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Anaphase bridges in fission yeast cells
Whitby lab
Lactose permease represented using bending cylinders in Bendix software
Caroline Dahl, Sansom lab
Epithelial cells in C. elegans showing a seam cell that failed to undergo cytokinesis
Serena Ding, Woollard lab
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Lu Yang, Davis lab
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Image showing the global movement of lipids in a model planar membrane
Matthieu Chavent, Sansom lab
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Newly solved structure to boost understanding of protein transport

A ten year collaborative effort to determine the structure of a key protein transporter has come to fruition with the publication of a recent paper in Nature (1).

Cartoon representation of the Aquifex aeolicus TatC protein. The position of the protein in the membrane bilayer was predicted on the basis of molecular dynamics simulations. The phospholipid head groups of the modelled membrane are depicted here as orange spheres, and a semi-ordered detergent molecule bound to TatC in the crystal is shown in purple

Cartoon representation of the Aquifex aeolicus TatC protein. The position of the protein in the membrane bilayer was predicted on the basis of molecular dynamics simulations. The phospholipid head groups of the modelled membrane are depicted here as orange spheres, and a semi-ordered detergent molecule bound to TatC in the crystal is shown in purple
(Click to enlarge)

Collaborative research between the groups of Professor Ben Berks in the Biochemistry department and Professor Susan Lea in the Sir William Dunn School of Pathology, together with other researchers, has determined the structure of TatC, the core of the Tat protein transport system. The system plays an important role in bacterial virulence and is essential for photosynthesis in plants.

The elucidation of the structure will underpin future work to understand how the Tat system – which has the challenging and unusual task of moving folded proteins across the membrane – operates.

Two protein transport pathways, Sec and Tat, are responsible for routine protein export across the cytoplasmic membrane of bacteria and archaea and the import of proteins across the internal thylakoid membrane of plant chloroplasts. Whilst the Sec pathway uses the conventional mechanism of threading unfolded proteins through a membrane-bound channel, the Tat pathway transports proteins in a folded state.

Proteins that must be transported in this way include periplasmic or extracellular proteins that need cofactors which can only be added in the cytoplasm.

‘There is quite a lot of metabolism that goes on in the periplasm,’ says Professor Berks. ‘This is where you find all the enzymes that are going to make the cell wall and outer membrane, as well as periplasmic binding proteins to capture nutrients and bring them in. And a lot of the energy metabolism using electron transport chains is done outside – partly for energetic reasons and partly because some of the compounds that are made or metabolised are quite toxic.’

The virulence of pathogenic bacteria such as Pseudomonas aeruginosa and Mycobacterium tuberculosis also depends on the Tat pathway. For example, some virulence factors use Tat to exit the cytoplasm and are then transported across the bacterial outer membrane into the environment. In plants, the Tat pathway moves protein components of Photosystem II into the thylakoid lumen.

Tat and Sec are parallel protein export pathways in the cytoplasmic membrane of prokaryotes. While proteins must be unstructured to be transported by the Sec apparatus, the Tat transport system transports proteins only after they have folded

Tat and Sec are parallel protein export pathways in the cytoplasmic membrane of prokaryotes. While proteins must be unstructured to be transported by the Sec apparatus, the Tat transport system transports proteins only after they have folded
(Click to enlarge)

TatC is the core organising component of the Tat transport system. It binds the protein substrate via a signal peptide, which triggers a change in its structure allowing it to form a complex with the much smaller TatB and TatA proteins. This complex allows the protein to pass through the membrane, accommodating a diverse range of protein shapes and sizes without allowing appreciable ion leakage across the membrane.

‘It’s not known how the protein gets through the membrane but the transporter is probably not functioning like a conventional channel,’ explains Professor Berks. ‘Other research we have carried out in collaboration with the Schnell and Sansom groups in Biochemistry suggests that the complex thins the bilayer and disorders the lipid, somehow making it easy to poke proteins through.’ (2)

Crystallisation of TatC to understand its atomic level structure has proved elusive for many years. As well as the general problems associated with trying to obtain crystals of membrane proteins, TatC crystallisation has been a challenge because very little of the protein lies outside the membrane making it difficult to form crystal contacts. As the protein is part of a bigger structure, it also has a tendency to oligomerise.

‘The trick was to find methods to get very homogeneous protein,’ says Professor Berks. Many researchers contributed to the work over a number of years, but a key advance was made by former postdoc Michael Tarry working in the laboratory of Professor Martin Högbom at the University of Stockholm. He showed that it was possible to obtain sufficient quantities of homogeneous TatC protein from the hyperthermophilic bacterium Aquifex aeolicus to crystallise.

Conceptual model for how TatC (red) forms an active translocation site with partner proteins TatA (grey) and TatB (yellow) in the presence of the protein to be transported (green). Only the transmembrane helices of the TatA proteins are depicted. The approximate position of the membrane is indicated by grey lines

Conceptual model for how TatC (red) forms an active translocation site with partner proteins TatA (grey) and TatB (yellow) in the presence of the protein to be transported (green). Only the transmembrane helices of the TatA proteins are depicted. The approximate position of the membrane is indicated by grey lines
(Click to enlarge)

Most of the structural studies since then have been carried out by Sara Rollauer, a student supervised jointly by Ben Berks and Susan Lea. She carried out tens of thousands of crystallisation trails on the A. aeolicus TatC protein purified in different detergents before she was able to produce crystals that enabled her to solve the TatC structure. `It took outstanding dedication and technical skill from Sarah to get the crystals we needed,’ Professor Berks comments.

Other researchers have contributed towards the interpretation of the structure including Phillip Stansfeld and Mark Sansom in the Department of Biochemistry who carried out the molecular dynamics simulations of the TatC protein in a membrane environment.

The TatC structure has allowed the researchers to determine where signal peptides bind to TatC and to infer the sites of interaction with the other components of the transport apparatus. Professor Berks comments that it provides the essential structural framework for determining the mechanism of Tat transport in future work.

The structure also has the potential to generate wider interest, for example in biotechnology or biomedical applications. The structure could be exploited to help improve secretion of commercially important proteins or could be used to design a mimic of the peptide recognition site that would block substrate binding as a potential antimicrobial strategy.

References

  1. Rollauer, S.E., Tarry. M.J., Graham, J.E., Jääskeläinen, M., Jäger, F., Johnson, S., Krehenbrink, M., Liu, S.M., Lukey, M.J., Marcoux, J., McDowell, M.A., Rodriquez, F., Roversi, P., Stansfeld, P.J., Robinson, C.V., Sansom, M.S., Palmer, T., Högbom, M., Berks, B.C. and Lea, S.M. ‘Structure of the TatC core of the twin-arginine protein transport system.’ Nature (2012) Dec 13; 492:210-4.  
  2. Rodriguez, F., Rouse, S.L., Tait, C., Harmer, J., de Riso, A., Timmel, C.R., Sansom, M.S.P., Berks, B.C., and Schnell, J.R. `Structural model for the protein-translocating element of the twin-arginine transport system.’ Proc.Natl.Acad.Sci.USA (2013)110: E1092–E1101

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