Lipid- and substrate-induced conformational and dynamic changes in a glycosyltransferase involved in E. coli LPS synthesis revealed by F-19 and P-31 NMR

WaaG is a glycosyltransferase (GT) involved in the synthesis of the bacterial cell wall, and in Escherichia coli it catalyzes the transfer of a glucose moiety from the donor substrate UDP-glucose onto the nascent lipopolysaccharide (LPS) molecule which when completed constitutes the major component of the bacterium's outermost defenses. Similar to other GTs of the GT-B fold, having two Rossman-like domains connected by a short linker, WaaG is believed to undergo complex inter-domain motions as part of its function to accommodate the nascent LPS and UDP-glucose in the catalytic site located in the cleft between the two domains. As the nascent LPS is bulky and membrane-bound, WaaG is a peripheral membrane protein, adding to the complexity of studying the enzyme in a biologically relevant environment. Using specific 5-fluoro-Trp labelling of native and inserted tryptophans and 19F NMR we herein studied the dynamic interactions of WaaG with lipids using bicelles, and with the donor substrate. Line-shape changes when bicelles are added to WaaG show that the dynamic behavior is altered when binding to the model membrane, while a chemical shift change indicates an altered environment around a tryptophan located in the C-terminal domain of WaaG upon interaction with UDP-glucose or UDP. A lipid-bound paramagnetic probe was used to confirm that the membrane interaction is mediated by a loop region located in the N-terminal domain. Furthermore, the hydrolysis of the donor substrate by WaaG was quantified by 31P NMR.

Automated summary

WaaG is a glycosyltransferase involved in bacterial cell wall synthesis. The study used 5-fluoro-Trp labelling and NMR techniques to investigate the dynamic interactions of WaaG with lipids and the donor substrate. The findings provide important insights into the function and structure of WaaG in a biologically relevant environment.