Human blood group A and B glycosyltransferases (GTA, GTB) are highly homologous glycosyltransferases. A number of high-resolution crystal structures is available showing that these enzymes convert from an open conformation into a catalytically active closed conformation upon substrate binding. However, the mechanism of glycosyltransfer is still under debate, and the precise nature as well as the time scales of conformational transitions are unknown. NMR offers a variety of experiments to shine more light on these unresolved questions. Therefore, in a first step we have assigned all methyl resonance signals in MILVA labeled samples of GTA and GTB, still a challenging task for 70 kDa homodimeric proteins. Assignments were obtained from methyl-methyl NOESY experiments, and from measurements of lanthanide-induced pseudocontact shifts (PCS) using high resolution crystal structures as templates. PCSs and chemical shift perturbations, induced by substrate analogue binding, suggest that the fully closed state is not adopted in the presence of lanthanide ions.
Donor and acceptor substrate binding to human blood group A and B glycosyltransferases (GTA, GTB) has been studied by a variety of protein NMR experiments. Prior crystallographic studies had shown these enzymes to adopt an open conformation in the absence of substrates. Binding either of the donor substrate UDP‐Gal or of UDP induces a semiclosed conformation. In the presence of both donor and acceptor substrates, the enzymes shift towards a closed conformation with ordering of an internal loop and the C‐terminal residues, which then completely cover the donor‐binding pocket. Chemical‐shift titrations of uniformly 2H,15N‐labeled GTA or GTB with UDP affected about 20 % of all crosspeaks in 1H,15N TROSY‐HSQC spectra, reflecting substantial plasticity of the enzymes. On the other hand, it is this conformational flexibility that impedes NH backbone assignments. Chemical‐shift‐perturbation experiments with δ1‐[13C]methyl‐Ile‐labeled samples revealed two Ile residues—Ile123 at the bottom of the UDP binding pocket, and Ile192 as part of the internal loop—that were significantly disturbed upon stepwise addition of UDP and H‐disaccharide, also revealing long‐range perturbations. Finally, methyl TROSY‐based relaxation dispersion experiments do not reveal micro‐ to millisecond timescale motions. Although this study reveals substantial conformational plasticity of GTA and GTB, the matter of how binding of substrates shifts the enzymes into catalytically competent states remains enigmatic.
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.
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