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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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Details of the modeled tertiary structure of the HbpR A-domain, showing amino acid residues that were mutated in this study and the region onto which 2-HBP is predicted to be bound.(A) Results of 1000 iterations of 2-HBP (in red) docking calculations using gramm onto the predicted HbpR A-domain protein surface, whilst indicating the position of residues altered to Phe. (B) Close-up of the same, but without the docked 2-HBP positions. (C) as for B, now highlighting the other changed residues. (D) Van der Waals-filled model slightly turned compared to A, in order to indicate the region of 2-HBP docked molecules. (E), as B, but with 2-HBP docked positions. (F) Turned van der Waals-filled model showing the tunnel from the other side of the entry.
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pone-0016539-g002: Details of the modeled tertiary structure of the HbpR A-domain, showing amino acid residues that were mutated in this study and the region onto which 2-HBP is predicted to be bound.(A) Results of 1000 iterations of 2-HBP (in red) docking calculations using gramm onto the predicted HbpR A-domain protein surface, whilst indicating the position of residues altered to Phe. (B) Close-up of the same, but without the docked 2-HBP positions. (C) as for B, now highlighting the other changed residues. (D) Van der Waals-filled model slightly turned compared to A, in order to indicate the region of 2-HBP docked molecules. (E), as B, but with 2-HBP docked positions. (F) Turned van der Waals-filled model showing the tunnel from the other side of the entry.

Mentions: The A-domain model for HbpR was then used as a template to predict the possible sites of interaction with its effector 2-HBP (Fig. 2A, D, E). Potential sites for 2-HBP interaction were calculated by using the program gramm, which uses Fast Fourier transformation to predict the energetically most favorable matches of a ligand on the modeled protein surface [28]. Interestingly, gramm calculations predicted that there would be an ‘interface’ region most favorable for interaction with 2-HBP rather than a single residue or active site, which upon closer inspection of the model seemed to provide a cavity (Fig. 2A). Among one thousand iterations, the program predicted almost exclusively interactions in this particular region. A number of amino acid residues such as E184 were located in this region (Fig. 2A, B), which upon mutation in XylR had been demonstrated to broaden effector-mediated induction [29]. In addition, a similar region had been predicted from the XylR A-domain model to be of potential interest to effector binding, even though few mutations had been generated in that part of the protein [22]. The main hypothesis in this work was therefore that this interface region would be critical for 2-HBP-mediated triggering of HbpR activation.


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)

Details of the modeled tertiary structure of the HbpR A-domain, showing amino acid residues that were mutated in this study and the region onto which 2-HBP is predicted to be bound.(A) Results of 1000 iterations of 2-HBP (in red) docking calculations using gramm onto the predicted HbpR A-domain protein surface, whilst indicating the position of residues altered to Phe. (B) Close-up of the same, but without the docked 2-HBP positions. (C) as for B, now highlighting the other changed residues. (D) Van der Waals-filled model slightly turned compared to A, in order to indicate the region of 2-HBP docked molecules. (E), as B, but with 2-HBP docked positions. (F) Turned van der Waals-filled model showing the tunnel from the other side of the entry.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0016539-g002: Details of the modeled tertiary structure of the HbpR A-domain, showing amino acid residues that were mutated in this study and the region onto which 2-HBP is predicted to be bound.(A) Results of 1000 iterations of 2-HBP (in red) docking calculations using gramm onto the predicted HbpR A-domain protein surface, whilst indicating the position of residues altered to Phe. (B) Close-up of the same, but without the docked 2-HBP positions. (C) as for B, now highlighting the other changed residues. (D) Van der Waals-filled model slightly turned compared to A, in order to indicate the region of 2-HBP docked molecules. (E), as B, but with 2-HBP docked positions. (F) Turned van der Waals-filled model showing the tunnel from the other side of the entry.
Mentions: The A-domain model for HbpR was then used as a template to predict the possible sites of interaction with its effector 2-HBP (Fig. 2A, D, E). Potential sites for 2-HBP interaction were calculated by using the program gramm, which uses Fast Fourier transformation to predict the energetically most favorable matches of a ligand on the modeled protein surface [28]. Interestingly, gramm calculations predicted that there would be an ‘interface’ region most favorable for interaction with 2-HBP rather than a single residue or active site, which upon closer inspection of the model seemed to provide a cavity (Fig. 2A). Among one thousand iterations, the program predicted almost exclusively interactions in this particular region. A number of amino acid residues such as E184 were located in this region (Fig. 2A, B), which upon mutation in XylR had been demonstrated to broaden effector-mediated induction [29]. In addition, a similar region had been predicted from the XylR A-domain model to be of potential interest to effector binding, even though few mutations had been generated in that part of the protein [22]. The main hypothesis in this work was therefore that this interface region would be critical for 2-HBP-mediated triggering of HbpR activation.

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