Salt bridge disruption in colicin Ib channel-forming domain enhances membrane translocation and bactericidal activity

Pore-forming colicins are bacteriocins produced by Escherichia coli to kill competing bacterial strains by forming ion-permeable channels in the inner membrane of target cells, leading to membrane depolarization, ion leakage, and ultimately cell death. While the crystal structures of their soluble form and membrane-perforating activities have been intensively studied, the structural rearrangements enabling outer membrane translocation and inner membrane pore formation remain puzzling. Here, we present the crystal structure of the channel-forming domain of colicin Ib (ColIb) and identify interhelical salt bridge networks that stabilize its tertiary structure. Comparative analysis shows that electrostatic interactions between helices H3–H7 and H4–H6 are conserved in E1-type but not A-type colicins. Disrupting these electrostatic interactions—either through alanine substitutions or acidic pH—produced a less compact structure with increased membrane association. Salt bridge mutations enhanced the bactericidal activity of full-length ColIb by at least an order of magnitude, and notably, introducing these mutations into the isolated C-domain conferred CirA-dependent cytotoxicity in the absence of the T- and R-domains. Protonation of the C-domain at pH 4.5 further amplified its killing capacity. Our findings reveal that destabilization of interhelical contacts facilitates unfolding and membrane association, providing a structural mechanism for CirA-mediated translocation and amplified killing efficiency.