Abstract
Protein bacteriocins are potent narrow spectrum antibiotics that exploit outer membrane porins to kill bacteria by poorly understood mechanisms. Here, we determine how colicins, bacteriocins specific for Escherichia coli, engage the trimeric porin OmpF to initiate toxin entry. The N-terminal ∼80 residues of the nuclease colicin ColE9 are intrinsically unstructured and house two OmpF binding sites (OBS1 and OBS2) that reside within the pores of OmpF and which flank an epitope that binds periplasmic TolB. Using a combination of molecular dynamics simulations, chemical trimerization, isothermal titration calorimetry, fluorescence microscopy, and single channel recording planar lipid bilayer measurements, we show that this arrangement is achieved by OBS2 binding from the extracellular face of OmpF, while the interaction of OBS1 occurs from the periplasmic face of OmpF. Our study shows how the narrow pores of oligomeric porins are exploited by colicin disordered regions for direction-specific binding, which ensures the constrained presentation of an activating signal within the bacterial periplasm.
Highlights
The asymmetric Gram-negative outer membrane (OM) is a robust protective barrier blocking entry of both hydrophilic and hydrophobic compounds into bacteria
Steered MD simulations were performed both for OBS1 and for OBS2, pulling each peptide from the extracellular side of OmpF into the periplasm and from the periplasm to the extracellular side of OmpF, with the average force required to pull the peptide calculated from three independent simulations (Figure S1)
Given the apparent preference of OBS1 for OmpF binding from the periplasmic side of the porin, we performed further MD simulations of this complex using OmpF embedded in a DPPC membrane
Summary
The asymmetric Gram-negative outer membrane (OM) is a robust protective barrier blocking entry of both hydrophilic and hydrophobic compounds into bacteria. Im9 binds the ColE9 DNase with a Kd value of 10−14 M.10 The heterocomplexes, referred to as OBS1TMR and OBS2TMR, were used to label OmpF-expressing cells in confocal fluorescence microscopy experiments (Figure S2).
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
