Nuclear magnetic resonance structural characterization of substrates bound to the alpha-2,6-sialyltransferase, ST6Gal-I.
The alpha-2,6-sialyltransferase (ST6Gal-I) is a key enzyme that regulates the distribution of sialic acid-containing molecules on mammalian cell surfaces. However, the fact that its native form is membrane-bound and glycosylated has made structural characterization by X-ray crystallography of this eukaryotic protein difficult. Its large size ( approximately 40 kDa for just the catalytic domain) also poses a challenge for complete structure determination by nuclear magnetic resonance (NMR). However, even without complete structure determination, there are NMR strategies that can return targeted information about select regions of the protein, including information about the active site as seen from the perspective of its bound ligands. Here, in a continuation of a previous study, a spin-labeled mimic of a glycan acceptor ligand is used to identify additional amino acids located in the protein active site. In addition, the spin-labeled donor is used to characterize the relative placement of the two bound ligands. The ligand conformation and protein-ligand contact surfaces are studied by transferred nuclear Overhauser effects (trNOEs) and saturation transfer difference (STD) experiments. The data afforded by the methods mentioned above lead to a geometric model of the bound substrates that in many ways carries an imprint of the ST6Gal-I binding site.
Complex Carbohydrate Research Center, University of Georgia, Athens, Georgia 30602, USA.
Shan
S
Liu
Lu
L
Meng
Kelley W
KW
Moremen
James H
JH
Prestegard
D020134:Catalytic Domain; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008970:Molecular Weight; D012799:Sialyltransferases / Q000737:chemistry* / Q000378:metabolism*; D013113:Spin Labels; D013379:Substrate Specificity; D000094802:beta-D-Galactoside alpha 2-6-Sialyltransferase
D008024:Ligands; D013113:Spin Labels; D012799:Sialyltransferases; D000094802:beta-D-Galactoside alpha 2-6-Sialyltransferase