Crystallization and preliminary X-ray analysis of the Man(alpha1-2)Man-specific lectin from Bowringia mildbraedii in complex with its carbohydrate ligand

Abstract

The lectin from Bowringia mildbraedii seeds crystallizes in the presence of the disaccharide Man(alpha1-2)Man. The best crystals grow at 293 K within four weeks after a pre-incubation at 277 K to induce nucleation. A complete data set was collected to a resolution of 1.90 A using synchrotron radiation. The crystals belong to space group I222, with unit-cell parameters a = 66.06, b = 86.35, c = 91.76 A, and contain one lectin monomer in the asymmetric unit.

Figure 1

Sequence alignment made with ClustalW using default settings (Thompson et al., 1994 ▶) of mannose-specific legume lectins: B. milbraedii (BMA), Canavalia ensiformis concanavalin A (ConA), Phaseolus vulgaris Flt3 receptor-interacting lectin (PvFRIL), L. ochrus isolectin I (LOL-I) and Pterocarpus angolensis seed lectin (PAL). The positions of the β-strands are indicated with arrows. Residues contributing to the monosaccharide-binding site are marked with a black background.

Figure 2

Purification of BMA. (a) Analytical gel-filtration profile of BMA on a Superdex 75-­HR column. The weights of the molecular-weight standards are indicated. (b) 12% SDS–PAGE under reducing conditions showing the purified lectin in lane 1 and a molecular-weight marker in lane 2. The molecular weights of the marker proteins are indicated in kDa.

Figure 3

Crystals of B. mildbraedii agglutinin. (a) Original microcrystals grown from 0.1 M MES pH 6.5, 12% PEG 20 000. (b) Crystals of BMA in the presense of trehalose. (c) Crystals of BMA in the presence of Man(α1-2)Man before optimization. (d) Typical crystals of the BMA–Man(α1-2)Man complex after optimization. The scale bar in each panel corresponds to 0.2 mm.

Figure 4

Diffraction pattern of BMA–Man(α1-2)Man crystals. The pattern is clean and extends to 1.90 Å resolution. The crystal was exposed at 100 K after soaking in 32.5% PEG 20 000 for cryoprotection. The rotation angle used was 0.7°, the crystal-to-detector distance was 165 mm and the wavelength was 0.8123 Å. The inset shows a detail of the diffraction pattern with spots visible near the edge of the detector.