Structure of the Tat protein translocase

All bacteria produce proteins that operate on the outside of the bacterial cell. Good examples of this are the toxins produced by bacterial pathogens. Since bacteria only make proteins inside the cell, the newly-synthesised extracellular proteins must be moved out of the cell across the normally impermeable cell membrane. This task is carried out by machines termed protein transporters that are located in the cell membrane.
The Tat protein transporter is found in many different bacteria including almost all those which cause human diseases such as Mycobacterium tuberculosis (which causes tuberculosis), and Salmonella (which causes food poisoning). In pathogenic bacteria the Tat transporter is essential for the bacterium to cause disease. Thus, scientists are becoming increasingly interested in developing drugs that prevent the Tat system from working.
In order to understand how the Tat machinery works, and ultimately to design new drugs to stop it working, this project aims to determine the structure of the Tat transport apparatus using X-ray crystallography.

The Tat protein transport system is essential for the virulence of most bacterial pathogens. Tat has the unusual and challenging task of exporting folded proteins across the cytoplasmic membrane. How Tat solves this problem is unknown.
The Tat system comprises the three integral proteins TatA, TatB, and TatC. TatC acts as the central organising element onto which the other two components assemble. Substrate proteins are recognized by specific signal peptides which bind to sites in TatC and TatB.
Determining the structure of the Tat transporter is essential if we are to elucidate the molecular mechanism of Tat transport. Indeed, structure determination alone should allow us to deduce the broad operating principles of the Tat apparatus. Building on our recent success in determining the crystal structure of the core TatC component [Nature (2012) 492: 210-214] we now aim to use X-crystallography to determine the structure of the full translocation site. Our approach will be to solve higher order Tat complexes built around the TatC core.
Specifically we aim to obtain structures of:
- TatB in complex with TatC
- Substrate signal peptide in complex with TatC or TatBC
- TatA in complex with TatBC, or TatBC plus signal peptide
The availability of these structures will transform our understanding of Tat transport.

The export of proteins by bacteria is a critical process that is essential for their survival and for the delivery of virulence factors during pathogenesis. The Tat pathway is a highly unusual protein transport system that exports folded proteins across the bacterial cytoplasmic membrane. The Tat system is found in most pathogenic bacteria (including the genera Salmonella, Escherichia, Shigella, Yersinia, Legionella, Vibrio, Helicobacter, Pseudomonas, Mycobacterium, Staphylococcus, Bacillus, Streptococcus, Haemophilus, Brucella, Campylobacter, Rickettsia, Bordetella, Burkholderia, Neisseria, and Klebsiella) and has been found to be essential for virulence in all tested cases. The Tat system is an essential pathway in some bacteria including Mycobacterium tuberculosis, the causative agent of tuberculosis. Since the Tat system is absent from human cells it represents a novel target for development of antibacterial compounds.
The aim of the work described here is to provide fundamental information on the mechanism of Tat transport through determination of high resolution structures of the transporter. This fundamental knowledge will:
- Improve our understanding of basic bacterial cell biology relevant to pathogenesis.
- Support drug discovery activities through characterisation of the target pathway.
- Underpin commercial efforts to exploit the ability of the Tat pathway to transport folded proteins for the production of proteins of theraputic relevance.
Although the proposed project does not encompass the development of antimicrobial compounds per se Lea has expertise in structure based drug design and so we are equipped to recognise and highlight those results of our work with promise in this area.
Communication with potential industrial beneficiaries will take place via the technology transfer infrastructures of the University of Oxford. Specifically, we will patent intellectual property arising from this research, and then seek to license or spin-out this technology with the support of Isis Innovation Ltd.
The primary mechanism for communication of this research will be through publication in peer review international journals. Open access publishing options will be used where available. We will liaise at the time of publication with the University of Oxford and BBSRC Press offices to ensure publicity of results of interest to the general public. Our results will also be made available on our regularly updated web sites. Note also that the Tat system is now featured in mainstream cell biology text books such as Molecular Biology of the Cell and so our data will potentially impact on future editions of standard texts.
The researchers employed on this grant will gain technical skills in cutting edge methodology in protein chemistry, protein engineering, and membrane protein crystallography. The crystallography researcher will develop computational skills through training in X-ray structure determination. The researchers will also gain writing, IT, and presentational skills. Researchers in our laboratories are expected to take part in Departmental Science Open Days (typically putting on practical demonstrations in protein science or bacteriology) and in the PI's science outreach activities at a local primary school .