Dynamics and selectivity of the NleB and SseK effectors studied by NMR spectroscopy and MD simulation

Hicks, Thomas (2022) Dynamics and selectivity of the NleB and SseK effectors studied by NMR spectroscopy and MD simulation. Doctoral thesis, University of East Anglia.

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The NleB and SseK effectors are a unique series of enzymes which act as glycosyltransferases (GT), transferring a single GlcNAc ring from donor UDP-GlcNAc to arginine residues. This activity is unique as GT activity is typically limited to serine and threonine, in the case of O-glycosyltransferases (O-GT), and asparagine, in the case of N-Glycosyltransferases (N-GT). These effectors are used by E. coli and S. enterica, affording the ability to disrupt the innate immune system response by inhibiting key protein-protein interactions in the NF-κB signalling pathway within host cells. To date a complete understanding of these effectors has not been found, with their selectivity, mechanism of action and dynamics not fully understood.

There are two main perspectives this thesis looks to explore. How do these effectors discern between acceptor substrates and how do they recognise their donor substrate? These two areas encompass the protein-protein and protein-ligand interactions that these enzymes have when performing catalysis. With an understanding of these interactions, it would be possible to begin to unravel the complexity of these enzymes and take steps towards disrupting their activity in the development of medicines to combat the bacteria that utilise them.

With this thesis, MD simulation, two-dimensional NMR and STD NMR were used to understand the dynamics and selectivity of these enzymes. Through a mutagenesis study it was possible to begin to understand the acceptor interface of NleB1, providing a structural rationale for a single tyrosine residue in the recognition of the acceptor substrate FADD. With the study of the binding of UDP-GlcNAc and other Leloir donors it is possible to see how they are recognised and discern different recognition profiles between the SseK effectors, SseK1 and SseK2. With these results, this thesis contributes to an understanding of the Dynamics and selectivity of the NleB and SseK effectors.

Item Type: Thesis (Doctoral)
Faculty \ School: Faculty of Science > School of Pharmacy
Depositing User: Kitty Laine
Date Deposited: 27 Jun 2023 10:18
Last Modified: 27 Jun 2023 10:18
URI: https://ueaeprints.uea.ac.uk/id/eprint/92506

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