Abstract
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.
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Acknowledgements
This project has been funded by the German Research Council DFG (DFG Pe494/11−1). We acknowledge continuing support from the University of Lübeck. Professor Monica Palcic (University of Victoria, BC, Canada) is thanked for many stimulating discussions. We would like to thank Professor Marcellus Ubbink (University of Leiden, The Netherlands) and Dr. Alvaro Mallagaray (University of Lübeck) or stimulating discussions. We would also like to thank Dr. Thorsten Biet for excellent support with the NMR experiments, and Wilfried Hellebrandt for providing some of the synthetic oligosaccharides. We are grateful for support within the iNEXT Program (PID 1483 and PID 2254) giving us access to the NMR high-field facilities at the Bijvoet Center in Utrecht. In particular, we would like to thank Dr. Hans Wienk for performing the 4D methyl–methyl NOESY experiment in Utrecht.
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Flügge, F., Peters, T. Complete assignment of Ala, Ile, Leu, Met and Val methyl groups of human blood group A and B glycosyltransferases using lanthanide-induced pseudocontact shifts and methyl–methyl NOESY. J Biomol NMR 70, 245–259 (2018). https://doi.org/10.1007/s10858-018-0183-4
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DOI: https://doi.org/10.1007/s10858-018-0183-4