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.
Espesset
D
D
Laboratoire d'Ingenierie et de Dynamics des Systèmes Membranaires, Marseilles, France.
Duché
D
D
Baty
D
D
Géli
V
V
D001426:Bacterial Proteins / Q000494:pharmacology*; D002462:Cell Membrane / Q000187:drug effects* / Q000378:metabolism; D003087:Colicins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000633:toxicity; D003553:Cystine / Q000502:physiology; D003902:Detergents / Q000494:pharmacology; D004926:Escherichia coli / Q000187:drug effects* / Q000378:metabolism; D007473:Ion Channels / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000494:pharmacology*; D017354:Point Mutation; D011133:Polysaccharide-Lyases / Q000235:genetics; D011485:Protein Binding; D011487:Protein Conformation; D021382:Protein Sorting Signals / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism*
D001426:Bacterial Proteins; D003087:Colicins; D003902:Detergents; D007473:Ion Channels; D008565:Membrane Proteins; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; C007104:colicin immunity proteins; D003553:Cystine; D011133:Polysaccharide-Lyases; C023911:pectate lyase