Structural and Functional Analysis of Multiprotein Complexes Required for Colicin Translocation

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. <P>
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.