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.