Understanding the ABCs of multi drug resistance - tying the knot on the antibacterial peptide ABC transporter McjD

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In recent years, outbreaks of E. coli infections in UK and Europe highlight the importance of understanding the disease mechanisms of such important pathogens and biomolecules. Bacteria have developed mechanisms to confer resistance to current antibiotics and one of the routes of resistance are membrane proteins that export drugs from the cell, called exporters. Membrane proteins represent around 30% of the proteomes of most organisms and more than 40% of drug targets and yet few structures of these molecules have been solved by X-ray crystallography. These proteins are usually embedded in oil like environment making them very difficult to work with. We first need to isolate them from the membrane using lipid mimics such as detergents. Membrane proteins usually have many functions and bacteria have developed mechanisms to utilise them in order to extrude antibiotics through these proteins. In order to develop new treatments it is important to understand the molecular mechanism of these important biological molecules. Using X-ray crystallography, we can gain a detailed understanding of the mechanism of these proteins; we need to grow crystals and expose them to X-rays in order to obtain the molecular structure. Certain drugs in the market are based on peptide chemistry and the bacteria have evolved these transporters to provide them with immunity by extruding them from inside the cell. One such family of proteins are the ATP-binding cassette transporters (ABC transporters) that are powered by the hydrolysis of ATP. Our group has recently solved the X-ray structure of an ABC exporter that is involved in antibacterial transport, McjD, which confers resistance to the antibacterial peptide MccJ25. We know that when the antibacterial peptide MccJ25 and ATP bind to McjD, the ABC exporter can adopt different conformations (a movement that mimics opening/closing of a gate) in order to extrude the toxic compound. In our crystal structure, McjD adopts a new conformation, nucleotide-bound outward occluded; the portion of the protein that sits in the membrane has a closed cavity where the MccJ25 can fit in. The structural information and biochemical experiments of this membrane protein will provide an important insight on the binding and recognition of antibacterial peptides and allow us to get a detailed picture of its mechanism. We aim to trap the transporter in different conformations and characterise it in the presence of the antibacterial peptides in order to reveal the mode of binding and transport. The biochemical experiments will try identifying (1) the binding and recognition of the peptide by McjD and (2) the conformational changes that McjD undergoes during peptide binding and transport (before ligand binding, occluded state and after the hydrolysis of ATP (resetting the system)). Understanding the structure and function of these important biological molecules will contribute to the understanding of the relationship between membrane protein and substrate recognition and provide valuable information to structural biology and pharmacology.