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 do main: the exported pore-forming domain was highly cytotoxic. We thus i ntroduced 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 cyt otoxicity 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-formin g domain, by using a co-immunoprecipitation procedure, in order to sho w that they interact, We showed both proteins to be co-localized in th e Escherichia coli inner membrane and subsequently co-immunoprecipitat ed them. The interaction required a functional immunity protein, The i mmunity 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 wi th the membrane-anchored channel domain; the interaction requires a fu nctional membrane-inserted immunity protein but does not require the c hannel to be in the open state.