Structure-function studies of the Tat protein translocation channel
Lead Research Organisation:
University of Oxford
Department Name: Biochemistry
Abstract
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Technical Summary
The membrane proteins TatA, TatB and TatC are the essential components of the Tat transport pathway. TatA and TatBC form separate, highly oligomeric, complexes. The TatBC complex functions as a receptor for the substrate protein. TatA is proposed to form the transmembrane protein translocation channel. This suggestion is supported by our recent low resolution structure of the TatA complex, obtained by negative stain electron microscopy, which shows a variable-diameter ring occluded at one end by a lid structure. This project aims to: (a) continue our studies of the structures of the Tat complexes by electron microscopy. Obtain structures for the TatBC complex by particle electron microscopy methods. Correlate structural features of the TatA complex with protein domains by truncation analysis. Produce higher resolution structures of TatA using single particle cryo-electron microscopy. (b) Carry out a multidisciplinary programme of research to test, exploit and expand the structural and mechanistic insights arising from our low resolution structure of the TatA complex. We will: Test models of channel operation by assessing the oligomeric state of native TatA molecules and examining whether TatA complexes exhibit transport-dependent exchange of TatA protomers. Assess using electron microscopy, atomic force microscopy and electrophysiological techniques whether the TatA complex contains an aqueous channel in the native membrane environment. Attempt to identify regions of the TatA protein that must move to allow Tat transport to occur. To do this we will analyse the effects of introduced disulfide bonds (identified in other work) on in vitro protein transport. Joint with BB/C516179/1
Organisations
Publications
Fronzes R
(2009)
Structure of a type IV secretion system core complex.
in Science (New York, N.Y.)
Krojer T
(2008)
Structural basis for the regulated protease and chaperone function of DegP.
in Nature
Orriss GL
(2007)
TatBC, TatB, and TatC form structurally autonomous units within the twin arginine protein transport system of Escherichia coli.
in FEBS letters
Tarry MJ
(2009)
Structural analysis of substrate binding by the TatBC component of the twin-arginine protein transport system.
in Proceedings of the National Academy of Sciences of the United States of America
| 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. A membrane bound receptor protein complex containing the proteins TatB and TatC recognises `signal peptides' on proteins that will be transported by the Tat system. Outcomes of this study: [1] Determination of the first three dimensional (3D) structure of the TatBC complex, using negative stain single particle electron microscopy. [2] Demonstration of separate TatB and TatC homo-oligomeric subcomplexes within the TatBC complex. [3] Trapping, purification, and electron microscopy structure determination of TatBC-substrate complexes. Analysis of these structures shows that: - Substrates bind to the periphery rather than the interior of the TatBC complex. - Substrate binding induces a large structural re-organization of the TatBC complex. - TatBC uses only one of the multiple potential substrate binding sites present in the complex. This suggests that some sites are privileged for binding, or that substrate binding is anti-cooperative. These results suggested significant alterations to the current model for Tat transport and prompted new approaches to unravel the Tat mechanism. In addition we: - made major contributions to the structural analysis of the Type IV Secretion System Core Complex found in bacterial pathogens, - determined the organisational changes undergone in response to the binding of unfolded protein binding by the major periplasmic protease/chaperone DegP. |
| Sectors | Pharmaceuticals and Medical Biotechnology |
| Description | Not yet used outside an academic context. |
| Description | X-ray crystallographic studies of the TatBC complex ? linchpin of the twin-arginine protein transport system |
| Amount | £379,467 (GBP) |
| Funding ID | BB/E023347/1 |
| Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
| Sector | Public |
| Country | United Kingdom |
| Start | 10/2007 |
| End | 08/2010 |