Protein-protein interactions in bacterial cell signalling and protein import
Co-workers: Nick Housden, Karthik Rajasekar, Justyna Wojdyla, Greg Papadakos,
Renata Kaminska, Colin Seepersad, Peter Holmes, Marie-Louise Francis, Patrice Rassam
In my laboratory we aim to understand how protein-protein interactions (PPIs) in bacteria underpin signalling within the cell envelope and cytoplasm, how changes in the environment modify these interactions to elicit different cellular responses and how such interactions are subverted by antibacterial proteins to catalyse their import into the cell. We study PPIs from a number of bacteria but predominantly from the model Gram-negative organism Escherichia coli. More broadly, we are interested in the molecular mechanisms that underpin specificity and affinity in PPIs, with a particular focus on the association mechanisms of intrinsically disordered proteins. Many of the PPIs we study are important in the virulence mechanisms of pathogens such as Salmonella typhimurium and Pseudomonas aeruginosa. We adopt a multidisciplinary approach in dissecting biological function that incorporates protein chemistry and engineering, molecular biophysics and structural biology
Colicin translocation Colicins are large (40-70 kDa) protein antibiotics that parasitize a variety of outer membrane and periplasmic proteins in Escherichia coli. Known generically as bacteriocins these toxins serve as important agents of competition between microbial communities. We focus on nuclease colicins (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, colicin translocation represents a highly simplified model for protein import into a cell.
Colicin-immunity protein interactions - A model system for studying PPIs Suicide of colicin-producing E. coli is avoided through the action of specific immunity proteins that bind and inactivate the toxin in the producing host but are jettisoned during colicin import. We are exploring the structural and energetic basis for the ultra-high affinity colicin DNase-immunity protein complexes (Kd~10-14 M) the specificities of which span the thermodynamic stability range of all known PPIs.
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 colicins subvert the assembly to initiate import across the outer membrane.ZAS complexes Extracytoplasmic function sigma factors along with their cognate anti-sigma factors represent one of the major cellular systems by which bacteria monitor and adapt to environmental change. We are investigating the structural mechanism(s) by which a group of Zinc-containing Anti-sigmafactor (ZAS) complexes from the antibiotic-producing bacterium Streptomyces coelicolor respond to environmental stress, focusing on the disulphide-stress regulator RsrA and its cognate sigma factor sigma R.
- Meenan, N.A.G., Sharma, A., Fleishman, S.J., MacDonald, C., Morel, B., Boetzel, R., Moore, G.R., Baker, D. & Kleanthous, C. (2010) The structural and energetic basis for high selectivity in a high affinity protein-protein interaction. Proc. Natl. Acad. Sci. USA 107, 10080-10085
- 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
- Wojdyla, J.A., Fleishman, S.J., Baker, D. & Kleanthous, C. (2012) Structure of the ultra-high affinity colicin E2 DNase-Im2 complex. J. Mol. Biol. 417, 79-94
- Papadakos, G., Housden, N.G., Lilly, K.J., Kaminska, R. & Kleanthous, C. (2012) Kinetic basis for the competitive recruitment of TolB by the intrinsically disordered translocation domain of colicin E9. J. Mol. Biol. 418, 269-280
- 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
Figure 1. Strategy for the disulphide entrapment of the ColE9 outer membrane translocon from E. coli-K12 cells (A) and its subsequent analysis by blue-native and SDS-PAGE (B, C) and native state electrospray ionisation mass spectrometry (D). The colicin translocon is composed of ColE9/Im9, BtuB, OmpF trimer and TolB in the periplasm. See Housden et al (2013) for further details.
Figure 2. Directed epitope delivery, a novel transmembrane signalling mechanism exploited by colicins. a, 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. Figure shows EM density for part of the isolated translcon. See Housden et al (2013) and (2010) for further details.
Figure 3. Kinetic analysis of colicin cell envelope protein-protein interactions. a, ColE9 binding to BtuB and OmpF in the outer membrane establishes the competitive recruitment of TolB from its complex with the lipoprotein Pal intiating cell entry. b, Stopped-flow fluorescence resonance energy transfer data using TAMRA-labelled ColE9 TBE and FAM-labelled TolB. The disordered TBE binds faster to TolB in the presence of Pal even though undergoing binding-induced folding. See Papadakos et al (2012) JMB and accompanying commentary by Uversky (2012) JMB.
Figure 4. 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.