Lockey, Christine (2017) Structural and ligand binding investigation of VanS, a protein involved in antibiotic resistance. PhD thesis, University of Warwick.

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Abstract

VanS is an integral membrane protein, and a receptor histidine kinase representing on half of the VanSR two component system. In the presence of vancomycin, as well as certain other antibiotics, VanS undergoes autophosphorylation at a conserved histidine residue and then transfers this phosphoryl group to a conserved aspartate on VanR. The phosphorylated form of VanR binds to the promoter region of the vanHAX operon, inducing expression of these genes, which confer glycopeptide antibiotic resistance to the cell.

Although the vancomycin resistance mechanism is mostly well-characterised, questions surrounding the structure and mode of activation of VanS remain unanswered. This thesis describes work towards the structural and ligand-binding characterisation of the extracellular \sensor" domain of VanS. Little is known of the structure of this domain, and while there exists evidence in the literature to support numerous theories surrounding the identity of the VanS ligand, no report has yet been made of a direct observation of a binding event involving any VanS protein and its ligand

To identify the ligand to VanS, the catalytically active, full-length VanS proteins of Enterococcus faecium (VanSA) and Streptomyces coelicolor (VanSSC) were heterologously expressed, solubilised and purified and binding studies were carried out by solution-state NMR. We present evidence of interactions between vancomycin and both VanS proteins, indicating that direct binding of vancomycin may be the mechanism by which both proteins are activated. The VanSSC extracellular domain was isolated as a synthetic peptide, its structure characterised, and the binding interface between this domain and vancomycin was investigated. Evidence of binding at the N-terminal end of the extracellular domain, in agreement with the findings of Koteva et al. (2010), is presented here. An interaction between vancomycin and DPC detergent micelles was also observed and characterised, and the biological significance of such an interaction is discussed

Item Type:	Thesis or Dissertation (PhD)
Subjects:	R Medicine > RS Pharmacy and materia medica
Library of Congress Subject Headings (LCSH):	Drugs -- Structure-activity relationships, Antibiotics, Drug resistance in microorganisms, Proteins, Enterococcus, Ligand binding (Biochemistry)
Official Date:	September 2017
Dates:	Date Event September 2017 Submitted
Institution:	University of Warwick
Theses Department:	Department of Chemistry
Thesis Type:	PhD
Publication Status:	Unpublished
Supervisor(s)/Advisor:	Dixon, Ann M. ; Roper, David I.
Sponsors:	Engineering and Physical Sciences Research Council, Molecular Organisation and Assembly in Cells
Extent:	xiv, 197 leaves : llustrations
Language:	eng

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