NMR Structural Characterization of Substrates Bound to N-Acetylglucosaminyltransferase V
N-Acetylglucosaminyltransferase V (GnT-V) is an enzyme involved in the biosynthesis of asparagine-linked oligosaccharides. It is responsible for the transfer of N-acetylglucosamine (GlcNAc) from the nucleotide sugar donor, uridine 5'-diphospho-N-acetylglucosamine (UDP-GlcNAc), to the 6 position of the α-1-6 linked Man residue in N-linked oligosaccharide core structures. GnT-V up-regulation has been linked to increased cancer invasiveness and metastasis and, appropriately, targeted for drug development. However, drug design is impeded by the lack of structural information on the protein and the way in which substrates are bound. Even though the catalytic domain of this type II membrane protein can be expressed in mammalian cell culture, obtaining structural information has proved challenging due to the size of the catalytic domain (95 kDa) and its required glycosylation. Here, we present an experimental approach to obtaining information on structural characteristics of the active site of GnT-V through the investigation of the bound conformation and relative placement of its ligands, UDP-GlcNAc and β-d-GlcpNAc-(1→2)-α-d-Manp-(1→6)-β-d-GlcpOOcty l. Nuclear magnetic resonance (NMR) spectroscopy experiments, inducing transferred nuclear Overhauser effect (trNOE) and saturation transfer difference (STD) experiments, were used to characterize the ligand conformation and ligand-protein contact surfaces. In addition, a novel paramagnetic relaxation enhancement experiment using a spin-labeled ligand analogue, 5'-diphospho-4-O-2,2,6,6-tetramethylpiperidine 1-oxyl (UDP-TEMPO), was used to characterize the relative orientation of the two bound ligands. The structural information obtained for the substrates in the active site of GnT-V can be useful in the design of inhibitors for GnT-V. © 2006 Elsevier Ltd. All rights reserved.
Publication Source (Journal or Book title)
Journal of Molecular Biology
Macnaughtan, M., Kamar, M., Alvarez-Manilla, G., Venot, A., Glushka, J., Pierce, J., & Prestegard, J. (2007). NMR Structural Characterization of Substrates Bound to N-Acetylglucosaminyltransferase V. Journal of Molecular Biology, 366 (4), 1266-1281. https://doi.org/10.1016/j.jmb.2006.12.015