Examination of the structural basis for O(H) blood group specificity by Ulex europaeus Lectin I

The structural basis for carbohydrate specificity of the first lectin from Ulex europaeus (UE-1) is reported. UE-I is a dimeric metalloglycoprotein that binds the H-type 2 human blood group determinant (alpha-L-Fucalpha(1-->2)-beta-DGalbeta(1-->4)-beta-D-GlcNAcalpha(-)), the blood group determinant present on the surface of O-type erythrocytes. The structural characteristics of UE-I involved in carbohydrate recognition have been examined using mass spectrometry (MS) and X-ray diffraction analysis. MS analysis allowed for discrimination between the different primary structures reported for UE-I. To examine the binding of the H-type 2 blood group determinant by UE-I, the methyl glycosides of the fucose monosaccharide (alpha-L-Fuc-OMe), known to exhibit primary binding specificity, and the H-type 2 trisaccharide (H-type 2-OMe) were, in two separate experiments, co-crystallized into the binding site of UE-I. The UE-I:alpha-L-Fuc-OMe complex crystallizes in the monoclinic space group P2(1), with unit cell dimensions a = 71.81, b = 69.00, and c = 119.02 Angstrom, and beta = 106.76degrees. Two UE-I dimers are observed to be present within the asymmetric unit, and the model has been refined to a R-value and R-Free of 0.202 and 0.289, respectively, to 2.3 Angstrom resolution. The preliminary model of the UE-I:H-type 2-OMe complex has been refined at 3.0 Angstrom resolution. The UE-I:H-type 2-OMe complex crystallizes in the orthorhombic space group C222(1), with unit cell dimensions a = 88.80, b = 164.75, and c = 77.42 Angstrom, and a single UE-I dimer is present within the asymmetric unit. The carbohydrate recognition domain of UE-I has been identified to be comprised of residues Glu44, Thr86, Asp87, Arg102, Ala103, Gly104, Gly105, Tyr106, Ile129, Val133, Asn134, Trp136, Tyr219, and Arg222. Several critical protein-carbohydrate interactions have been identified, including the role of the hydrophobic interaction between the Thr86 side chain and C-5-CH3 of the alpha-L-Fuc-OMe. The role of these interactions in carbohydrate recognition-binding by UE-I, as well as differences between the observed and previously modeled complexes, are discussed.