Limits...
Characterisation of the putative effector interaction site of the regulatory HbpR protein from Pseudomonas azelaica by site-directed mutagenesis.

Vogne C, Bisht H, Arias S, Fraile S, Lal R, van der Meer JR - PLoS ONE (2011)

Bottom Line: Where the chemical effector interacts with the transcription regulator protein to achieve activation is still largely unknown.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.This suggests that they are important for the process of effector activation, but not necessarily for effector specificity recognition.

View Article: PubMed Central - PubMed

Affiliation: Department of Fundamental Microbiology, University of Lausanne, Lausanne, Switzerland.

ABSTRACT
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.

Show MeSH

Related in: MedlinePlus

EGFP expression from the HbpR-dependent PC-promoter in E. coli in the presence (black bars) or absence (grey bars) of 2-HBP as inducer measured for the different HbpR A-domain mutants after 2 h induction time, and either complemented with a second plasmid carrying wild-type HbpR (pHB240) or not.Note the partial ‘rescue’ of the abolished phenotype in T52F, I180F, E184F, C187F and V182T by wild-type HbpR, suggesting that the mutant proteins are not dominantly negative over the wild-type. Results indicate the mean of triplicate incubations, plus the calculated standard deviation.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3040749&req=5

pone-0016539-g006: EGFP expression from the HbpR-dependent PC-promoter in E. coli in the presence (black bars) or absence (grey bars) of 2-HBP as inducer measured for the different HbpR A-domain mutants after 2 h induction time, and either complemented with a second plasmid carrying wild-type HbpR (pHB240) or not.Note the partial ‘rescue’ of the abolished phenotype in T52F, I180F, E184F, C187F and V182T by wild-type HbpR, suggesting that the mutant proteins are not dominantly negative over the wild-type. Results indicate the mean of triplicate incubations, plus the calculated standard deviation.

Mentions: Next, we tested whether the created HbpR mutations were dominant over wild-type HbpR, which would be a further indication for their activity in E. coli, since we previously demonstrated that an HbpR mutant devoid of the A-domain was dominant negative on wild-type HbpR [17]. Hereto, the A-domain mutant strains of HbpR in E. coli were complemented with a plasmid expressing wild-type hbpR from its native promoter (pHBP124). For all type I mutants (loss of induction), complementation with wild-type HbpR restored 2-HBP inducible activity although not to the level of wild type response (Fig. 6). Mutants C187F and I180F reverted to 3/4th of the lost activity upon complementation with wild-type. This might be the result of formation of heteromultimers between wild-type HbpR and mutant protomers, which do not fully restore functionality. It is worth noticing that for mutations W205H, I185T and Q188E, which did not affect the activity of the protein, complementation reduced the response to 2-HBP. This effect could also be seen with the semi-constitutive mutants A202S, V181F and S32F; complementation with the wild-type HbpR decreased the response upon induction (Fig. 6). Such mutations may therefore affect heterodimer formation.


Characterisation of the putative effector interaction site of the regulatory HbpR protein from Pseudomonas azelaica by site-directed mutagenesis.

Vogne C, Bisht H, Arias S, Fraile S, Lal R, van der Meer JR - PLoS ONE (2011)

EGFP expression from the HbpR-dependent PC-promoter in E. coli in the presence (black bars) or absence (grey bars) of 2-HBP as inducer measured for the different HbpR A-domain mutants after 2 h induction time, and either complemented with a second plasmid carrying wild-type HbpR (pHB240) or not.Note the partial ‘rescue’ of the abolished phenotype in T52F, I180F, E184F, C187F and V182T by wild-type HbpR, suggesting that the mutant proteins are not dominantly negative over the wild-type. Results indicate the mean of triplicate incubations, plus the calculated standard deviation.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3040749&req=5

pone-0016539-g006: EGFP expression from the HbpR-dependent PC-promoter in E. coli in the presence (black bars) or absence (grey bars) of 2-HBP as inducer measured for the different HbpR A-domain mutants after 2 h induction time, and either complemented with a second plasmid carrying wild-type HbpR (pHB240) or not.Note the partial ‘rescue’ of the abolished phenotype in T52F, I180F, E184F, C187F and V182T by wild-type HbpR, suggesting that the mutant proteins are not dominantly negative over the wild-type. Results indicate the mean of triplicate incubations, plus the calculated standard deviation.
Mentions: Next, we tested whether the created HbpR mutations were dominant over wild-type HbpR, which would be a further indication for their activity in E. coli, since we previously demonstrated that an HbpR mutant devoid of the A-domain was dominant negative on wild-type HbpR [17]. Hereto, the A-domain mutant strains of HbpR in E. coli were complemented with a plasmid expressing wild-type hbpR from its native promoter (pHBP124). For all type I mutants (loss of induction), complementation with wild-type HbpR restored 2-HBP inducible activity although not to the level of wild type response (Fig. 6). Mutants C187F and I180F reverted to 3/4th of the lost activity upon complementation with wild-type. This might be the result of formation of heteromultimers between wild-type HbpR and mutant protomers, which do not fully restore functionality. It is worth noticing that for mutations W205H, I185T and Q188E, which did not affect the activity of the protein, complementation reduced the response to 2-HBP. This effect could also be seen with the semi-constitutive mutants A202S, V181F and S32F; complementation with the wild-type HbpR decreased the response upon induction (Fig. 6). Such mutations may therefore affect heterodimer formation.

Bottom Line: Where the chemical effector interacts with the transcription regulator protein to achieve activation is still largely unknown.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.This suggests that they are important for the process of effector activation, but not necessarily for effector specificity recognition.

View Article: PubMed Central - PubMed

Affiliation: Department of Fundamental Microbiology, University of Lausanne, Lausanne, Switzerland.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus