with this carbohydrate. The differences between the affinities of these mannosides may reflect how the protein binds to receptors related to lectin-mediated responses in plants or in other organisms. Therefore, solving the structures of CGL and CML complexed with dimannosides may be important to understanding many of their biological activities. Our data may clarify the understanding of how the interactions between the dimannosides and the hydrophobic subsite formed by Tyr12, Tyr100 and Leu99 occur.