Structural and functional studies of Gram -Negative Bacteria Phospholipid Transport Proteins and Human SHP2 Protein

Coleman, James (2020) Structural and functional studies of Gram -Negative Bacteria Phospholipid Transport Proteins and Human SHP2 Protein. Doctoral thesis, University of East Anglia.

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Abstract

Gram-Negative bacteria have an envelope composed of an Inner Membrane (IM) and Outer Membrane (OM) with a periplasmic space between the two. The OM is an asymmetrical membrane which forms an important protective layer for the cell and is a contributing factor to resistances to toxic compounds and antibiotic agents. The OM consists of an outer leaflet made up of Lipopolysaccharides (LPS), and an inner leaflet constructed from Phospholipids (PLs), predominantly Phosphatidylethanolamine (PE), Phosphatidylglycerol (PG) and Cardiolipin (CA) in Escherichia coli. The manner by which LPS is transported between the IM and the OM has been well studied, with the Lipopolysaccharide transport (Lpt) protein complex responsible for their trafficking from IM to OM. In contrast, the pathways by which Phospholipids are transported between the two membranes remain unclear. The Maintenance of lipid asymmetry (Mla) pathway has been partially characterised and is thought to shuttle PLs from the OM to the IM (the retrograde direction), although this directionality is a point of contention. The YebST and PqiABC pathways have also been implicated in PL trafficking, but the structures of these proteins and the associated mechanisms by which they potentially transport PLs are unknown. The work in this thesis uses the technique of Protein X-ray Crystallography to gain structural and functional insights into the putative PL transport pathways YebST and PqiABC. In the following work, a novel structure for PqiC is determined, with preliminary in vitro and in vivo assays suggesting functionally and structurally important residues, as well as the protein’s capability of binding PLs. Successful purification and crystallisation of the protein YebT is also outlined, in addition to a purification technique and initial crystallisation trials for the IM-localised YebS protein, highlighting it as a target for future structural studies.

Further to this work, Protein X-ray Crystallography is also here used to characterise the binding between the Homo sapiens SHP2 protein and the compound Methylene Blue (MB). SHP2 is the product of a protooncogene and appears to allow cancer cells to evade the immune response through cooperation with the regulatory protein PD-1. The SHP2-MB interaction was identified by the work of collaborators and was shown to prevent SHP2 binding to PD-1. A concurrent investigation into the interaction between SHP2 and PD-1 was also carried out. While SHP2 was successfully purified and crystallised, the work here shows that MB does not bind SHP2 directly, and is instead likely binds to PD-1, an insight which will inform further study into the potential therapeutic uses of MB in PD-1-mediated immune evasion.

Item Type: Thesis (Doctoral)
Faculty \ School: Faculty of Medicine and Health Sciences > Norwich Medical School
Depositing User: Chris White
Date Deposited: 17 Nov 2021 11:34
Last Modified: 17 Nov 2021 11:34
URI: https://ueaeprints.uea.ac.uk/id/eprint/82149
DOI:

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