Functional dynamics of glycosyltransferases : Solution-state NMR studies of peripheral membrane proteins involved in glycolipid biosynthesis in bacteria

Abstract: Antibiotic resistance is an existential threat enabled by bacterial adaptation and fuelled by inappropriate use of medication. The ensuing shortage of effective treatments has led to a rise in deaths linked to resistant bacterial pathogens. Disrupting cell wall biosynthesis can undermine bacterial defences, so new insights into the dynamic function of the enzymes involved could facilitate new therapies.Glycosyltransferases (GTs), enzymes forming glycosidic bonds, build molecules by transferring a sugar group from a donor to an acceptor. In Gram-negative bacteria, an enzymatic assembly line constructs membrane-anchored virulence factor lipopolysaccharide (LPS), which dominates the outer membrane, forming a protective layer. In mycobacteria, phosphatidyl-myo-inositol mannosides (PIMs) ensure the stability and impermeability of the inner membrane, and are constructed by a similar array of enzymes. In this thesis, bacterial GTs that work at the cytoplasmic leaflet of the inner membrane were investigated.PimA is an essential mycobacterial enzyme involved in constructing PIMs. It exists in multiple conformations, implying that it undergoes complex conformational changes, including a fold-switch. Associated motions were characterised with NMR dynamics experiments, revealing donor substrate-dependent population shifts and dynamic changes. At least four different states co-exist in solution, regardless of whether or not the enzyme is bound to substrate.WaaG performs one step in the biosynthesis of LPS in bacteria including E. coli and P.  aeruginosa. As it is not an essential enzyme, EcWaaG-deficient E. coli survive, but are more vulnerable to antibiotics. 19F NMR was employed to detect conformational and dynamic changes in EcWaaG. Upon interaction with bicelle-bound lipids and its donor substrate, UDP-glucose, EcWaaG was shown to experience a dynamic change, while a part of the protein was shown to experience slow conformational change. Hydrolysis of the donor substrate was quantified using 31P NMR. WaaG from P. aeruginosa was also investigated, focusing on the functional mechanism. NMR experiments determined that only UDP-GalNAc was hydrolysed by PaWaaG. When the active site was mutated to resemble that of EcWaaG, it was shown by 31P NMR that the mutated enzyme instead hydrolysed the donor substrate of EcWaaG, UDP-glucose. However, PaWaaG cannot be substituted for EcWaaG in vivo, underlining the importance of the interaction with the lipid-bound acceptor substrate.Both WaaG and PimA function adjacent to membrane. As larger objects give rise to broader signals, solution-state NMR imposes constraints on the detection of protein-lipid interactions. Small membrane mimetics like lipid bicelles can be used to mimic a membrane, but while they permit detection of effects on protein signals, detecting the effects on lipid signals requires further optimization, as further concentration-dependent challenges arise in multi-component experiments. Thus, lipid dynamics in bicelles designed to exist at low concentrations were characterized using 1H and 13C NMR. Upon binding spin-labelled PimA, paramagnetic relaxation enhancement of the lipids could be observed.This thesis thus widens the toolkit available to study membrane-associated proteins. It demonstrates that, far from being static structures, biomolecules like lipids and proteins are highly flexible objects whose function can only be understood if dynamics are taken into account.