STRUCTURE OF A SELECTIN-LIKE MUTANT OF MANNOSE-BINDING PROTEIN COMPLEXED WITH SIALYLATED AND SULFATED LEWIS(X) OLIGOSACCHARIDES

Rat serum mannose-binding protein in which residues 211-213 have been changed to the Lys-Lys-Lys sequence found in E-selectin binds HL-60 ce lls and the oligosaccharide 3'-NeuAc-Le(x). To understand how this mut ant, designated K3, mimics the carbohydrate-binding properties of E-se lectin, structures of K3 alone and in complexes with 3'-NeuAc-Le(x), 3 '-sulfo-Le(x), and 4'-sulfo-Le(x) have been determined at 1.95-2.1 Ang strom resolution by X-ray crystallography, The region of K3 that inter acts with bound oligosaccharides superimposes closely with the corresp onding region of unliganded E-selectin, In each of the oligosaccharide -protein complexes, the 2- and 3-OH of Fuc coordinate Ca2+ and form a network of cooperative hydrogen bonds with amino acid side chains that also coordinate the Ca2+. Lys(211) of the K3 mutant, which correspond s to Lys(111) of E-selectin, interacts with each of the three bound li gands: the N zeta atom donates a hydrogen bond to the 4-OH of Gal in 3 '-NeuAc-Le(x), forms a water-mediated hydrogen bond with the 4-OH of G al in 3'-sulfo-Le(x), and forms a salt bridge with the sulfate group o f 4'-sulfo-Le(x), Lys(213) packs against an otherwise exposed aromatic residue and forms a water-mediated hydrogen bond with Lys(211) which may help to position that residue for interactions with bound oligosac charides, These structures are consistent with previous mutagenesis an d chemical modification studies which demonstrate the importance of th e Ca2+ ligands as well as Lys(111) and Lys(113) for carbohydrate bindi ng in the selectins, and they provide a structural basis for understan ding the selective recognition of negatively charged Le(x) derivatives by the selectins.