Protein-protein interactions in bacterial cell signalling and protein import
Co-workers: Nick Housden, Patrice Rassam, Renata Kaminska, Sejeong Lee, Katarina Jansen, Peter Holmes, Paul White, Connor Sharp, Marie-Louise Francis, Yana Demyanenko
In my laboratory we aim to understand how protein-protein interactions (PPIs) in bacteria underpin organisation of the bacterial outer membrane and Tol-Pal signalling across the bacterial cell envelope and how these signals are subverted by antibacterial proteins (bacteriocins) to catalyse their import into the cell. More broadly, we are interested in developing bacteriocins as antibiotics to target Gram-negative pathogens. We adopt a multidisciplinary approach in dissecting biological function that incorporates protein chemistry and engineering, molecular biophysics, structural biology and in vivo imaging.
Bacteriocin translocation – Bacteriocins are species-specific protein antibiotics that parasitize a variety of outer membrane and periplasmic proteins in Gram-negative bacteria. Much of our work has focused on the entry mechanism of colicins, which target Escherichia coli, but we have also begun studies on pyocins, which target Pseudomonas aeruginosa. These toxins serve as important agents of competition within microbial communities. We focus on nuclease bacteriocins (DNases, rRNases and tRNases), which use their network of PPIs within the cell envelope to establish a translocon complex that delivers a toxic domain into the cell. Hence, bacteriocin translocation represents a highly simplified model for cellular protein import.
Supramolecular assembly of outer membrane proteins – The asymmetric bilayer of the outer membrane contains outer membrane proteins (OMPs) and lipoproteins. Using colicins as OMP-specific probes, we recently discovered that OMPs cluster to form ‘OMP islands’ in the membrane and that this clustering behaviour underpins their turnover. We also demonstrated that the clustering behaviour of OMPs could be replicated in vitro using supported bilayers. Through the creation of a variety of OMP-specific labels we are dissecting the organisation of OMP islands and investigating the ramifications of these micro-domains to bacterial physiology.
The Tol-Pal assembly – Tol-Pal is a little understood complex that is required for the stable maintenance of the Gram-negative outer membrane and which is recruited to the septation apparatus during cell division. We are trying to uncover the native function of the Tol-Pal assembly, how and why it is coupled to the proton-motive force across the inner membrane and the mechanism by which bacteriocins subvert the assembly to initiate import across the outer membrane.PPIs of intrinsically disordered proteins – Intrinsically disordered sequences within bacteriocins play an important role in their import used, for example, to pass through the narrow pores of porins in order to contact Tol-Pal proteins in the periplasm. This has led us to address more fundamental questions about how intrinsically disordered proteins interact with binding partners.
1. Rassam, P., Copeland, N.A., Birkholz, O., Tóth, C., Chavent, M., Duncan, A.L., Cross, S.J., Housden, N.G., Kaminska, R., Seger, U., Quinn, D.M., Garrod, T.J., Sansom, M.S.P., Piehler, J., Baumann, C.G. & Kleanthous, C. (2015) Supramolecular assemblies underpin turnover of outer membrane proteins in bacteria. Nature 523, 333-336.
2. Papadakos, G., Sharma, A., Lancaster, L.E., Kaminska, R., Leech, A.P., Walker, D., Redfield, C. & Kleanthous, C. (2015) The consequences of inducing intrinsic disorder in a high affinity protein-protein interaction. J. Am Chem. Soc. 137, 5252-5255.
3. Wojdyla, J.A., Cutts, E., Papadakos, G., Hopper, J.T.S., Kaminska, R., Staunton, D., Stansfeld, P.J., Robinson, C.V. & Kleanthous, C. (2015) Structure and function of the Escherichia coli Tol-Pal stator protein TolR. J. Biol. Chem. 290, 26675-26687
4. Housden, N.G., Hopper, J.T.S., Lukoyanova, N., Rodriguez-Larrea, D., Wojdyla, J.A., Klein, A., Kaminska, R., Bayley, H., Saibil, H.R., Robinson, C.V. & Kleanthous, C. (2013) Intrinsically disordered protein threads through the bacterial outer membrane porin OmpF. Science 340, 1570-1574.
5. Housden, N.G., Wojdyla, J.A., Korczynska, J., Grishkovskaya, I., Kirkpatrick, N., Brzozowski, A.M. & Kleanthous, C. (2010) Directed epitope delivery across the Escherichia coli outer membrane through the porin OmpF. Proc. Natl. Acad. Sci. USA 107, 21412-21417.
Figure 1. Directed epitope delivery, a novel transmembrane signalling mechanism exploited by colicins. Colicin E9 (ColE9) houses two OmpF-binding sites within its 83-residue intrinsically unstructured translocation domain (IUTD). The colicin manages to capture TolB on the other side of the membrane by threading its IUTD through two pores of an OmpF trimer, one going into the cell the other coming back out again. In this way TolB is held in a defined orientation relative to the TolQRA inner membrane complex contact with which triggers entry of the toxin. See ref. 4 for further details.
Figure 2. OMPs co-localize in supramolecular assemblies in the outer membrane of E. coli. Figure adapted from ref. 1. Left-hand panel, engineering colicins E9 and Ia as specific, high affinity labels of the OMPs BtuB and Cir. BtuB is the transporter for vitamin B12 while Cir is a siderophore transporter. The two colicins were inactivated by internal disulphide bonds prior to labelling with fluorophores. Right-hand panel, E. coli OMP islands imaged by total internal reflection fluorescence microscopy and showing the co-localization of BtuB (labelled with ColE9-AF488, green) and Cir (labelled with ColIa-TMR, red). Scale bar is 1 mm.
Figure 3. Activation of the disulphide-stress sensing σR/RsrA-Zn complex. Under reducing conditions, RsrAred-Zn2+ inhibits the activity of σR. Disulphide stress induces the formation of intramolecular disulphides in RsrA (RsrAox) causing it to dissociate from σR. σR binds to RNA polymerase (RP) resulting in the activation of a regulon that re-establish redox homeostasis.