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Directional Porin Binding of Intrinsically Disordered Protein Sequences Promotes Colicin Epitope Display in the
Nicholas G Housden1, Patrice Rassam1, Sejeong Lee2
1Department of Biochemistry , University of Oxford , South Parks Road , Oxford OX1 3QU , U.K.
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.
Insights
Colicins, bacterial protein antibiotics, use specific binding sites within outer membrane porins like OmpF to enter Escherichia coli. This study reveals how these disordered binding regions ensure targeted delivery into the bacterial periplasm.
Area of Science:
- Microbiology
- Structural Biology
- Biochemistry
Background:
- Protein bacteriocins, like colicins, are narrow-spectrum antibiotics targeting bacteria.
- Their entry mechanism into bacteria often involves outer membrane porins, but is poorly understood.
- Colicins specific to Escherichia coli, such as ColE9, utilize the OmpF porin for entry.
Purpose of the Study:
- To elucidate the mechanism by which colicins engage the OmpF porin for bacterial entry.
- To determine the specific binding interactions of colicin ColE9's N-terminal region with OmpF.
Main Methods:
- Molecular dynamics simulations
- Chemical trimerization assays
- Isothermal titration calorimetry
- Fluorescence microscopy
- Planar lipid bilayer measurements
Main Results:
- Colicin ColE9 possesses two OmpF binding sites (OBS1 and OBS2) within its intrinsically unstructured N-terminal region.
- OBS2 binds to OmpF from the extracellular side, while OBS1 binds from the periplasmic side.
- This dual-sided binding within the narrow OmpF pore directs the colicin's interaction.
Conclusions:
- Colicins exploit the narrow pores of oligomeric porins like OmpF for direction-specific binding.
- The disordered regions of colicins facilitate precise interaction with porins, ensuring signal presentation in the periplasm.
- This mechanism is crucial for initiating colicin-mediated bacterial killing.
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