concanavalin-a and mannobiose

Plant lectins are carbohydrate-binding proteins with various biomedical applications. Concanavalin A (Con A) holds promise in treating cancerous tumors. To better understand the Con A carbohydrate binding specificity, we obtained a room-temperature neutron structure of this legume lectin in complex with a disaccharide Manα1-2Man, mannobiose. The neutron structure afforded direct visualization of the hydrogen bonding between the protein and ligand, showing that the ligand is able to alter both protonation states and interactions for residues located close to and distant from the binding site. An unprecedented low-barrier hydrogen bond was observed forming between the carboxylic side chains of Asp28 and Glu8, with the D atom positioned equidistant from the oxygen atoms having an O···D···O angle of 101.5°.

Topics: Amino Acid Sequence; Binding Sites; Concanavalin A; Hydrogen Bonding; Mannans; Protein Conformation

The complex between concanavalin A (Con A) and alpha1-2 mannobiose (mannose alpha1-2 mannose) has been refined to 1.2 A resolution. This is the highest resolution structure reported for any sugar-lectin complex. As the native structure of Con A to 0.94 A resolution is already in the database, this gives us a unique opportunity to examine sugar-protein binding at high resolution. These data have allowed us to model a number of hydrogen atoms involved in the binding of the sugar to Con A, using the difference density map to place the hydrogen atoms. This map reveals the presence of the protonated form of Asp208 involved in binding. Asp208 is not protonated in the 0.94 A native structure. Our results clearly show that this residue is protonated and hydrogen bonds to the sugar. The structure accounts for the higher affinity of the alpha1-2 linked sugar when compared to other disaccharides. This structure identifies different interactions to those predicted by previous modelling studies. We believe that the additional data presented here will enable significant improvements to be made to the sugar-protein modelling algorithms.

Topics: Concanavalin A; Crystallography, X-Ray; Fabaceae; Hydrogen Bonding; Mannans; Models, Molecular; Plant Lectins; Plants, Medicinal; Protein Conformation; Protons; Water

A small grid of conditions has been developed for co-crystallization of the plant lectin concanavalin A (conA) and polysaccharides. Crystals have been obtained of complexes of conA with alpha1-2 mannobiose, 1-methyl alpha1-2 mannobiose, fructose, a trisaccharide and a pentasaccharide. The crystals diffract to resolutions of 1. 75-2.7 A using a copper rotating-anode source. The crystals are grown in the presence of polyethylene glycol 6K [10-20%(w/v)] at around pH 6.0. Optimization for each particular carbohydrate requires small adjustments in the conditions; however, all complexes give some crystalline precipitate in this limited grid. The alpha1-2 mannobiose complex crystals diffract to 1.75 A with space group I222 and cell dimensions a = 91.7, b = 86.8, c = 66.6 A. One monomer is present in the asymmetric unit. The 1-methyl alpha1-2 mannobioside complex crystallizes in space group P212121, cell dimensions a = 119. 7, b = 119.7, c = 68.9 A and diffract to 2.75 A. One tetramer is present in the asymmetric unit. Two crystal forms of the conA-fructose complex have been obtained. The first has space group P212121, cell dimensions a = 121.7, b = 119.9, c = 67.3 A with a tetramer in the asymmetric unit and diffracts to 2.6 A. The second crystallizes in space group C2221, cell dimensions a = 103.3, b = 117.9, c = 254.3 A with two dimers in the asymmetric unit and diffracts to 2.42 A. Structures and crystallization of the trisaccharide-conA and pentasaccharide-conA complexes have already been reported. In all complexes, the protein is found as a tetramer, although varying combinations of non-crystallographic and crystallographic symmetry are involved in generating the tetramer. The precise packing of the tetramer varies from crystal to crystal and it is likely that this variability facilitates crystallization.

Topics: Carbohydrate Sequence; Carbohydrates; Concanavalin A; Crystallization; Crystallography, X-Ray; Fructose; Macromolecular Substances; Mannans; Methods; Molecular Sequence Data; Protein Conformation

Three-dimensional structures of the complexes of concanavalin A (ConA) with alpha(1-2) linked mannobiose, triose and tetraose have been generated with the X-ray crystal structure data on native ConA using the CCEM (contact criteria and energy minimization) method. All the constituting mannose residues of the oligosaccharide can reach the primary binding site of ConA (where methyl-alpha-D-mannopyranose binds). However, in all the energetically favoured complexes, either the non-reducing end or middle mannose residues of the oligosaccharide occupy the primary binding site. The middle mannose residues have marginally higher preference over the non-reducing end residue. The sugar binding site of ConA is extended and accommodates at least three alpha(1-2) linked mannose residues. Based on the present calculations two mechanisms have been proposed for the binding of alpha(1-2) linked mannotriose and tetraose to ConA.

Topics: Concanavalin A; Hydrogen Bonding; Mannans; Models, Molecular; Molecular Conformation; Oligosaccharides; Trisaccharides; X-Ray Diffraction

The association constants for binding of methyl alpha-D-mannopyranoside (I), mannobiose (II) and mannotriose (III) to concanavalin A were determined in the temperature range 285-313 K by a substitution titration, using 4-methylumbelliferyl alpha-D-mannopyranoside as a carbohydrate-specific and fluorescent indicator. All binding equilibria are simple, but establish extremely slowly with II and III. At 298.3 K, K increases moderately from I to III: (6.4 +/- 0.5) x 10(3), (1.2 +/- 0.1) x 10(4) and (1.10 +/- 0.05) x 10(5) M-1. For binding of I, II and III, the - delta H degree values are constant (36 +/- 2 kJ mol-1) and equal to the average value (36.1 +/- 0.6 kJ mol-1) obtained for the three corresponding 4-methylumbelliferyl alpha-D-manno-oligosaccharides [Van Landschoot, A., Loontiens, F. G., and De Bruyne, C. K (1978) Eur. J. Biochem. 83, 277-285]. The data are interpreted as arising from specific binding to a single mannopyranosyl residue in (alpha 1 leads to 2)-linked manno-oligosaccharides.

Topics: Concanavalin A; Fluorescent Dyes; Hymecromone; Kinetics; Ligands; Mannans; Mannose; Mannosides; Mathematics; Methylglycosides; Methylmannosides; Oligosaccharides; Protein Binding; Structure-Activity Relationship; Thermodynamics; Trisaccharides