Search tips
Search criteria 


Logo of actafjournal home pagethis articleInternational Union of Crystallographysearchsubscribearticle submission
Acta Crystallogr Sect F Struct Biol Cryst Commun. 2006 November 1; 62(Pt 11): 1100–1103.
Published online 2006 October 20. doi:  10.1107/S1744309106038887
PMCID: PMC2225211

New crystal forms of Diocleinae lectins in the presence of different dimannosides


Studying the interactions between lectins and sugars is important in order to explain the differences observed in the biological activities presented by the highly similar proteins of the Diocleinae subtribe. Here, the crystallization and preliminary X-­ray data of Canavalia gladiata lectin (CGL) and C. maritima lectin (CML) complexed with Man(α1-2)Man(α1)OMe, Man(α1-3)Man(α1)OMe and Man(α1-4)Man(α1)OMe in two crystal forms [the complexes with Man(α1-3)Man(α1)OMe and Man(α1-4)Man(α1)OMe crystallized in space group P32 and those with Man(α1-2)Man(α1)OMe crystallized in space group I222], which differed from those of the native proteins (P21212 for CML and C222 for CGL), are reported. The crystal complexes of ConA-like lectins with Man(α1-4)Man(α1)OMe are reported here for the first time.

Keywords: lectin–sugar interactions, Dioocleinae lectins

1. Introduction

Lectins are carbohydrate-binding proteins which function as cognate receptors for various cell-surface glycoproteins, resulting in several important cellular-mediated events ranging from mitogenic processes to plant defence mechanisms (Weis & Drickamer, 1996 [triangle]).

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 [triangle]). 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 [triangle]).

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 [triangle]). 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 [triangle]) 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 [triangle]).

Native CGL and CML have been crystallized and their structures have been solved in space groups C222 and P21212, respectively (Moreno et al., 2004 [triangle]; Gadelha et al., 2005 [triangle]). 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 [triangle]).

We report here the crystallization and preliminary data of six dimannoside-complexed crystals of CGL and CML in two different space groups, including the first crystal of a ConA-like lectin with the dimannoside Man(α1-4)Man(α1)OMe.

2. Material and methods

2.1. Crystallization

CML and CGL were purified according to the methods of Moreira & Cavada (1984 [triangle]) and Ceccatto et al. (2002 [triangle]), respectively. Each dimannoside [Man(α1-2)Man(α1)OMe, Man(α1-3)Man(α1)OMe and Man(α1-4)Man(α1)OMe; purchased from Sigma–Aldrich] was added in a 0.08 molar proportion to 10 mg ml−1 of the pure lectin dissolved in 20 mM Tris–HCl pH 7.6 containing 5 mM CaCl2/MnCl2 and incubated for 24 h at room temperature.

Crystallization trials were performed based on two different strategies: (i) varying the ammonium sulfate concentration from 0.25 to 3.0 M combined with different buffers varying from pH 3.5 to 9.0 (strategy supplied by McPherson, 2003 [triangle]) and (ii) varying the sodium formate concentration from 0.5 to 7.0 M with Tris–HCl pH 7.6. Both strategies were carried out at 293 K using the hanging-drop vapour-diffusion method in Linbro plates. The drops were composed of equal volumes (2 µl) of protein solution and reservoir solution and were equilibrated against 500 µl of the latter.

2.2. Data collection and processing

X-ray diffraction data were collected at a wavelength of 1.43 Å using a synchrotron-radiation source (MX1 station, Laboratório Nacional de Luz Síncrotron, Campinas, Brazil) and a CCD detector (MAR Research) at 100 K. To avoid freezing, crystals were soaked in a cryoprotectant solution containing between 30 and 50% glycerol in mother liquor. Data were processed, indexed and integrated using MOSFLM and scaled using SCALA (Collaborative Computational Project, Number 4, 1994 [triangle]; Leslie, 1992 [triangle]).

3. Results and discussion

Crystallization conditions, space groups and the number of molecules per asymmetric unit of the dimannoside complexes and native crystals are listed in Table 1 [triangle]. The preliminary crystallographic data for CGL complexes is presented in Table 2 [triangle] and for CML complexes in Table 3 [triangle]. The crystal complexes are depicted in Fig. 1 [triangle].

