Binding of N-acetylglucosamine (GlcNAc) β1-6-branched oligosaccharide acceptors to β4-galactosyltransferase I reveals a new ligand binding mode.

N-acetyllactosamine is the most prevalent disaccharide moiety in the glycans on the surface of mammalian cells and often found as repeat units in the linear and branched polylactosamines, known as i- and I-antigen, respectively. The β1-4-galactosyltransferase-I (β4Gal-T1) enzyme is responsible for the synthesis of the N-acetyllactosamine moiety. To understand its oligosaccharide acceptor specificity, we have previously investigated the binding of tri- and pentasaccharides of N-glycan with a GlcNAc at their nonreducing end and found that the extended sugar moiety in these acceptor substrates binds to the crevice present at the acceptor substrate binding site of the β4Gal-T1 molecule. Here we report seven crystal structures of β4Gal-T1 in complex with an oligosaccharide acceptor with a nonreducing end GlcNAc that has a β1-6-glycosidic link and that are analogous to either N-glycan or i/I-antigen. In the crystal structure of the complex of β4Gal-T1 with I-antigen analog pentasaccharide, the β1-6-branched GlcNAc moiety is bound to the sugar acceptor binding site of the β4Gal-T1 molecule in a way similar to the crystal structures described previously; however, the extended linear tetrasaccharide moiety does not interact with the previously found extended sugar binding site on the β4Gal-T1 molecule. Instead, it interacts with the different hydrophobic surface of the protein molecule formed by the residues Tyr-276, Trp-310, and Phe-356. Results from the present and previous studies suggest that β4Gal-T1 molecule has two different oligosaccharide binding regions for the binding of the extended oligosaccharide moiety of the acceptor substrate.