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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.

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Circular dichroism spectra of purified His6-tagged HbpR wild-type protein or of sixteen purified HbpR A-domain mutants, between 200 and 250 nm, at a protein concentration of ≈0.3 mg/ml.Spectra were normalized to Δε, as indicated in the Experimental Procedures section, and grouped to reveal similar dichroism trends. (A) HbpR wild-type and mutants V182T, L207F and T52F (type II effects with delayed and lower induction by 2-HBP). (B) Mutants with similar dichroisms as HbpR wild-type. (C) Mutants with the most strong aberration of the wild-type circular dichroism trace, of which C187F and E184L completely abolished activation by 2-HBP, but E42F and E203P having no major effect on 2-HBP dependent induction in E. coli.
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pone-0016539-g005: Circular dichroism spectra of purified His6-tagged HbpR wild-type protein or of sixteen purified HbpR A-domain mutants, between 200 and 250 nm, at a protein concentration of ≈0.3 mg/ml.Spectra were normalized to Δε, as indicated in the Experimental Procedures section, and grouped to reveal similar dichroism trends. (A) HbpR wild-type and mutants V182T, L207F and T52F (type II effects with delayed and lower induction by 2-HBP). (B) Mutants with similar dichroisms as HbpR wild-type. (C) Mutants with the most strong aberration of the wild-type circular dichroism trace, of which C187F and E184L completely abolished activation by 2-HBP, but E42F and E203P having no major effect on 2-HBP dependent induction in E. coli.

Mentions: Western blotting with an anti-HbpR M13-displayed VHH camel antibody suggested (within the accuracy of this technique) that most HbpR mutant proteins were produced to the same level in E. coli (Fig. 4), except for L207F (lower than expected) and E203P (higher than expected). This indicated that differential EGFP expression in E. coli carrying a mutant hbpR gene was not due to complete misfolding or degradation of the protein, but rather due to a critical amino acid change in the effector binding region. In particular E184L, I180T, I180F, T52F and C187F, which were the mutations causing the largest decrease of 2-HBP-dependent EGFP expression from PC, resulted in HbpR mutant proteins that were expressed in E. coli within the normal range observed for all (Fig. 4). To corroborate this further, we purified a number of (mutant) HbpR proteins and compared their circular dichroism spectra between 200 and 250 nm. Sixteen mutant HbpR proteins and HbpR wild-type (all tagged with His6) were hereto purified by Ni-NTA chromatography, dialysed and diluted to 0.3 mg protein per ml (Fig. 5). For reasons of protein stability, it was not possible to completely omit traces of EDTA and glycerol from the dialysis buffer. As a result no reliable spectra below 198 nm could be recorded (not shown).


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)

Circular dichroism spectra of purified His6-tagged HbpR wild-type protein or of sixteen purified HbpR A-domain mutants, between 200 and 250 nm, at a protein concentration of ≈0.3 mg/ml.Spectra were normalized to Δε, as indicated in the Experimental Procedures section, and grouped to reveal similar dichroism trends. (A) HbpR wild-type and mutants V182T, L207F and T52F (type II effects with delayed and lower induction by 2-HBP). (B) Mutants with similar dichroisms as HbpR wild-type. (C) Mutants with the most strong aberration of the wild-type circular dichroism trace, of which C187F and E184L completely abolished activation by 2-HBP, but E42F and E203P having no major effect on 2-HBP dependent induction in E. coli.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3040749&req=5

pone-0016539-g005: Circular dichroism spectra of purified His6-tagged HbpR wild-type protein or of sixteen purified HbpR A-domain mutants, between 200 and 250 nm, at a protein concentration of ≈0.3 mg/ml.Spectra were normalized to Δε, as indicated in the Experimental Procedures section, and grouped to reveal similar dichroism trends. (A) HbpR wild-type and mutants V182T, L207F and T52F (type II effects with delayed and lower induction by 2-HBP). (B) Mutants with similar dichroisms as HbpR wild-type. (C) Mutants with the most strong aberration of the wild-type circular dichroism trace, of which C187F and E184L completely abolished activation by 2-HBP, but E42F and E203P having no major effect on 2-HBP dependent induction in E. coli.
Mentions: Western blotting with an anti-HbpR M13-displayed VHH camel antibody suggested (within the accuracy of this technique) that most HbpR mutant proteins were produced to the same level in E. coli (Fig. 4), except for L207F (lower than expected) and E203P (higher than expected). This indicated that differential EGFP expression in E. coli carrying a mutant hbpR gene was not due to complete misfolding or degradation of the protein, but rather due to a critical amino acid change in the effector binding region. In particular E184L, I180T, I180F, T52F and C187F, which were the mutations causing the largest decrease of 2-HBP-dependent EGFP expression from PC, resulted in HbpR mutant proteins that were expressed in E. coli within the normal range observed for all (Fig. 4). To corroborate this further, we purified a number of (mutant) HbpR proteins and compared their circular dichroism spectra between 200 and 250 nm. Sixteen mutant HbpR proteins and HbpR wild-type (all tagged with His6) were hereto purified by Ni-NTA chromatography, dialysed and diluted to 0.3 mg protein per ml (Fig. 5). For reasons of protein stability, it was not possible to completely omit traces of EDTA and glycerol from the dialysis buffer. As a result no reliable spectra below 198 nm could be recorded (not shown).

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