Characterisation of the E.coli and Pseudomonas aeruginosa TolA-TolB interaction

The Tol-Pal complex of Gram-negative bacteria is a highly conserved family of interacting proteins that span the periplasm, from inner to outer membrane. Despite decades of work on this protein complex, only the structure of a small part of the Tol complex in E.coli (TolB, Pal, TolB-Pal complex, TolA domain 3) as well as part of TolA from Pseudomonas aeruginosa have been resolved, and the native function of the Tol-Pal system remains elusive. A key interaction of the Tol-Pal system is between TolA and TolB. These two proteins bridge the periplasm, linking the inner membrane complex of TolQ, TolR and TolA with the outer membrane through the outer membrane bound Pal and periplasmic TolB. The structure of the TolA-TolB complex is not known, and although the TolA binding epitope of TolB has been localised to the intrinsically disordered N-terminus of TolB, the binding site on TolA is also unknown (Bonsor et al. 2009). The aim of this work is to address a number of questions regarding a fundamental part of the Tol-Pal system: the interaction between TolA and TolB. This thesis reports that not only is the short 22 residue N-terminus of TolB important for its interaction with TolA domain 3, but that it is the sole site of interaction between E.coli TolA and TolB. This was shown by engineering the E.coli TolB N-terminus onto another protein to create a novel interaction with E.coli TolA. In addition, a synthetic peptide of the N-terminus of TolB binding TolA can recapitulate and serve as a model for the native interaction. This work also reports that the TolA-TolB interaction is conserved between Pseudomonas aeruginosa TolA and TolB, and that the N-terminus is also important for this interaction, the first work to suggest this. Finally, through use of nuclear magnetic resonance spectroscopy, residues perturbed on Pseudomonas aeruginosa TolA domain 3 through the binding of a synthetic peptide representing Pseudomonas aeruginosa TolB have been mapped onto the TolA protein to determine the potential binding site of TolB on TolA, something which until now has been unknown.