Structural and functional analysis of multiprotein complexes required for colicin translocation

Bacterial cells can export many proteins from the cytoplasm to the outside of the cell, including toxins that are capable of causing damage to humans that come into contact with them. The mechanisms of protein export have been well characterised in the last 20 years. In contrast the import of proteins into bacterial cells from the outside is a rare event that is limited to colicins; protein antibiotics produced by bacteria to kill closely related species in order to gain a selective advantage. An example is colicin E9 which has a C-terminal cytotoxic domain that has deoxyribonuclease (DNase) activity. If delivered into E.coli cells the DNase attacks and degrades their DNA in the cytoplasm and leads to cell death. The process by which the cytotoxic DNase domain is delivered from outside the cell into the cytoplasm is called translocation, and involves the interaction of the N-terminal translocation domain of colicin E9 with a number of E.coli proteins. The molecular details of how the protein-protein interactions involving the translocation domain result in the delivery of the DNase domain into the cytoplasm are poorly understood. This project aims to generate data so that better models of the translocation process can be formulated. Our proposed studies have potential implications in the development of new antibiotics and improving our understanding of the process by which complexes of proteins are formed.

To provide a competitive advantage for nutrient acquisition or colonisation of new environments many Escherichia coli cells secrete a ribosomally synthesised bactericidal colicin to kill closely related non-immune bacteria. The majority of colicins exert their lethality by a C-terminal cytotoxic domain that either causes membrane depolarisation of the cytoplasmic membrane (the pore-forming colicins) or degradation of RNA or DNA (the enzymatic colicins). Import of the cytotoxic domain requires the co-operation of two other separate structural domains. The first is a centrally located receptor binding domain that targets the colicin to the bacterial outer membrane and the second is an N-terminal translocation domain that makes contact with several proteins in the outer membrane and periplasmic space of the cell, and is believed to act as a signalling mechanism for entry of the cytotoxic domain. Colicin translocation can occur via the tol-dependent translocation system which is used by Group A colicins such as the enzymatic E colicins and requires a specific pentapeptide sequence in the N-terminal translocation region (TolB box), that is intrinsically disordered and is known to interact with the periplasmic protein TolB. In contrast the pore-forming colicin A has a TolA box in the translocation domain that interacts with TolA, as well as having a TolB box. In this project we will carry out structural and functional analysis of the protein-protein interactions of colicin A and colicin E9 with all the translocation proteins in order to better understand the translocation process.