Exploiting the structure of the twin-arginine protein translocase core

Lead Research Organisation: University of Dundee
Department Name: School of Life Sciences


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Technical Summary

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

Planned Impact

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
In addition the work will contribute to the development of methods to analyse the structural organisation of integral membrane protein complexes, in particular the emerging technology of membrane protein native MS being pioneered by CI Robinson.
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 in Oxford. Note that CI Robinson has experience in patent applications.
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 and protein biophysics 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 on BBSRC grants in our laboratories have the opportunity to take part in Departmental Science Open Days (typically putting on practical demonstrations in protein science or bacteriology).
Description Bacteria possess proteins located outside the membrane that surrounds the cell. 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. Our study concerned one such transporter, the Tat system, which is able to move folded proteins across the cell membrane. The Tat protein transport system is found not only 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 transporter is made up of three proteins called TatA, TatB, and TatC, all of which are all located in the cell membrane. Multiple copies of TatB and TatC come together to form a receptor for the protein that is to be transported. Once the protein to be transported is bound to the TatBC receptor many copies of the TatA protein are then recruited to form the active transporter. At the outset of the work we had determined the molecular structures of the individual TatA and TatC proteins, and the structure of TatB was also available. The project aimed to use this information to elucidate how the individual Tat components come together to form the translocation site and help understand how the Tat machinery works. The main outcomes were:
[1] We applied the emerging technique of sequence co-evolution analysis to the Tat components to work out how the TatB and TatC assemble together to form the receptor complex. The model we built for the complex was then confirmed experimentally. This work demonstrates the power of sequence co-evolution analysis in the prediction of protein-protein interactions.
[2] We identified a previously unrecognized site in the TatBC complex that is crucial for Tat transport.
[3] We worked out that TatA and TatB compete for the same binding site on TatC. We deduced that TatA displaces TatB during the crucial switch point in the Tat mechanism where substrate protein binding to the TatBC complex triggers TatA recruitment to form the active transporter.
[4] We used disulphide crosslinking to show that TatA is a component of the resting TatBC receptor complex and identified its binding site.
[5] We showed that interaction of a signal peptide with the TatABC receptor complex resulted in Tata and TatB switching their binding sites.
[6] We identified mutations that lock the transporter in the fully assembled state. This opens the way to further analysis of the structure of the otherwise transiently assembled transporter.
Exploitation Route This is basic science that will underpin exploitation of the Tat pathway in the biotechnological production of proteins and as a possible target of novel antimicrobial agents.
Sectors Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

URL http://www.bioch.ox.ac.uk/aspsite/index.asp?pageid=1368
Description BBSRC Responsive Mode
Amount £467,534 (GBP)
Funding ID BB/N014545/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 08/2016 
End 08/2019
Description BCB 
Organisation University of Oxford
Department Department of Biochemistry
Country United Kingdom 
Sector Academic/University 
PI Contribution Joint grant holder.
Collaborator Contribution Sharing reagents and protocols. Exchange of personnel. Sharing results before publication
Impact Many joint papers and joint grant funding.
Start Year 2006
Description Magnificent Microbes 3 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact A large public engagement event entitled 'Magnificent Microbes 3' held in conjunction with Sensation, Dundee's Science Centre. Teachers were engaged prior to the event and were enrolled as members of the Society for General Microbiology. Children were challenged prior to the event about what they associated with the word microbe. On the day we had 10 hands on stands (Day 1 was the schoolchildren, day two was the general public). I had a stall entitled 'where our medicines come from' and children isolated soil microbes. After the event the children went away and with their teachers performed microbiology experiments. About six weeks later the children returned to visit us at the University of Dundee for an open evening where they presented posters outlining their experiment and art work of microbes. Word association showed that the children had assimilated information from the event and that their perception of microbes had changed. The event featured in local press and on local radio.

Word association showed that the children had assimilated information from the event and that their perception of microbes had changed.
Year(s) Of Engagement Activity 2014
Description Magnificent Microbes 4 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact Two day event with multiple stands introducing 180 primary 7 school pupils (day one) and members of the general public (day two) to how microbes shape our world, including the microbiome, biofuels, plant diseases, antibiotics, food etc
Year(s) Of Engagement Activity 2016