X-ray crystallographic studies of the TatBC complex - linchpin of the twin-arginine protein transport system

Lead Research Organisation: University of Oxford
Department Name: Biochemistry


Some bacterial proteins operate on the outside of the cell, for example the toxins produced by bacterial pathogens. Since all proteins are made inside the bacterium the 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. 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 protein transport system is not only found in bacteria it is also present in the chloroplasts of plants where it is essential to form and maintain the proteins required to carry out photosynthesis. The heart of the Tat system is a complex formed by two proteins called TatB and TatC. This complex sits in the cell membrane and recognises and binds the proteins that are to be transported. It then binds another protein called TatA to form the active transporter. We aim to determine a detailed structure of the complex containing TatB and TatC. We will also try to determine the structure of TatC alone since this may be easier and TatC appears to form the stable functional core of the Tat system. To obtain these structures we first need to grow crystals of the purified proteins. This is likely to be quite challenging because it is not easy to form crystals of proteins found in membranes. For this reason we have proposed a number or different crystallization approaches to maximize our chances of success. We will also make full use of the latest high-throughput technologies to speed the work. Determination of a structure for TatBC and/or TatC is essential if we are to understand the unusual mechanism of transport by the Tat system. The Tat system is a possible drug target because it is required for bacterial pathogenesis but is not found in humans. It is also of biotechnological interest because it could be utilised to secrete useful protein products.

Technical Summary

The membrane proteins TatA, TatB and TatC are the essential components of the Tat protein transport pathway. The complex formed by the TatB and TatC proteins is the linchpin of the Tat pathway. It is the membrane receptor for substrate proteins and it recruits TatA to form the active translocation machinery. We aim to determine a high resolution structure for the key TatBC complex, and/or the potentially more crystallographically amenable TatC core complex, using X-ray crystallographic methods. Building on our success in the overexpression and purification of the TatBC complex of Escherichia coli our initial efforts will be concentrated on crystallizing TatBC and TatC from this source. We will utilise not only the native complexes but also engineered variants and complexes putatively stabilized by site-specific disulfide crosslinks or by the presence of a substrate signal peptide. In addition to the crystallization trials with the E. coli proteins we will attempt to increase the chances of successful structure determination by undertaking high throughput heterologous expression and crystallization trials of TatC and TatBC from a range of other prokaryotes.


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Description The twin arginine (Tat) protein transport system is involved in moving folded proteins across the cytoplasmic membrane of prokaryotes and the thylakoid membrane of plant chloroplasts. The Tat system is of potential use as pathway to produce proteins of industrial or pharmacological utility.
The TatC protein is the largest and most conserved component of the Tat protein transport system. A multimer of TatC complex forms the core of the translocation site and is responsible for substrate binding and dynamic organisation of other components of the pathway. We identified TatC proteins from different bacterial sources that express sufficiently well in our protein production host to be used in structural studies. Using this set of TatC proteins we identified protein/detergent combinations that yield homogeneous, well-behaved, pure TatC complexes suitable for crystallization trials. We were able to identify, and partially optimize, multiple conditions that allow crystallization of the TatC complex from the hyperthermophilic bacterium Aquifex aeolicus. These crystals are close to the quality needed for meaningful structure determination and indicate that further optimization/screening will lead to a TatC structure (and this was achieved in follow-on work).
Exploitation Route In follow-on work we were able to determine the structure of the Aquifex aeolicus TatC protein at atomic resolution. This provides a possible basis for structure-based design of Tat pathway inhibitors as novel antimicrobials and a molecular basis for the engineering of the Tat pathway for industrial protein production.
Sectors Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

Description Not yet used outside an academic context.
Description School science demonstrations 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Engagement of children with science.
Year(s) Of Engagement Activity 2009,2010,2011,2012,2013