Assembling a protein transporter
How can you obtain high resolution structural information about protein complexes when you cannot determine the structure directly by techniques such as X-ray crystallography? This was the problem faced by Ben Berks' group in their work on the Tat protein transport system.
(Fig 1.) High precision evolutionary contacts between Tat proteins predict subunit packing interfaces. Reproduced from .
Bacteria use the Tat pathway to export folded proteins across their cell membrane. Tat transport is catalysed by three small membrane proteins called TatA, TatB, and TatC. During the transport cycle multiple copies of each of these proteins come together to transiently form the protein transporter . Berks and collaborators have previously determined the structures of the individual Tat components [2,3]. However, they did not know how these individual proteins assemble together in the translocation machine. Without this information it would be very difficult to understand how the transporter works. Unfortunately the Tat complexes are exceedingly challenging to work with and attempts to determine their structures by standard techniques have so far been unsuccessful. A novel approach was therefore required to determine the inter-protein contacts within the Tat apparatus.
The Berks group turned for help to Computational Biochemistry Research Fellow Phillip Stansfeld who applied the emerging bioinformatics technique of sequence co-evolution analysis to the problem. This approach relies on the principle that substitution of an amino acid at a tight packing interface will result in selection of compensatory changes in nearby amino acid side chains that re-optimise the interface. Thus, if two amino acids are in contact in the three dimensional structure of a protein, sequence changes at one position will tend to be coupled with sequence changes at the other position. Using these sequence correlations between Tat proteins Stansfeld was able to construct a model for the multisubunit TatBC core of the translocation apparatus at molecular-level resolution.
(Fig 2.) Molecular models for the TatBC core of the Tat translocation apparatus based on evolutionary contact analysis and biochemical experimentation. Models based on either three (left) or four (right) TatBC repeats are shown.
A key novel prediction of the analysis was that TatB and TatC are held together by a previously unsuspected cluster of intramembrane hydrogen bonds between polar side chains. A second unexpected outcome was the prediction that TatA associates with TatC through the same polar binding site used by TatB. These predictions were experimentally validated by Dr.Felicity Alcock in the Berks group with additional contributions from collaborator Dr Tracy Palmer at the University of Dundee. These new insights led the authors to infer a structural model for assembly of the active Tat transporter in which substrate binding triggers exchange of TatA for TatB at the polar binding site on TatC.
This work, which has been published in the journal eLife , demonstrates the power of co-evolution analysis to predict protein interfaces in multi-subunit complexes at molecular level resolution and represents a major step forward in elucidating the structure and mechanism of the Tat transport system.
 Alcock, F., Baker, M.A.B., Greene, N.P., Palmer, T., Wallace, M.I., and Berks, B.C. (2013) Live cell imaging shows reversible assembly of the TatA component of the twin-arginine protein transport system. Proc Natl Acad Sci USA 110: E3650-E3659.
 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. (2013) Structural model for the protein-translocating element of the twin-arginine transport system. Proc Natl Acad Sci USA 110: E1092-E1101.
 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., Roversi, P., Stansfeld, P.J., Robinson, C.V., Sansom, M.S.P., Palmer, T., Högbom, M., Berks, B.C., and Lea, S.M. (2012) Structure of the TatC core of the twin arginine protein transport system. Nature 492: 210-214.
 Alcock, F., Stansfeld, P.J., Basit, H., Habersetzer, J., Baker, M.A.B., Palmer, T., Wallace, M.I., and Berks, B.C. (2016) Assembling the Tat protein translocase. eLife, https://elifesciences.org/content/5/e20718 .