Substrate Binding Drives Active‐Site Closing of Human Blood Group B Galactosyltransferase as Revealed by Hot‐Spot Labeling and NMR Spectroscopy Experiments

Abstract

Crystallography has shown that human blood group A (GTA) and B (GTB) glycosyltransferases undergo transitions between "open", "semiclosed", and "closed" conformations upon substrate binding. However, the timescales of the corresponding conformational reorientations are unknown. Crystal structures show that the Trp and Met residues are located at "conformational hot spots" of the enzymes. Therefore, we utilized N side-chain labeling of Trp residues and C-methyl labeling of Met residues to study substrate-induced conformational transitions of GTB. Chemical-shift perturbations (CSPs) of Met and Trp residues in direct contact with substrate ligands reflect binding kinetics, whereas the CSPs of Met and Trp residues at remote sites reflect conformational changes of the enzyme upon substrate binding. Acceptor binding is fast on the chemical-shift timescale with rather small CSPs in the range of less than approximately 20 Hz. Donor binding matches the intermediate exchange regime to yield an estimate for exchange rate constants of approximately 200-300 Hz. Donor or acceptor binding to GTB saturated with acceptor or donor substrate, respectively, is slow (<10 Hz), as are coupled protein motions, reflecting mutual allosteric control of donor and acceptor binding. Remote CSPs suggest that substrate binding drives the enzyme into the closed state required for catalysis. These findings should contribute to better understanding of the mechanism of glycosyl transfer of GTA and GTB.

Insights into Allosteric Control of Human Blood Group A and B Glycosyltransferases from Dynamic NMR

“…The 13 C‐methyl group of Met 214 responds strongly to binding of donor‐type as well as acceptor‐type ligands, although Met 214 is not in the immediate proximity of any ligand. This sensitivity to ligand binding has already been described before and has been linked to a close proximity of the Phe 341 side chain …”

“…Building on these assignments we studied the binding kinetics of donor‐ and acceptor substrates and analogs thereof in more detail. As reported, depending on the ligand combination, exchange may be fast, intermediate, or slow on the chemical shift time scale . Knowledge of the corresponding on‐ and off‐rate constants would provide a link to the conformational changes associated with substrate binding as observed by crystallography and would assist in understanding the mechanisms of allosteric control.…”

Novel NMR Avenues to Explore the Conformation and Interactions of Glycans

“…Apart from the effect of the attached sugars on the glycoprotein conformations, the influence of the natural, external glycan substrates upon binding can also lead to notorious changes in the 3D structure of the protein counterpart, as exemplified by the human blood group A and B glycosyltransferases (GTA and GTB). 38 Both enzymes experience transitions between “open”, “semi-closed”, and “closed” conformations when interacting with the corresponding substrates, the histo-blood group H (HBG-H) and the sugar donor (UDP-Gal/GalNAc). For this particular system, “hot-spot labeling” enabled conformational studies through the observation of the HN side chain from 15 N-Trp and the ε-methyl of 13 C-Met.…”