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Acta Crystallogr Sect F Struct Biol Cryst Commun. 2006 February 1; 62(Pt 2): 166–168.
Published online 2006 January 27. doi:  10.1107/S1744309106001801
PMCID: PMC2150952

Crystallization and preliminary X-ray diffraction analysis of the lectin from Dioclea rostrata Benth seeds


Lectins from the Diocleinae subtribe (Leguminosae) are highly similar proteins that promote various biological activities with distinctly differing potencies. The structural basis for this experimental data is not yet fully understood. Dioclea rostrata lectin was purified and crystallized by hanging-drop vapour diffusion at 293 K. The crystal belongs to the orthorhombic space group I222, with unit-cell parameters a = 61.51, b = 88.22, c = 87.76 Å.  Assuming the presence of one monomer per asymmetric unit, the solvent content was estimated to be about 47.9%. A complete data set was collected at 1.87 Å resolution.

Keywords: lectins, Dioclea rostrata

1. Introduction

Lectins are carbohydrate-binding proteins or glycoproteins of non-immune origin found in all types of living organisms that decipher the glycocodes encoded in the structure of glycans attached to soluble and integral cell-membrane glycoconjugates (Gabius & Gabius, 1997 [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]).

Although the Diocleinae subtribe lectins sequenced to date show a high degree of similarity, they present significant differences in the potency/efficacy of their many biological activities (Alencar et al., 1999 [triangle]; Andrade et al., 1999 [triangle]; Assreuy et al., 1999 [triangle]; Havt et al., 2003 [triangle]; Lopes et al., 2005 [triangle]). For instance, Dioclea rostrata lectin (DRL) has been found to be more efficient in promoting the synthesis of IFN-γ and histamine secretion than other lectins from the same subtribe (e.g. D. grandiflora and D. guianensis). On the other hand, DRL is less efficient than other Diocleinae lectins in stimulating the synthesis of TNF-α and IL-10 (Cavada et al., 2001 [triangle]). Several factors contribute to these differences in the activities of Diocleinae lectins: for example, the pH-dependent oligomerization that some of these lectins present and the relative position of the carbohydrate-binding site (Wah et al., 2001 [triangle]). The distinct biological activities presented by ConA and ConBr have been shown to be a consequence of a non-conservative substitution that affects the carbohydrate binding site, making it more open in ConBr than it is in ConA (Sanz-Aparicio et al., 1997 [triangle]). Canavalia maritima lectin presents decreased nitric oxide release compared with ConA, an effect that was explained by a difference in only one amino-acid residue (Gadelha et al., 2005 [triangle]). The present study reports the crystallization and X-ray diffraction of the D. rostata lectin.

2. Material and methods

2.1. Purification and crystallization

D. rostrata seeds were collected in Ceará state in northeast Brazil. The seeds were ground to a fine powder in a coffee mill and the soluble proteins were extracted at 298 K by continuous stirring with 0.15 M NaCl [1:10(w:v)] for 1 h, followed by centrifugation at 10 000g at 277 K for 20 min. The supernatant was applied onto a Sephadex G-­50 column (10 × 50 cm) previously equilibrated with 0.15 M NaCl containing 5 mM CaCl2 and 5 mM MnCl2 as described by Cavada et al. (1996 [triangle]). The unbound material was eluted with 0.15 M NaCl at a flow rate of 45 ml h−1 until the absorbance at 280 nm of the effluent stabilized at 0.05. The retained fraction was eluted with 0.1 M glycine pH 2.6 containing 0.15 M NaCl and dialyzed exhaustively against Milli-Q water. This fraction was freeze-dried, resuspended in 0.075 M Tris pH 9.3 (5 mg ml−1) and loaded onto a MonoP pre-packed column connected to an HPLC/FPLC AKTA system (GE Healthcare). The lectin (DRL) was eluted with Polybuffer 96 pH 6.0. The purity of all DRL preparations was monitored by SDS–PAGE (Laemmli, 1970 [triangle]). The purified lectin was diluted in Tris pH 7.5 containing 5 mM CaCl2 and 5 mM MnCl2 to perform the crystallization experiments. The crystallization trials were carried out by the hanging-drop vapour-diffusion method at 293 K. 1 µl lectin solution was mixed with 1 µl reservoir solution and equilibrated against 300 µl reservoir solution. Initial screening was performed using a sparse-matrix method (Jancarik & Kim, 1991 [triangle]) with Crystal Screens I and II (Hampton Research, Riverside, CA, USA).

