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Detection of Toxin Translocation into the Host Cytosol by Surface Plasmon Resonance
Published on: January 3, 2012
Discovery of critical Tol A-binding residues in the bactericidal toxin colicin N: a biophysical approach
E M Raggett1, G Bainbridge, L J Evans
1Department of Biochemistry and Genetics, The Medical School, University of Newcastle Upon Tyne, UK.
Abstract:
Colicins translocate across the Escherichia coli outer membrane and periplasm by interacting with several receptors. After first binding to outer membrane surface receptors via their central region, they interact with TolA or TonB proteins via their N-terminal regions. Finally, the toxic C-terminal region is inserted into or across the cytoplasmic membrane. We have measured the binding of colicin N to TolA by isothermal titration microcalorimetry (ITC) and tryptophan fluorescence. The isolated N-terminal domain exhibits a higher affinity for TolA (Kd = 1 microM) than does the whole colicin (18 microM), and similar behaviour has been observed when the N-terminal domain of the g3p protein of the bacteriophage fd, which also binds TolA, is examined in isolation and in situ. This may indicate a similar mechanism in which a cryptic TolA binding site is revealed after primary receptor binding. The isolated colicin N N-terminal domain appears to be unstructured in circular dichroism and fluorescence studies. We have used mutagenesis and ITC to characterize the TolA binding site and have shown it to be of a different sequence and much further from the N-terminus than previously thought.
Insights
Colicin N binds TolA with higher affinity when isolated, suggesting a cryptic binding site revealed upon receptor interaction. This study characterizes the TolA binding site on colicin N.
Area of Science:
- Molecular Biology
- Biochemistry
- Microbiology
Background:
- Colicins are bacteriocins produced by Escherichia coli that translocate across the bacterial envelope to exert their toxic effects.
- Colicin translocation involves interactions with outer membrane receptors, periplasmic proteins like TolA or TonB, and finally insertion into the cytoplasmic membrane.
- The N-terminal region of colicins is crucial for interacting with periplasmic proteins, but the precise binding mechanism and site remain incompletely understood.
Purpose of the Study:
- To investigate the binding interaction between colicin N and the periplasmic protein TolA.
- To characterize the TolA binding site on colicin N using biophysical and genetic methods.
- To compare the binding behavior of isolated colicin N domains with the full-length protein.
Main Methods:
- Isothermal titration microcalorimetry (ITC) was employed to quantify the binding affinity between colicin N and TolA.
- Tryptophan fluorescence spectroscopy was used to monitor conformational changes and binding events.
- Circular dichroism spectroscopy assessed the secondary structure of the isolated N-terminal domain.
- Site-directed mutagenesis combined with ITC was utilized to map the TolA binding site.
Main Results:
- The isolated N-terminal domain of colicin N exhibited a significantly higher binding affinity for TolA (Kd = 1 µM) compared to the full-length colicin (Kd = 18 µM).
- The isolated N-terminal domain of colicin N appeared unstructured in solution, suggesting a potential role for primary receptor binding in inducing a structured TolA-binding conformation.
- Mutagenesis and ITC studies revealed that the TolA binding site on colicin N is located further from the N-terminus and has a different sequence composition than previously hypothesized.
Conclusions:
- The binding of colicin N to TolA is modulated by its interaction with outer membrane receptors, likely through the exposure of a cryptic TolA binding site.
- The N-terminal domain of colicin N plays a critical role in TolA interaction, and its binding site is distinct and more C-terminal than initially predicted.
- These findings provide insights into the intricate mechanism of colicin translocation and protein-protein interactions within the bacterial periplasm.