Figure 1
Crystals of (a) CGL–Man(α1-2)Man(α1)OMe, (b) CML–Man(α1-2)Man(α1)OMe, (c) CGL–Man(α1-3)Man(α1)OMe, (d) CML–Man(α1-3)Man(α1)OMe, (e) CGL–Man(α1-4)Man(α1)OMe ...
Table 1
Crystallization conditions, space groups and number of molecules per asymmetric unit for native CGL and CML and for their complexes with Man(α1-2)­Man(α1)OMe, Man(α1-3)Man(α1)OMe and Man(α1-4)Man(α1)OMe ...
Table 2
Statistics of data collection for CGL complexes
Table 3
Statistics of data collection for CML complexes

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 [triangle]). 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 [triangle]).

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 [triangle]). 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.

The complex crystals were obtained in space groups that differed from those of the native lectins. Since crystal packing has an influence on the protein conformation (Kanellopoulos et al., 1996 [triangle]), our work may be important in revealing interactions distinct from those in the native structures.


This work was supported by Fundação Cearense de Apoio ao Desenvolvimento Científico e Tecnológico (FUNCAP), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação de Amparo à Pesquisa do estado de São Paulo (FAPESP), Universidade Regional do Cariri (URCA), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and National Synchrotron Light Laboratory (LNLS), Brazil. BSC and WFA are senior investigators of CNPq.


  • Bouckaert, J., Hamelryck, T. W., Wyns, L. & Loris, R. (1999). J. Biol. Chem.274, 29188–29195. [PubMed]
  • Brinda, K. V., Mitra, N., Surolia, A. & Vishveshwara, S. (2004). Protein Sci.13, 1735–1749. [PubMed]
  • Ceccatto, V. M, Cavada, B. S., Nunes, E. P., Nogueira, N. A., Grangeiro, M. B., Moreno, F. B., Teixeira, E. H., Sampaio, A. H., Alves, M. A., Ramos, M. V., Calvete, J. J. & Grangeiro, T. B. (2002). Protein Pept. Lett.9, 67–73. [PubMed]
  • Collaborative Computational Project, Number 4 (1994). Acta Cryst. D50, 760–­763. [PubMed]
  • Delatorre, P., Nascimento, K. S., Melo, L. M., de Souza, E. P., da Rocha, B. A. M., Benevides, R. G., de Oliveira, T. M., Bezerra, G. A., Bezerra, M. J. L. B., da Cunha, R. M. S., Freire, V. N. & Cavada, B. S. (2006). Acta Cryst. F62, 166–168. [PMC free article] [PubMed]
  • Gadelha, C. A. A., Moreno, F. B. M. B., Santi-Gadelha, T., Cajazeiras, J. B., da Rocha, B. A. M., Rustiguel, J. K. R., Freitas, B. T., Canduri, F., Delatorre, P., de Azevedo, W. F. Jr & Cavada, B. S. (2005). Acta Cryst. F61, 87–89. [PMC free article] [PubMed]
  • Hamelryck, T. W, Loris, R., Bouckaert, J. & Lode, W. (1998). Trends Glycosci. Glycotechnol.10, 349–360.
  • Kanellopoulos, P. N., Tucker, P. A., Pavlou, K., Agianian, B. & Hamodrakas, S. J. (1996). J. Struct. Biol.117, 16–23. [PubMed]
  • Leslie, A. G. W. (1992). Jnt CCP4/ESF–EACBM Newsl. Protein Crystallogr.26
  • Lis, H. & Sharon, N. (1998). Chem. Rev.98, 637–674. [PubMed]
  • McPherson, A. (2003). Crystallization of Biological Macromolecules. New York: Cold Spring Harbor Laboratory Press.
  • Moothoo, D. N., Canan, B., Field, R. A. & Naismith, J. H. (1999). Glycobiology, 9, 539–545. [PubMed]
  • Moreira, R. A. & Cavada, B. S. (1984). Biol. Plant.26, 113–120.
  • Moreno, F. B. M. B., Delatorre, P., Freitas, B. T., Rocha, B. A. M., Souza, E. P., Faco, F., Canduri, F., Cardoso, A. L. H., Freire, V. N., Lima, J. L., Sampaio, A. H., Calvete, J. J., de Azevedo, W. F. Jr & Cavada, B. S. (2004). Acta Cryst. D60, 1493–1495. [PubMed]
  • Sharon, N. & Lis, H. (1990). FASEB J.4, 3198–3208. [PubMed]
  • Van Damme, E. J. M., Peumans, W. J., Barre, A. & Rougé, P. (1998). Crit. Rev. Plant Sci.17, 575–692.
  • Weis, W. I. & Drickamer, K. (1996). Annu. Rev. Biochem.65, 441–473. [PubMed]

Articles from Acta Crystallographica Section F: Structural Biology and Crystallization Communications are provided here courtesy of International Union of Crystallography