Substrate-receptor interactions in the Tat protein transport pathway

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 all pathogenic bacteria tested 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 will use cutting edge technology to elucidate how the transport machinery recognises the targeting tags present on the proteins that are to be transported. We will also test the hypothesis that the Tat transporter works by moving the tag across the membrane and that this pulls the passenger protein after it.

The Tat system in the model organism Escherichia coli comprises the three integral proteins TatA, TatB, and TatC. Multiple copies of TatB and TatC form a large complex that acts both as the substrate receptor for the Tat system and as the central organising element of the translocation site. Tat substrate proteins possess a signal peptide containing a twin-arginine consensus motif that is recognised by the TatC protein. Although all of the multiple TatC subunits in the TatBC complex are chemically identical TatBC complexes contain two different types of signal peptide binding site: a single high affinity, non-exchangeable site, and multiple low affinity, readily-exchangeable, sites.
Building on this work we now aim to:
- Probe the molecular nature of the two types of signal peptide binding sites.
- Test models for the relationship between these two sites.
- Elucidate the molecular events triggered by signal peptide binding to the TatBC complex.
Methods to be used include analysis of signal peptide-TatBC interactions in solution using isothermal titration calorimetery and fluorescence labelling, as well as analysis of substrate-transporter interactions in membranes using site-directed photoaffinity crosslinking and in vitro transport assays. These studies will be assisted by the characterization and use of a range of engineered Tat proteins. Further insight into the mechanism of TatBC will be obtained by isolating genetic suppressors of mutations that affect substrate-translocase interactions.
These data will provide crucial information on how the substrate protein interacts with the Tat system during the translocation cycle. This fundamental knowledge will support drug discovery activities and underpin commercial efforts to exploit the Tat pathway in the production of proteins of theraputic relevance.

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 and in a separate project we are currently screening for small molecules that interfere with the operation of the Tat pathway.
The aim of the work described here is to provide fundamental information on the mechanism of Tat transport. This fundamental knowledge will support drug discovery activities. It is also relevant in underpinning commercial efforts to exploit the ability of the Tat pathway to transport folded proteins in the production of proteins of theraputic relevance.
Communication with potential industrial beneficiaries will take place via the technology transfer infrastructures of the University of Oxford and Dundee. 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 in Oxford and the technology transfer office in Dundee. CI Palmer has an active programme through the Dundee Drug Discovery Unit of screening for inhibitors of the Tat pathway for use both in mechanistic studies and as lead compounds for antimicrobial development.
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 biophysics, membrane transport assays, and bacterial molecular genetics and in the application of such techniques in complex systems involving integral membrane systems. The biophysical parts of the study will provide a training in quantitative data analysis and the researchers will also gain writing, IT, and presentational skills. Researchers in our laboratories take part in Departmental Science Open Days (typically putting on practical demonstrations in protein science or bacteriology).