Crystallographic elucidation of the saccharide binding mode in wheat germ agglutinin and its biological significance

Abstract

The specific saccharide binding sites in wheat germ agglutinin have been characterized by difference Fourier analysis of a number of WGA † † Abbreviations used: WGA, wheat germ agglutinin; WGA2, isolectin 2 of wheat germ agglutinin; GLA, glutaraldehyde; ConA, concanavalin A; GlcNAc, N-acetyl- d-glucosamine; NeuNAc, N-acetyl- d-neuraminic acid; INAG, 6-iodo-1,4-dimethyl- N-acety1- d-glucosaminc; MUC, O-4-methylumbelliferyl-di- N-acetyl-β-chitobioside; NeuLac, N-acetyl- d-neuraminic acid lactose (neuramin lactose); NeuLacNAc, N-acetyl- d-neuraminic acid lactosamine; n.m.r. nuclear magnetic resonance; cd., circular dichroism; CHO, Chinese hamster ovary; Bkg, background; F CMP, structure factors for WGA: sugar complexes; F PAR, structure factors for either native or glutaraldehyde-treated WGA crystals; WGM, wheat germ mitogen; MIR, multiple isomorphous replacement. -sugar complexes. All four domains (A,B,C,D) of each WGA protomer (I and II) were found to contribute to sugar binding at four locations in the protomer/protomer contact region: B I C II , B II C I , I D II , A II D I . The two equivalent binding sites involving the B and C domains have been shown to bind N-acetylneuraminic acid, a common cell surface sialic acid (Wright, 1980 a). These two sites, termed the primary binding location, are readily accessible in the crystal to all three saccharides investigated: di- N-acetylglucosamine (2.8 Å data), 6-iodo-1, 4-dimethyl- N-acetylglucosamine (3 Å data) and N-acetylneuraminic acid lactose (4 Å data). The other two sites involving A and D domains are designated as the secondary binding locations, since they are only poorly occupied by di- N-acetylglucosamine in glutaraldehyde-crosslinked crystals and not at all by the sialic acid sugar. The early observation that native WGA crystals disintegrate in the presence of N-acetylglucosamine, is attributed to interference of sugar binding at this secondary site with molecular lattice interactions. Despite differences in their accessibility in the crystal and specificity toward the two types of acetylated sugars, the two unique binding locations exhibit very similar saccharide binding modes. The main contribution to binding of oligosaccharides comes from interactions of the acetamido and one hydroxyl group of the non-reducing terminal sugars with the protein at subsite 1, whereas little or no contribution is provided by subsites 2 and 3. Moreover, oligosaccharides binding at the primary binding location which possess terminal sialic acid, assume a distinctly different orientation from that of N-acetylglucosamine oligomers, although they share subsite 1. This is due to a difference in position of their glycosidic hydroxyl groups. Space limitations in crosslinked crystals prevent oligomers of N-acetylglucosamine larger than the dimer from binding either at the primary or secondary binding locations. These results suggest that differing accessibility and specificity determinants for the two unique binding sites in WGA, as well as protein self-association as seen in the crystal lattice, could explain (1) the co-operative binding behavior of WGA binding to various cells, and (2) the rapid release of cell-bound WGA when N-acetylglucosamine is introduced.