Structural investigation of interactions within the tripartite drug-efflux pumps in Gram-negative bacteria.

Tripartite multidrug efflux-pumps are key contributors to the rising global problem of multidrug resistance in Gram-negative bacteria. These systems are composed of tree components spanning both the outer and inner membranes of the Gram-negative cell, namely the outer-membrane proteins (OMPs), the inner-membrane proteins (IMPs) and the membrane-fusion proteins (MFPs) providing a link between the two and thus forming a continuous conduit from the cytoplasm to the cell exterior. The OMPs belonging to the TolC family are central conduits for a number of efflux systems and hence an attractive drug target. However, the development of novel antibacterials is hindered by our lack of understanding of mechanics of pump assembly and despite recent advances in their structural characterisation, even such fundamental questions as pump stoichiometry and the mechanism of opening of the OMP channel remain unclear, and inaccessible by the standard biochemical and structural biology techniques.
This project seeks to address the gap in our current understanding of these clinically relevant systems by employing an integrative structural biology approach, combining a number of state-of-the-art technologies.
These include a radically new approach to the problem, namely using oxidative labelling of solvent-accessible protein residues combined with quantitative mass-spectrometry [3] to map the protein-protein interaction surfaces within the tripartite pumps, focussing on the interaction between the outer membrane channel and its membrane fusion-partner.
Primary objective would be identification of binding determinants and their effect on pump function with the view of disrupting and modulating its activity. This work will be invaluable for future design of novel classes of antibacterial therapeutics based on interference of protein-protein interactions.
Additional objective will be to resolve the long-standing question of the stoichiometry of the pump and to differentiate between the currently proposed conflicting models [6,7,8] of binding modes of the MFP to TolC.
This will be achieved by means of oxidative footprinting combined with quantitative mass-spectrometry [3] and the interaction-residues indentified in these primary screenings will be further characterised and validated using informed site-directed mutagenesis followed by high-throughput in vivo functional characterisation and promising candidates investigated further by additional structural biology and biophysical approaches.
The protein-protein interactions indentified will be used for creation of detailed structural models of the complete assembly and interaction maps, which will involve homology modelling and protein-docking simulations [2,5,8].
Furthermore, these detailed interaction maps will serve as a platform for rational design of novel efflux-pump inhibitors and peptidomimetics acting on the level of pump assembly.
The project will involve large-scale recombinant protein production of membrane-proteins and in vitro reconstitution of the complexes, as well as in vivo functional and cross-linking assays. The candidate will make extensive use of advanced mass-spectrometry technology and will spend a significant amount of time performing data analysis. If sufficient quantity of proteins is available, further crystallographic investigations of the complexes will be pursued.
A number of tripartite pump assemblies will be investigated, including the prototypical RND-transporter based tripartite pump AcrA/B-TolC from E.coli [5,7,8], as well as homologous system from Neisseria gonorrhoeae MtrCDE [2] and the ABC-transporter based MacAB-TolC.

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Please find additional funding text below. For further funding details, please see the ‘Funding’ section.
The School of Biosciences offers a number of UK Research Council (e.g. BBSRC, NERC) PhD studentships each year. Fully funded research council studentships are normally only available to UK nationals (or EU nationals resident in the UK) but part-funded studentships may be available to EU applicants resident outside of the UK. The deadline for applications for research council studentships is typically at the end of January each year.

Each year we also have a number of fully funded Darwin Trust Scholarships. These are provided by the Darwin Trust of Edinburgh and are for non-UK students wishing to undertake a PhD in the general area of Molecular Microbiology. The deadline for this scheme is also is typically at the end of January each year.