The lectin Cbol was purified to apparent homogeneity by affinity chromatography on Sephadex G-50, in which Cbol was quantitatively retained in the cross-linked dextran gel column and was desorbed with
d-glucose, providing strong evidence of its carbohydrate-binding properties (Fig. 1
a). This procedure has widely been used for the purification of Diocleinae lectins (Cavada
et al., 2001
![[triangle]](/corehtml/pmc/pmcents/rtrif.gif)
). About 80 mg purified lectin was obtained from 1 g powdered seeds. The purified protein showed haemagglutination activity towards native and enzyme-treated rabbit erythrocytes. SDS–PAGE confirmed the purity of the lectin and its similarity to other Diocleinae lectins, showing a main α chain and β and γ fragments (Fig. 1
b). These results suggest that Cbol is a ConA-like lectin and undergoes the same post-translational process of circular permutation as first described by Carrington
et al. (1985
). This process involves proteolytic cleavage of the precursor at an internal site, resulting in the γ and β fragments. These fragments are religated in an inverse DNA-coded order, producing the active α chain (α = β + γ; Carrington
et al., 1985
; Chrispeels
et al., 1986
; Min & Jones, 1994
). The β and γ fragments observed in the SDS–PAGE (Fig. 1
b) are non-religated products of this process and the α chain is the mature protein. The Diocleinae lectins used for comparison, ConBr and CGL, have previously been characterized and their three-dimensional structures have been determined (PDB code
1azd, Sanz-Aparicio
et al., 1997
; PDB code
2d7f, Delatorre
et al., 2007
).
Crystals were obtained after a week using condition Nos. 30, 31, 34 and 38 of Hampton Crystal Screen I. Condition No. 34 was chosen to be optimized; it contained 0.1
M sodium acetate trihydrate pH 4.6 and 2.0
M sodium formate. To optimize the crystallization conditions, the concentration of sodium formate was modified from 1 to 6
M and buffers of differing pH were tested. Crystals suitable for diffraction experiments (Fig. 2) were obtained under the condition 0.1
M HEPES pH 7.5 and 3.0
M sodium formate. The crystals obtained provided a data set extending to 1.5 Å resolution, which was scaled in the resolution range 46.56–1.60 Å. The
C. boliviana lectin crystal belongs to the centred monoclinic space group
C2, with unit-cell parameters
a = 126.7,
b = 66.6,
c = 64.9 Å, α = 90.0, β = 120.8, γ = 90.0°. The calculated value of the Matthews coefficient (Matthews, 1968
), based on the molecular weight of 25.5 kDa, indicated a solvent content of 46.7%, which corresponds to the presence of a dimer in the asymmetric unit. Data-collection statistics are shown in Table 1. The preliminary crystal structure of Cbol was determined by standard molecular-replacement methods using the program
MOLREP (Vagin & Teplyakov, 1997
). Various monomers were tested for molecular replacement and the best result was obtained using CGL (PDB code
2d7f; Delatorre
et al., 2007
) as a structural model. The best solution had a final correlation coefficient of 0.705 and an
R factor of 0.424. After placing the molecule in the unit cell, rigid-body refinement was performed using the
REFMAC5 program. Refinement resulted in a model with an
R
free of 0.363 and an
R factor of 0.359. Primary sequence determination by mass spectrometry and Edman degradation as well as solution of the crystal structure are in progress.
| Table 1Statistics of data collection |
