Colicin translocation: investigating the topology of the translocon and potential applications

Colicins are proteins produced by some strains of E. coli to achieve competitive advantage over related species. With MW in the range between 30-75 kDa they consist of receptor, translocator and cytotoxic domains. After being released from producing cells, colicins cross the outer and, in some cases, inner membrane to kill susceptible cells. This ability to cross the membrane in a species-specific manner has been extensively studied in hope to use the acquired knowledge to develop new antimicrobials. It is known that using the unstructured N-terminus and receptor domain, colicins contact common porins at the outer membrane such as OmpF and BtuB and components of Tol and Ton machinery in the periplasm assembling a translocon. Although several crystal structures of colicin fragments with effector proteins (BtuB, TolB, OmpF) have been published, the architecture of the translocon remains unclear. To address this issue, we’ve studied colicin E9 in complex with its receptor and translocator proteins BtuB, OmpF and TolB by crosslinking mass spectrometry. As well as numerous internal cross-links within individual proteins, seven inter-protein crosslinks were identified. These data largely agree with the available crystal structures and previous MD simulations. However, some inter- and intra-molecular links cannot be explained with our current knowledge of the complex, so in this work I suggest four alternative models of the translocon structure. Clear functional separation between the domains makes colicins attractive candidates to use as the delivery chassis for other peptide and protein antimicrobials, which cannot otherwise cross the membrane or are not specific to pathogenic bacteria. To test whether this is feasible, I used the intrinsically disordered N- terminus of colicin N as a vehicle to deliver a BamD-binding peptide into the periplasm of E. coli. The delivery of the BamD epitope to the periplasm was successful, however the cytotoxic effect of this delivery was limited.