Concanavalin A distorts the GlcNAc b1-2 Man linkage of the pentasaccharide core upon binding

Carbohydrate recognition by proteins is a key event in many biological processes. Concanavalin A is known to specifically recognize the pentasaccharide core (beta-GlcNAc-(1--&gt;2)-alpha-Man-(1--&gt;3)-[beta-GlcNAc-(1--&gt;2)-alpha-Man-(1--&gt;6)]- Man) of N-linked oligosaccharides with a K-a of 1.41 x 10(6) M-1. We have determined the structure of concanavalin A bound to beta-GlcNAc-(1--&gt;2)-alpha-Man-(1--&gt;3)-[beta-GlcNAc-(1--&gt;2)-alpha-Man- (1--&gt;6)]-Man to 2.7 Angstrom. In six of eight subunits there is clear density for all five sugar residues and a well ordered binding site. The pentasaccharide adopts the same conformation in all eight subunits. The binding site is a continuous extended cleft on the surface of the protein. Van der Waals interactions and hydrogen bonds anchor the carbohydrate to the protein. Both GlcNAc residues contact the protein. The GlcNAc on the 1--&gt;6 arm of the pentasaccharide makes particularly extensive contacts and including two hydrogen bonds. The binding site of the 1--&gt;3 arm GlcNAc is much less extensive. Oligosaccharide recognition by Con A occurs through specific protein carbohydrate interactions and does not require recruitment of adventitious water molecules. The beta-GlcNAc-(1--&gt;2)-Man glycosidic linkage PSI torsion angle on the 1--&gt;6 arm is rotated by over 50 degrees from that observed in solution. This rotation is coupled to disruption of interactions at the monosaccharide site. We suggest destabilization of the monosaccharide site and the conformational strain reduces the free energy liberated by additional interactions at the 1--&gt;6 arm GlcNAc site.</p

Concanavalin A distorts the GlcNAc b1-2 Man linkage of the pentasaccharide core upon binding

Carbohydrate recognition by proteins is a key event in many biological processes. Concanavalin A is known to specifically recognize the pentasaccharide core (beta-GlcNAc-(1--&gt;2)-alpha-Man-(1--&gt;3)-[beta-GlcNAc-(1--&gt;2)-alpha-Man-(1--&gt;6)]- Man) of N-linked oligosaccharides with a K-a of 1.41 x 10(6) M-1. We have determined the structure of concanavalin A bound to beta-GlcNAc-(1--&gt;2)-alpha-Man-(1--&gt;3)-[beta-GlcNAc-(1--&gt;2)-alpha-Man- (1--&gt;6)]-Man to 2.7 Angstrom. In six of eight subunits there is clear density for all five sugar residues and a well ordered binding site. The pentasaccharide adopts the same conformation in all eight subunits. The binding site is a continuous extended cleft on the surface of the protein. Van der Waals interactions and hydrogen bonds anchor the carbohydrate to the protein. Both GlcNAc residues contact the protein. The GlcNAc on the 1--&gt;6 arm of the pentasaccharide makes particularly extensive contacts and including two hydrogen bonds. The binding site of the 1--&gt;3 arm GlcNAc is much less extensive. Oligosaccharide recognition by Con A occurs through specific protein carbohydrate interactions and does not require recruitment of adventitious water molecules. The beta-GlcNAc-(1--&gt;2)-Man glycosidic linkage PSI torsion angle on the 1--&gt;6 arm is rotated by over 50 degrees from that observed in solution. This rotation is coupled to disruption of interactions at the monosaccharide site. We suggest destabilization of the monosaccharide site and the conformational strain reduces the free energy liberated by additional interactions at the 1--&gt;6 arm GlcNAc site.</p

A general method for co-crystallisation of concanavalin A with carbohydrates.

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 alpha 1-2 mannobiose, 1-methyl alpha 1-2 mannobiose, fructose, a trisaccharide and a pentasaccharide. The crystals diffract to resolutions of 1.75-2.7 Angstrom 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 alpha 1-2 mannobiose complex crystals diffract to 1.75 Angstrom with space group I222 and cell dimensions a = 91.7, b = 86.8, c = 66.6 Angstrom . One monomer is present in the asymmetric unit. The 1-methyl alpha 1-2 mannobioside complex crystallizes in space group P2(1)2(1)2(1), cell dimensions a = 119.7, b = 119.7, c = 68.9 Angstrom and diffract to 2.75 Angstrom. One tetramer is present in the asymmetric unit. Two crystal forms of the conA-fructose complex have been obtained. The first has space group P2(1)2(1)2(1), cell dimensions a = 121.7, b = 119.9, c = 67.3 Angstrom with a tetramer in the asymmetric unit and diffracts to 2.6 Angstrom. The second crystallizes in space group C222(1), cell dimensions a = 103.3, b = 117.9, c = 254.3 Angstrom with two dimers in the asymmetric unit and diffracts to 2.42 Angstrom. 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.</p

A general method for co-crystallisation of concanavalin A with carbohydrates.

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 alpha 1-2 mannobiose, 1-methyl alpha 1-2 mannobiose, fructose, a trisaccharide and a pentasaccharide. The crystals diffract to resolutions of 1.75-2.7 Angstrom 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 alpha 1-2 mannobiose complex crystals diffract to 1.75 Angstrom with space group I222 and cell dimensions a = 91.7, b = 86.8, c = 66.6 Angstrom . One monomer is present in the asymmetric unit. The 1-methyl alpha 1-2 mannobioside complex crystallizes in space group P2(1)2(1)2(1), cell dimensions a = 119.7, b = 119.7, c = 68.9 Angstrom and diffract to 2.75 Angstrom. One tetramer is present in the asymmetric unit. Two crystal forms of the conA-fructose complex have been obtained. The first has space group P2(1)2(1)2(1), cell dimensions a = 121.7, b = 119.9, c = 67.3 Angstrom with a tetramer in the asymmetric unit and diffracts to 2.6 Angstrom. The second crystallizes in space group C222(1), cell dimensions a = 103.3, b = 117.9, c = 254.3 Angstrom with two dimers in the asymmetric unit and diffracts to 2.42 Angstrom. 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.</p