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.

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.

The glycosyltransferase WaaG in Pseudomonas aeruginosa (PaWaaG) is involved in the synthesis of the core region of lipopolysaccharides. It is a promising target for developing adjuvants that could help in the uptake of antibiotics. Herein, we have determined structures of PaWaaG in complex with the nucleotide-sugars UDP-glucose, UDP-galactose, and UDP-GalNAc. Structural comparison with the homolog from Escherichia coli (EcWaaG) revealed five key differences in the sugar-binding pocket. Solution-state NMR analysis showed that WT PaWaaG specifically hydrolyzes UDP-GalNAc and unlike EcWaaG, does not hydrolyze UDP-glucose. Furthermore, we found that a PaWaaG mutant (Y97F/T208R/N282A/T283A/T285I) designed to resemble the EcWaaG sugar binding site, only hydrolyzed UDP-glucose, underscoring the importance of the identified amino acids in substrate specificity. However, neither WT PaWaaG nor the PaWaaG mutant capable of hydrolyzing UDP-glucose was able to complement an E. coli ΔwaaG strain, indicating that more remains to be uncovered about the function of PaWaaG in vivo. This structural and biochemical information will guide future structure-based drug design efforts targeting PaWaaG.

Solution-state NMR can be used to study protein–lipid interactions, in particular, the effect that proteins have on lipids. One drawback is that only small assemblies can be studied, and therefore, fast-tumbling bicelles are commonly used. Bicelles contain a lipid bilayer that is solubilized by detergents. A complication is that they are only stable at high concentrations, exceeding the CMC of the detergent. This issue has previously been addressed by introducing a detergent (Cyclosfos-6) with a substantially lower CMC. Here, we developed a set of bicelles using this detergent for studies of membrane-associated mycobacterial proteins, for example, PimA, a key enzyme for bacterial growth. To mimic the lipid composition of mycobacterial membranes, PI, PG, and PC lipids were used. Diffusion NMR was used to characterize the bicelles, and spin relaxation was used to measure the dynamic properties of the lipids. The results suggest that bicelles are formed, although they are smaller than “conventional” bicelles. Moreover, we studied the effect of MTSL-labeled PimA on bicelles containing PI and PC. The paramagnetic label was shown to have a shallow location in the bicelle, affecting the glycerol backbone of the lipids. We foresee that these bicelles will be useful for detailed studies of protein–lipid interactions. 

Chitin synthases are vital for growth in certain oomycetes as chitin is an essential component in the cell wall of these species. In Saprolegnia monoica, two chitin synthases have been found, and both contain a Microtubule Interacting and Trafficking (MIT) domain. The MIT domain has been implicated in lipid interaction, which in turn may be of significance for targeting of chitin synthases to the plasma membrane. In this work we have investigated the lipid interacting properties of the MIT domain from chitin synthase 1 in Saprolegnia monoica. We show by fluorescence spectroscopy techniques that the MIT domain interacts preferentially with phosphatidic acid (PA), while it does not interact with phosphatidylglycerol (PG) or phosphatidylcholine (PC). These results strongly suggest that the specific properties of PA are required for membrane interaction of the MIT domain. PA is negatively charged, binds basic side chains with high affinity and its small headgroup gives rise to membrane packing defects that enable intercalation of hydrophobic amino acids. We propose a mode of lipid interaction that involves a combination of basic amino acid residues and Trp residues that anchor the MIT domain specifically to bilayers that contain PA.

The twin-arginine translocase (Tat) mediates the transport of already-folded proteins across membranes in bacteria, plants and archaea. TatA is a small, dynamic subunit of the Tat-system that is believed to be the active component during target protein translocation. TatA is foremost characterized as a bitopic membrane protein, but has also been found to partition into a soluble, oligomeric structure of yet unknown function. To elucidate the interplay between the membrane-bound and soluble forms we have investigated the oligomers formed by Arabidopsis thaliana TatA. We used several biophysical techniques to study the oligomeric structure in solution, the conversion that takes place upon interaction with membrane models of different compositions, and the effect on bilayer integrity upon insertion. Our results demonstrate that in solution TatA oligomerizes into large objects with a high degree of ordered structure. Upon interaction with lipids, conformational changes take place and TatA disintegrates into lower order oligomers. The insertion of TatA into lipid bilayers causes a temporary leakage of small molecules across the bilayer. The disruptive effect on the membrane is dependent on the liposome's negative surface charge density, with more leakage observed for purely zwitterionic bilayers. Overall, our findings indicate that A. thaliana TatA forms oligomers in solution that insert into bilayers, a process that involves reorganization of the protein oligomer.

Fold-switch pathways remodel the secondary structure topology of proteins in response to the cellular environment. It is a major challenge to understand the dynamics of these folding processes. Here, we conducted an in-depth analysis of the α-helix–to–β-strand and β-strand–to–α-helix transitions and domain motions displayed by the essential mannosyltransferase PimA from mycobacteria. Using ¹⁹F NMR, we identified four functionally relevant states of PimA that coexist in dynamic equilibria on millisecond-to-second timescales in solution. We discovered that fold-switching is a slow process, on the order of seconds, whereas domain motions occur simultaneously but are substantially faster, on the order of milliseconds. Strikingly, the addition of substrate accelerated the fold-switching dynamics of PimA. We propose a model in which the fold-switching dynamics constitute a mechanism for PimA activation.