2.2. Data collection and processing

X-ray data were collected from a single crystal cooled to a temperature of 100 K. Crystals were soaked in a cryoprotectant solution consisting of the reservoir solution containing 30% glycerol and submitted to data collection at a wavelength of 1.43 Å using a synchrotron-radiation source [CPr station, Laboratório Nacional de Luz Síncrotron (LNLS), Campinas, Brazil]. A complete data set was obtained using a CCD (MAR Research) in 59 frames with an oscillation range of 1.5°. The data set was indexed, integrated and scaled using MOSFLM (Leslie, 1992 [triangle]) and the CCP4 program SCALA (Collaborative Computational Project, Number 4, 1994 [triangle]). Sequence alignment of the lectins was performed using ClustalW (Thompson et al., 1994 [triangle]).

3. Results and discussion

Crystals were obtained after a week using condition No. 29 (0.1 M Na HEPES pH 7.5, 0.8 M sodium potassium tartrate tetrahydrate) of Hampton Crystal Screen I. To optimize the crystallization conditions, the concentration of tartrate was modified and different buffers in various pH ranges were tested. Crystals of good size for diffraction (Fig. 1 [triangle]) were obtained under the conditions 0.1 M Na HEPES pH 7.5–8.5, 0.4–1.2 M sodium potassium tartrate.

Figure 1
Crystals of D. rostrata lectin.

Surprisingly, the crystals obtained (Fig. 1 [triangle]) provided a data set extending to 1.87 Å resolution which was scaled in the resolution range 33.150–2.05 Å. The D. rostrata lectin crystal belongs to the orthorhombic space group I222, with unit-cell parameters a = 61.51, b = 88.22, c = 87.76 Å. The calculated value for the Matthews coefficient (Matthews, 1968 [triangle]) based on the molecular weight of 25.5 kDa is 2.4 Å3 Da−1, resulting in a solvent content of 47.9%, which corresponds to the presence of a monomer in the asymmetric unit. Data-collection statistics are shown in Table 1 [triangle]. Crystal structure solution is under way.

Table 1
Statistics of data collection

Sequence alignment of fragment 2 from D. rostrata lectin (SWISS-PROT code P58908) with D. guianensis lectin (PDB code 1h9p, chain A; Calvete et al., 1999 [triangle]) and D. grandiflora lectin (PDB code 1dgl, chain B; Rozwarski et al., 1998 [triangle]) showed that DRL contains an Asn residue in position 131, while D. grandiflora lectin contains a His residue in this position (Fig. 2 [triangle]). Comparison of the structures of D. guianensis lectin, which presents pH-dependent oligomerization, and D. grandiflora lectin, which does not display a dimer–tetramer equilibrium, indicated that substitution of His131 for Asn drastically reduces interdimer contacts and disorders the loop comprising residues 117–123 which, in its ordered conformation, stabilizes the pH-independent tetrameric association of the D. grandiflora lectin (Wah et al., 2001 [triangle]). The solution of the DRL crystal structure may be useful to determine whether DRL presents the same features as D. guianensis regarding the dimer–tetramer association. Since only the tetravalent form is able to cause cross-linking of receptors on the cell surface, the different ratio of divalent and tetravalent lectin species at a given pH may contribute to the variability of biological functions.

Figure 2
Sequence alignment of the lectin segment 119–142 from D. rostrata (DRL), D. guianensis (Dgui) and D. grandiflora (DGL). Asterisks (*) related to conserved residues and colons (:) to semi-conserved residues.


We thank Dr Juan J. Calvete from Institututo de Biomedicina de Valencia, Spain for technical support. This work was supported by Fundação Cearense de Apoio ao Desenvolvimento Científico e Tecnológico (FUNCAP), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), FAPESP, Universidade Regional do Cariri (URCA), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), National Synchrotron Light Laboratory (LNLS), Brazil. BSC and VNF are senior investigators of CNPq.


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