VanS is a sensor histidine kinase which forms part of a Two-Component System, with the response regulator, VanR. This system regulates inducible transcription of genes responsible for glycopeptide resistance in enterococci and actinomycetes. In the presence of an antibiotic inducer, VanS autophosphorylates at a conserved histidine residue, and transfers this phosphate to VanR. This phosphorylated form of VanR activates transcription of vanHAX genes, which confer resistance to glycopeptide antibiotics. This research investigates the structure and function of VanS proteins derived from E. faecium and S. coelicolor, which exhibit different antibiotic resistance phenotypes. The focus is on improving understanding of how glycopeptide antibiotics can either, directly or indirectly, induce VanS activity in each species, and if direct, to identify any ligand binding sites. To date, only one study has shown a direct binding between VanS (derived from S. coelicolor) and a vancomycin glycopeptide (Koteva et al., 2010), so this protein (termed VanSSC) was chosen as a control in ligand binding assays, alongside VanS proteins derived from E. faecium (termed VanSA). Full-length VanS proteins were purified in a functionally-active state and analysed for their structure and ligand binding properties by NMR spectroscopy. High resolution 2D NMR spectra of the isolated VanS sensor domains in each protein have been collected for the first time, and provided a platform for conducting NMR-based ligand titrations. Chemical Shift Perturbation Analysis of the resulting NMR titration data indicates several residues in VanS involved in binding to vancomycin. In conjunction with fluorescence assays, this data newly suggests that both VanSA and VanSSC can interact directly with vancomycin. This challenges consensus belief in the literature that VanSA should only be activated indirectly. NMR-based assays outlined here now pave the way for further in-depth studies of the ligand binding mechanism and putative identification of other ligands in this Two-Component System.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:644924 |
| Date | January 2014 |
| Creators | Edwards, Richard James |
| Publisher | University of Warwick |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://wrap.warwick.ac.uk/67112/ |
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