Fig. 5: Model depicting how the KlebC toxin crosses the OM through TolC.

The N-terminal domain of KlebC (blue) exists as a folded hairpin in solution, with a disordered N-terminal domain containing a TonB-box sequence (magenta). The helical hairpin is likely in equilibrium with an unfurled counterpart, which is the binding-competent form of KlebC. We speculate that electrostatic attraction brings the positively charged N-terminus of KlebC into the negatively charged TolC channel. Thereafter, KlebC snakes through the TolC channel, resulting in ultra-slow association kinetics for the complex. The disordered N-terminal region of KlebC then passes through the periplasmic entrance of TolC, which likely requires flexing of the iris helices to accommodate a disordered polypeptide. Around 70 residues at the N-terminus of KlebC escape the iris leaving the helical regions of KlebC docked within the TolC channel. The disordered nature of these residues means that a wide search radius is possible. Toxin translocation is driven by TonB, which associates with the KlebC TonB box (residues 16–20), in conjunction with the PMF-linked inner membrane stator complex ExbB-ExbD. Previous work on TonB-dependent bacteriocins has shown that this protein–protein interaction is sufficient to drive entry into the cell¹³. How the nuclease then translocates to the cytoplasm is not known but may involve the inner membrane AAA⁺ATPase/protease FtsH, as has been shown for E. coli-specific colicins^{52, 53}.