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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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Overview of the hbp regulatory system and tertiary structure modeling of the HbpR A-domain using a previously established XylR model as template.(A) Organization of the hbp genes and the location of the HbpR binding sites (UAS, upstream activating sequences) in front of the PC and PD promoters. HbpR domains are depicted to scale according to the predictions by Jaspers et al [7]. (B) to (E) Fitting used swiss-model and was performed on XylR A-domain PDB coordinates as calculated by Devos et al [22]. (B) Ribbon model of HbpR A-domain residues 11–209, with predicted coils, alpha-helices and beta-sheets indicated. (C) Superposition of the predicted HbpR and XylR A-domains in the same configuration as A. (D) Tertiary structure model of HbpR A-domain with calculated molecular surface at 1.4Å and 40% transparency, in order to see the helical, coil and sheets. Model turned into a position which enables visualization of the proposed tunnel entry (b). C-terminal end of coil ending the A-domain indicated with an arrow at (a). Pinkish region in the centre of the A-domain illustrates a predicted cavity within the A-domain. (E), as C but now for the XylR A-domain, with exception of the ten most C-terminal residues, which otherwise are predicted to occlude the tunnel.
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pone-0016539-g001: Overview of the hbp regulatory system and tertiary structure modeling of the HbpR A-domain using a previously established XylR model as template.(A) Organization of the hbp genes and the location of the HbpR binding sites (UAS, upstream activating sequences) in front of the PC and PD promoters. HbpR domains are depicted to scale according to the predictions by Jaspers et al [7]. (B) to (E) Fitting used swiss-model and was performed on XylR A-domain PDB coordinates as calculated by Devos et al [22]. (B) Ribbon model of HbpR A-domain residues 11–209, with predicted coils, alpha-helices and beta-sheets indicated. (C) Superposition of the predicted HbpR and XylR A-domains in the same configuration as A. (D) Tertiary structure model of HbpR A-domain with calculated molecular surface at 1.4Å and 40% transparency, in order to see the helical, coil and sheets. Model turned into a position which enables visualization of the proposed tunnel entry (b). C-terminal end of coil ending the A-domain indicated with an arrow at (a). Pinkish region in the centre of the A-domain illustrates a predicted cavity within the A-domain. (E), as C but now for the XylR A-domain, with exception of the ten most C-terminal residues, which otherwise are predicted to occlude the tunnel.

Mentions: The goal of the current work was to identify the residues critical for effector-mediated triggering in the HbpR protein from P. azelaica [7], [17]. In its native host, the hbpR gene product regulates expression from two promoters, called the PC and PD promoters, which are located in front of two small operons (hbpCA and hbpD) encoding the enzymes for initial steps of 2-hydroxybiphenyl (2-HBP) degradation (Fig. 1A) [24]. The hbpR gene is located directly upstream of and is divergently oriented from the hbpCA genes. HbpR displays only 37% amino acid sequence identity with XylR, and in contrast to XylR and DmpR, is responsive to biaromatic compounds such as 2-HBP, 2,2′-dihydroxybiphenyl, 2-aminobiphenyl and 2-hydroxydiphenylmethane [7]. XylR and HbpR display detectable but little crossbinding to each other's DNA binding sites although hybrid promoters can be produced that are activated by both XylR and HbpR in the same cell [25]. In contrast to XylR and DmpR, the Q-linker of HbpR is shorter and A-domain deletions of HbpR result in a constitutive repressor protein [17]. Since such A-domain deletions are made without any protein structure basis, it is possible that they accidentally produce different effects in HbpR and XylR or DmpR.


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)

Overview of the hbp regulatory system and tertiary structure modeling of the HbpR A-domain using a previously established XylR model as template.(A) Organization of the hbp genes and the location of the HbpR binding sites (UAS, upstream activating sequences) in front of the PC and PD promoters. HbpR domains are depicted to scale according to the predictions by Jaspers et al [7]. (B) to (E) Fitting used swiss-model and was performed on XylR A-domain PDB coordinates as calculated by Devos et al [22]. (B) Ribbon model of HbpR A-domain residues 11–209, with predicted coils, alpha-helices and beta-sheets indicated. (C) Superposition of the predicted HbpR and XylR A-domains in the same configuration as A. (D) Tertiary structure model of HbpR A-domain with calculated molecular surface at 1.4Å and 40% transparency, in order to see the helical, coil and sheets. Model turned into a position which enables visualization of the proposed tunnel entry (b). C-terminal end of coil ending the A-domain indicated with an arrow at (a). Pinkish region in the centre of the A-domain illustrates a predicted cavity within the A-domain. (E), as C but now for the XylR A-domain, with exception of the ten most C-terminal residues, which otherwise are predicted to occlude the tunnel.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3040749&req=5

pone-0016539-g001: Overview of the hbp regulatory system and tertiary structure modeling of the HbpR A-domain using a previously established XylR model as template.(A) Organization of the hbp genes and the location of the HbpR binding sites (UAS, upstream activating sequences) in front of the PC and PD promoters. HbpR domains are depicted to scale according to the predictions by Jaspers et al [7]. (B) to (E) Fitting used swiss-model and was performed on XylR A-domain PDB coordinates as calculated by Devos et al [22]. (B) Ribbon model of HbpR A-domain residues 11–209, with predicted coils, alpha-helices and beta-sheets indicated. (C) Superposition of the predicted HbpR and XylR A-domains in the same configuration as A. (D) Tertiary structure model of HbpR A-domain with calculated molecular surface at 1.4Å and 40% transparency, in order to see the helical, coil and sheets. Model turned into a position which enables visualization of the proposed tunnel entry (b). C-terminal end of coil ending the A-domain indicated with an arrow at (a). Pinkish region in the centre of the A-domain illustrates a predicted cavity within the A-domain. (E), as C but now for the XylR A-domain, with exception of the ten most C-terminal residues, which otherwise are predicted to occlude the tunnel.
Mentions: The goal of the current work was to identify the residues critical for effector-mediated triggering in the HbpR protein from P. azelaica [7], [17]. In its native host, the hbpR gene product regulates expression from two promoters, called the PC and PD promoters, which are located in front of two small operons (hbpCA and hbpD) encoding the enzymes for initial steps of 2-hydroxybiphenyl (2-HBP) degradation (Fig. 1A) [24]. The hbpR gene is located directly upstream of and is divergently oriented from the hbpCA genes. HbpR displays only 37% amino acid sequence identity with XylR, and in contrast to XylR and DmpR, is responsive to biaromatic compounds such as 2-HBP, 2,2′-dihydroxybiphenyl, 2-aminobiphenyl and 2-hydroxydiphenylmethane [7]. XylR and HbpR display detectable but little crossbinding to each other's DNA binding sites although hybrid promoters can be produced that are activated by both XylR and HbpR in the same cell [25]. In contrast to XylR and DmpR, the Q-linker of HbpR is shorter and A-domain deletions of HbpR result in a constitutive repressor protein [17]. Since such A-domain deletions are made without any protein structure basis, it is possible that they accidentally produce different effects in HbpR and XylR or DmpR.

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