Exploiting the structure of the twin-arginine protein translocase core

Some proteins in bacteria are located outside the membrane that surrounds the cell, for example the toxins produced by bacterial pathogens. Because all proteins are made inside the bacterium the external proteins have to be moved out of the cell across the normally impermeable cell membrane by machines termed protein transporters. One type of transporter moves unfolded proteins, threading them across the membrane like string through the eye of a needle. By contrast, a second type of transporter, which we term the Tat system, moves folded proteins across the membrane. This is much more challenging than threading and so it is thought that the Tat system operates by an unusual mechanism. The Tat system is required for many bacterial processes including energy generation, cell division, nutrient acquisition, pathogenesis, and the nitrogen-fixing symbiosis of soil bacteria with plants. The Tat protein transport system is not only found in bacteria but is also present in the chloroplasts of plants where it is essential to form and maintain the proteins required to carry out photosynthesis.
The Tat system is a possible drug target because it is required for bacterial pathogenesis but is not found in humans or animals. It is also of biotechnological interest because it could be utilised to secrete useful protein products.
We have recently determined the molecular structure of the core part of the Tat protein translocation apparatus. This project aims to exploit this structural data to help us understand how the Tat machinery works.

The Tat system of bacteria and chloroplasts carries out the unusual, and mechanistically challenging, task of moving folded proteins across biological membranes. Substrates of the Tat transport system are responsible for a wide range of cellular processes in bacteria and are essential for plant photosynthesis. The mechanism of Tat transport remains to be elucidated.
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.
We have recently succeeded in determining the crystal structure of the core TatC component [Nature (2012) 492: 210-214]. This breakthrough transforms our ability to experimentally address the mechanism of Tat transport because for the first time we have a structural context to guide experimentation and interpretation. We now propose a programme of studies to exploit the TatC structure with the aim of producing a molecular-level understanding of the mechanism of Tat transport.
We have defined the signal peptide binding site on TatC, and the site of interaction between the TatB transmembrane helix and TatC. We will now use biochemical, biophysical, genetic, and computational methods to:
- Determine where TatA interacts with TatC.
- Determine the location of the cytoplasmic domain of TatB in the TatBC complex and gain insight into the function of this domain.
- Analyse the role of the conserved polar Glu/Gln residue exposed to the centre of the membrane bilayer in the TatC central cavity.
- Explore the conformational dynamics of TatC that may link signal peptide binding to TatA recruitment.
- Determine the interfaces by which TatC proteins interact to form the functional substrate receptor complex.
This experimental programme is expected to lead to fundamental advances in our understanding of the mechanism

This is hypothesis driven research. However, our results will be relevant in underpinning commercial efforts to exploit the Tat pathway
- for production of proteins of therapeutic and industrial relevance
- as an analytical tool for quality control of protein folding
- as a target for novel antimicrobials
The work, therefore, has relevance to the BBSRC Strategic Priority `New strategic approaches to industrial biotechnology'. In addition there may be relevance to the BBSRC Strategic Priority `Bioenergy; generating new replacement fuels for a greener, sustainable future' because the hydrogenase enzymes being explored for use in biocells are Tat substrates.
Although the proposed project does not encompass the development of antimicrobial compounds per se, Palmer works with the University of Dundee Drug Discovery Unit and is thus 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 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.
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, University of Dundee, 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 departmental 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, genetics, protein engineering, and in the application of such techniques in complex systems involving integral membrane systems. The researchers will also gain writing, IT, and presentational skills. Researchers in the PI's group at Oxford 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 (`bacteria' and `cold'). Researchers in the CI's group at Dundee will participate in the `Magnificent Microbes' public understanding of science events planned for 2014 and 2016.