Exploiting the structure of the twin-arginine protein translocase core

Lead Research Organisation: University of Oxford
Department Name: Biochemistry

Abstract

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.

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
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.

Publications

10 25 50
publication icon
Huang Q (2017) A signal sequence suppressor mutant that stabilizes an assembled state of the twin arginine translocase. in Proceedings of the National Academy of Sciences of the United States of America

publication icon
Alcock F (2016) Assembling the Tat protein translocase. in eLife

publication icon
Stansfeld PJ (2017) Computational studies of membrane proteins: from sequence to structure to simulation. in Current opinion in structural biology

publication icon
Berks BC (2014) Structural biology of Tat protein transport. in Current opinion in structural biology

publication icon
Berks BC (2015) The twin-arginine protein translocation pathway. in Annual review of biochemistry

 
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 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.
[5] Others have proposed a model for the structure of the TatA assembly in the active transporter has been proposed using structures called `charge zippers'. We obtained evidence that shows this model is incorrect.
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 Investigator Award
Amount £1,252,987 (GBP)
Funding ID 107929/Z/15/Z 
Organisation Wellcome Trust 
Department Wellcome Trust Senior Investigator Award
Sector Charity/Non Profit
Country United Kingdom
Start 07/2016 
End 07/2021
 
Description Science Open Day demonstration 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact School students (and some parents) took part in a demonstration of the science associated with the funded project including hands-on experience of imaging live bacterial cells. The research and research approaches were discussed with the grant RAs.

None
Year(s) Of Engagement Activity 2013,2014
 
Description Science open day 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact Presentation of Tat protein transport work via live cell microscopy and video demonstration.
Year(s) Of Engagement Activity 2016,2017,2018,2019