Domain cross-talk during effector binding to the multidrug binding TTGR regulator
Craig Daniels1, Abdelali Daddaoua, Duo Lu
1Department of Environmental Protection, CSIC, E-18008 Granada, Spain.
The Journal of Biological Chemistry
|May 4, 2010
Summary
Bacterial antibiotic resistance involves efflux pumps regulated by TtgR protein. Mutating TtgR
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- Antibiotic resistance is a major global health threat.
- Bacterial efflux pumps actively expel toxic compounds, contributing to resistance.
- The TtgR protein regulates the ttgABC efflux pump in Pseudomonas putida.
Purpose of the Study:
- To investigate the structure-function relationship of the TtgR protein.
- To understand how ligand binding affects TtgR's interaction with its DNA target.
- To elucidate the mechanism of transcriptional repression by TtgR.
Main Methods:
- Site-directed mutagenesis of TtgR ligand binding sites.
- Electrophoretic mobility shift assays (EMSAs) to assess DNA binding.
- Isothermal titration calorimetry (ITC) for binding thermodynamics.
- In vitro and in vivo expression assays.
- Crystal structure determination of a TtgR mutant.
Main Results:
- Mutants H70A, H72A, R75A failed to dissociate from DNA with chloramphenicol.
- Mutants L66A and L66AV96A showed increased DNA binding affinity.
- Mutant H67A exhibited decreased DNA binding affinity.
- In vivo assays correlated with in vitro binding, showing altered antibiotic tolerance.
- Crystal structure of TtgR H67A revealed structural insights.
Conclusions:
- Ligand binding to TtgR modulates its DNA binding affinity.
- Inter-domain communication is crucial for TtgR function.
- Understanding TtgR interactions can inform strategies against antibiotic resistance.
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