Crystallization of succinylated concanavalin A bound to a synthetic bivalent ligand and preliminary structural analysis

Abstract

Crystals have been obtained of succinylated concanavalin A complexed to a novel bidentate synthetic ligand. The crystals are the first example of a lectin with a synthetic multivalent ligand and the first report of crystallization of succinylated concanavalin A. The crystals were obtained by sitting-drop vapour diffusion equilibrating with a solution of 20% polyethylene glycol, pH 5,293.5 K. Crystals are orthorhombic, belonging to space group C222~ with unit-cell dimensions of a = 99.1, b = 127.4, c = 118.9 A. The asymmetric unit contains a dimer, with over 65% of the volume occupied by water. The ligand cross links concanavalin A monomers. Succinylated concanavalin A is known to be a dimcr in solution, yet it is found as the typical concanavalin A tetramer in the crystal. The contacts holding together the tetramer appear extensive and suggest that a fine balance between dimer and tetramers exists. Data to 2.65 A, have been collected and the structure determined by the molecular replacement method.

Protein-Carbohydrate Interactions

“…This conformation lies 1 kcalmoi'^ above the 120 energy minima (Imberty et a i, 1991). A distortion of similar size in a protein bound oligosaccharide was also observed in the pentasaccharide -con A complex which placed the glycosidic linkage -3 kcalmol'^ above the global minimum (Moothoo and Naismith, 1998). In this case a distortion in the interaction between the sugar and the monosaccharide binding site was also observed.…”

“…As seen in the other carbohydrate bound con A structures Naismith et al, 1994;Naismith and Field, 1996;Moothoo and Naismith, 1998) (see also Chapters 2, 3 and 4), there is no change in metal coordination in this complex. The average metal to ligand distances and ligand temperature factors are given in Table 5.16.…”

“…The first structure of a lectin with a synthetic polyvalent ligand has recently been reported (Moothoo and Naismith, 1998b;Dimick et al, 1998). The ligand is a bidentate ligand ring are less extensive than observed for the reducing sugar in the trimannose bound and pentasaccharide bound complexes, but are more than predicted by molecular modelling studies.…”

“…The thermodynamics of con A binding to pyranose sugars has been well documented (Chervenak and Toone, 1994;Chervenak and Toone, 1996;Brewer et al" 1997; Gupta et a l, 1997), and the structural basis of recognition is being investigated (Derewenda et a l, 1989;Harrop et a l, 1996;Naismith and Field, 1996;Moothoo and Naismith, 1998;Bradbrook et al, 1998; Chapters 2, 3 and 4 of this thesis). The binding of con A to furanose sugars has not been as well documented.…”

“…There are eight sites per dimer, four of which are unique. In the complex o f WGA with a sialylglycopeptide (Wright, 1992) (Figure 1.5.h) the carbohydrate binds to the protein so that the a2-6 linked sugar occupies the binding site in one domain o f one dimer and the a2-3 linked sugar binds a different domain of a crystallographically related dimer.The first structure of a lectin with a synthetic polyvalent ligand has recently been reported (Moothoo and Naismith, 1998b;Dimick et al, 1998). The ligand is a bidentate ligand ring are less extensive than observed for the reducing sugar in the trimannose bound and pentasaccharide bound complexes, but are more than predicted by molecular modelling studies.…”