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Characterisation of the putative effector interaction site of the regulatory HbpR protein from Pseudomonas azelaica by site-directed mutagenesis.
Bacterial transcription activators of the XylR/DmpR subfamily exert their expression control via σ(54)-dependent RNA polymerase upon stimulation by a chemical effector, typically an aromatic compound. Where the chemical effector interacts with the transcription regulator protein to achieve activation is still largely unknown. Here we focus on the HbpR protein from Pseudomonas azelaica, which is a member of the XylR/DmpR subfamily and responds to biaromatic effectors such as 2-hydroxybiphenyl. We use protein structure modeling to predict folding of the effector recognition domain of HbpR and molecular docking to identify the region where 2-hydroxybiphenyl may interact with HbpR. A large number of site-directed HbpR mutants of residues in- and outside the predicted interaction area was created and their potential to induce reporter gene expression in Escherichia coli from the cognate P(C) promoter upon activation with 2-hydroxybiphenyl was studied. Mutant proteins were purified to study their conformation. Critical residues for effector stimulation indeed grouped near the predicted area, some of which are conserved among XylR/DmpR subfamily members in spite of displaying different effector specificities. This suggests that they are important for the process of effector activation, but not necessarily for effector specificity recognition.
Amino Acid Substitution/genetics, Amino Acid Substitution/physiology, Bacterial Proteins/chemistry, Bacterial Proteins/genetics, Binding Sites/genetics, Computational Biology, Forecasting, Models, Biological, Models, Molecular, Mutagenesis, Site-Directed/methods, Organisms, Genetically Modified, Protein Binding/genetics, Protein Folding, Protein Interaction Mapping/methods, Protein Structure, Tertiary/genetics, Pseudomonas/genetics, Trans-Activators/chemistry, Trans-Activators/genetics
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