Structural studies of a bacterial membrane bound antibiotic transporter

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. Bacterial membrane proteins are essential for antibiotic resistance since they are involved in the export of the drugs from the cell. 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. 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. In order to 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. We have obtained such crystals of a bacterial membrane protein antibiotic transporter. The structural information of this membrane protein will provide an important insight on the binding and recognition of antibiotics from this new family of proteins. We are also going to understand the biochemistry of this protein in the presence of antibiotics. Having all these information, the structure and biochemical analysis, will have pharmacological importance since it will allow the development of novel antibiotics that can fight bacterial resistance.

The SbmA protein plays an important role in the uptake of structurally unrelated microcins. They are secreted under conditions of nutrient exhaustion and exert potent antibacterial activity against closely related species. Microcins can inhibit the growth of a variety of Gram-negative bacteria with minimal inhibitory concentrations in the nanomolar range. Microcins act by inhibiting the DNA gyrase and RNA polymerase action, thus causing cell death. The microcins that are transported by SbmA are structurally unrelated. There is no structural information on the SbmA protein or any of its homologues, and obtaining the crystal structure of this protein will give an in-depth and valuable understanding on the recognition mechanism for microcins. We have grown three-dimensional crystals for this membrane protein that can diffract X-rays up to 3.9A resolution using different strategies, an important step towards the elucidation of the structure of this transporter. We also want to obtain the structure of the transporter with different antibiotics as wells as measure their ability to inhibit cell growth. We also want to obtain the structure of SbmA with different antibiotics in order to understand the recognition mechanism for the substrates. Using reconstitution, uptake and binding assays we will measure the binding constants for the different substrates as well as phenotypic effect of antibiotic mimics to the cell. We also want to investigate if the driving force for uptake is ATP or Na+/H+ driven. The overall aim is to understand how these antibiotics are uptaken, how they interact with the transporter, and how they can become more efficient. This information will be of great assistance to synthetic chemists for the synthesis of inhibitors capable of crossing the membrane and targeting the DNA gyrase or RNA polymerase or even a new pathway.

The elucidation of the structure and biochemical characterisation of the SbmA protein from E.coli will greatly benefit the academic community. The industrial sector will benefit as a result of the structure elucidation, since it can provide useful information on the binding and transport of the antimicrobial peptides. This in turn can help them design new drus for fighting bacterial infections. In long term, the general public will benefit from the development and release of any new drugs or therapies from pharmaceutical companies. If any potential drugs are developed as a result of our research, then the wider public will benefit, as they will improve their health during bacterial infection. These drugs may cut down the time of incubation of the pathogen or minimise the side affects from the infection. Any industrial potential will be realised once we have the structure with the peptides bound and the biochemical data. Working with membrane proteins, expression, purification and crystallisation, will allow the PDRA to develop specialised lab skills that he can transfer to his next post, either academic or industrial environment and in turn train more scientists. Many labs have start targeting membrane proteins, and one of their requirements during recruitment is to have experience working on membrane molecules. The PDRA with his training from this post, will put him in a very strong position for future jobs. The successful outcome of this proposal depends a lot on collaborations. We have collaboration with two internationally recognised scientists in their field who will greatly facilitate the research. In our application we have requested funding to visit our collaborator in Italy in order to setup experiments and discuss the results. We also requested funding for the EM work at the University of Manchester.