The channel domain of colicin A is inhibited by its immunity protein through direct interaction in the Escherichia coli inner membrane.

A bacterial signal sequence was fused to the colicin A pore‐forming domain: the exported pore‐forming domain was highly cytotoxic. We thus introduced a cysteine‐residue pair in the fusion protein which has been shown to form a disulfide bond in the natural colicin A pore‐forming domain between alpha‐helices 5 and 6. Formation of the disulfide bond prevented the cytotoxic activity of the fusion protein, presumably by preventing the membrane insertion of helices 5 and 6. However, the cytotoxicity of the disulfide‐linked pore‐forming domain was reactivated by adding dithiothreitol into the culture medium. We were then able to co‐produce the immunity protein with the disulfide linked pore‐forming domain, by using a co‐immunoprecipitation procedure, in order to show that they interact. We showed both proteins to be co‐localized in the Escherichia coli inner membrane and subsequently co‐immunoprecipitated them. The interaction required a functional immunity protein. The immunity protein also interacted with a mutant form of the pore‐forming domain carrying a mutation located in the voltage‐gated region: this mutant was devoid of pore‐forming activity but still inserted into the membrane. Our results indicate that the immunity protein interacts with the membrane‐anchored channel domain; the interaction requires a functional membrane‐inserted immunity protein but does not require the channel to be in the open state.