Most colicins kill Escherichia coli cells by membrane pore formation or nuclease activity and, superficially, the mechanisms are similar; receptor binding, translocon recruitment, periplasmic receptor binding and membrane insertion. However, in detail they employ a wide variety of molecular interactions that reveal a high degree of evolutionary diversification. Group A colicins bind to members of the TolQRAB complex in the periplasm and heterotrimeric complexes of Colicin-TolA-TolB have been observed for both colicins A and E9. Colicin N, the smallest and simplest pore -forming colicin, binds only to TolA and we show here that it uses the binding site normally used by TolB, effectively preventing formation of the larger complex used by other colicins. Colicin N binding to TolA is by β-strand addition with a Kd of 1uM compared to 40 µM for the TolA-B interaction. The β-strand addition and Colicin N activity can be abolished by single proline point mutations in TolA, which each remove one backbone hydrogen bond. By also blocking TolA-TolB binding these point mutations confer a complete tol phenotype which destabilises the outer membrane, prevents both colicin A and E9 activity and abolishes phage protein binding to TolA. These are the only point mutations known to have such pleiotropic effects and show that the TolA-TolB β-strand addition is essential for Tol function. This formation of this simple binary colicin N-TolA complex provides yet more evidence of a distinct translocation route for Colicin N and may help to explain the unique toxicity of its N terminal domain.