REFINED STRUCTURE OF CONCANAVALIN-A COMPLEXED WITH METHYL ALPHA-D-MANNOPYRANOSIDE AT 2.0 ANGSTROM RESOLUTION AND COMPARISON WITH THE SACCHARIDE-FREE STRUCTURE

The three-dimensional structure of the complex between methyl alpha-D-mannopyranoside and concanavalin A has been refined at 2.0 Angstrom resolution. Diffraction data were recorded from a single crystal (space group P2(1)2(1)2(1), a = 123.7, b = 128.6, c = 67.2 Angstrom) using synchrotron radiation at a wavelength of 1.488 Angstrom. The final model has good geometry and an R factor of 19.9% for 58871 reflections (82% complete), within the resolution limits of 8 to 2 Angstrom, with F > 1.0 sigma(F). The asymmetric unit contains four protein subunits arranged as a dimer of dimers with approximate 222 point symmetry. monomer binds one saccharide molecule. Each sugar is bound to the protein by hydrogen bonds and van der Waals contacts. Although the four subunits are not crystallographically equivalent, the protein-saccharide interactions are nearly identical in each of the four binding sites. The differences that do occur between the four sites are in the structure of the water network which surrounds each saccharide; these networks are involved in crystal packing. The structure of the complex is compared with a refined saccharide-free concanavalin A structure. The saccharide-free structure is composed of graphically identical subunits, again as a dimer of dimers, but with, exact 222 symmetry. In the saccharide complex the tetramer association is different in that the monomers tend to separate resulting in fewer intersubunit interactions. The average temperature factor of the mannoside complex is considerably higher than that of the saccharide-free protein. The binding site in the saccharide-free structure is occupied by three ordered water molecules and the side chain of Asp71 from a neighbouring molecule in the crystal. These occupy positions similar to those of the four saccharide hydroxyls which are hydrogen bonded to the site. Superposition of the saccharide-binding site from each structure shows that the major changes on binding involve expulsion of these ordered solvents and the reorientation of the side chain of Tyr100. Overall the surface accessibility of the saccharide decreases from 370 to 100 Angstrom(2) when it binds to the protein. This work builds upon the earlier studies of Derewenda el al. [Derewenda, Yariv, Helliwell, Kalb (Gilboa), Dodson, Papiz, Wan and Campbell (1989). EMBO J. 8, 2198-2193] at 2.9 Angstrom resolution, which was the first detailed study of lectin-saccharide interactions.