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Conformational and thermodynamic changes of the repressor/DNA operator complex upon monomerization shed new light on regulation mechanisms of bacterial resistance against beta-lactam antibiotics.

Boudet J, Duval V, Van Melckebeke H, Blackledge M, Amoroso A, Joris B, Simorre JP - Nucleic Acids Res. (2007)

Bottom Line: To understand the loss of the high DNA affinity of the truncated repressor, we have determined the different dissociation constants of the system and solved the solution structure of the B. licheniformis monomeric repressor complexed to the semi-operating sequence OP1 of blaP (1/2OP1blaP) by using a de novo docking approach based on inter-molecular nuclear Overhauser effects and chemical-shift differences measured on each macromolecular partner.Although the N-terminal domain of the repressor is not subject to internal structural rearrangements upon DNA binding, the molecules adopt a tertiary conformation different from the crystallographic operator-repressor dimer complex, leading to a 30 degrees rotation of the monomer with respect to a central axis extended across the DNA.These results open new insights for the repression and induction mechanisms of bacterial resistance to beta-lactams.

View Article: PubMed Central - PubMed

Affiliation: Institut de Biologie Structurale Jean-Pierre Ebel CEA-CNRS-UJF, 41 Avenue Jules Horowitz, 38027 Grenoble Cedex 1, France.

ABSTRACT
In absence of beta-lactam antibiotics, BlaI and MecI homodimeric repressors negatively control the expression of genes involved in beta-lactam resistance in Bacillus licheniformis and in Staphylococcus aureus. Subsequently to beta-lactam presence, BlaI/MecI is inactivated by a single-point proteolysis that separates its N-terminal DNA-binding domain to its C-terminal domain responsible for its dimerization. Concomitantly to this proteolysis, the truncated repressor acquires a low affinity for its DNA target that explains the expression of the structural gene for resistance. To understand the loss of the high DNA affinity of the truncated repressor, we have determined the different dissociation constants of the system and solved the solution structure of the B. licheniformis monomeric repressor complexed to the semi-operating sequence OP1 of blaP (1/2OP1blaP) by using a de novo docking approach based on inter-molecular nuclear Overhauser effects and chemical-shift differences measured on each macromolecular partner. Although the N-terminal domain of the repressor is not subject to internal structural rearrangements upon DNA binding, the molecules adopt a tertiary conformation different from the crystallographic operator-repressor dimer complex, leading to a 30 degrees rotation of the monomer with respect to a central axis extended across the DNA. These results open new insights for the repression and induction mechanisms of bacterial resistance to beta-lactams.

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Determination of dissociation constants using chemical shift titration obtained by NMR. Weighted sum of the 1H,15N chemical shift variation measured for the protein on 15N-HSQC is plotted as a function of the DNA/protein ratio for the different complexes. (A) B. licheniformis BlaI-NTD with the B. licheniformis 1/2-operator of the blaP gene and (B) with the S. aureus 1/2-operator of the mecAgene, (C) S. aureus MecI-NTD with the 1/2-operator of the mecA gene and (D) with the B. licheniformis 1/2-operator of the blaP gene. By fitting the curves, the different dissociation constants Kd were obtained: Kd1 [BLBlaI-NTD][1/2OP1blaP] = 190 ± 50 μM, Kd2 [BLBlaI-NTD][1/2OPmecA] = 170 ± 50 μM, Kd3 [SAMecI-NTD][1/2OP1blaP] = 860 ± 80 μM and Kd4 [SAMecI-NTD][1/2OPmecA] = 160 ± 60 μM.
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Figure 2: Determination of dissociation constants using chemical shift titration obtained by NMR. Weighted sum of the 1H,15N chemical shift variation measured for the protein on 15N-HSQC is plotted as a function of the DNA/protein ratio for the different complexes. (A) B. licheniformis BlaI-NTD with the B. licheniformis 1/2-operator of the blaP gene and (B) with the S. aureus 1/2-operator of the mecAgene, (C) S. aureus MecI-NTD with the 1/2-operator of the mecA gene and (D) with the B. licheniformis 1/2-operator of the blaP gene. By fitting the curves, the different dissociation constants Kd were obtained: Kd1 [BLBlaI-NTD][1/2OP1blaP] = 190 ± 50 μM, Kd2 [BLBlaI-NTD][1/2OPmecA] = 170 ± 50 μM, Kd3 [SAMecI-NTD][1/2OP1blaP] = 860 ± 80 μM and Kd4 [SAMecI-NTD][1/2OPmecA] = 160 ± 60 μM.

Mentions: Titration of 15N-labeled BLBlaI or SAMecI truncated repressor with progressive amounts of unlabeled DNA half-dyads was performed by monitoring changes in 1H-15N HSQC spectra. Significant chemical-shift changes for correlation peaks in the 1H-15N HSQC spectra were observed when the 1/2OP1blaP and the 1/2OPmecA were added to protein samples. [DNA]/[Protein] molar ratios were varied from 0 to 10 for [BLBlaI-NTD]/[1/2OP1blaP], [BLBlaI-NTD]/[1/2OPmecA], [SAMecI-NTD]/[1/2OPmecA] and from 0 to 50 for [SAMecI-NTD]/[1/2OP1blaP]. The chemical-shift changes observed upon complexes formation reached a plateau over 7 [DNA]/[protein] molar ratios for [BLBlaI-NTD]/[1/2OP1blaP], [BLBlaI-NTD]/[1/2OPmecA], [SAMecI-NTD]/[1/2OPmecA] and a plateau over 30 for [SAMecI-NTD]/[1/2OP1blaP] (Figure 2). These results confirm the formation of stable intermolecular interactions with saturating DNA quantities. At any point during the titration, specific single correlation peaks were detected, suggesting that the truncated monomeric repressors/semi-operators complexes are in fast exchange regarding the NMR time scale. Only well-resolved correlation peaks with a chemical-shift cut-off equal or superior to 0.03 ppm were used in the four experiments. The affinity constants were calculated from a non-linear fit of the significant chemical-shift variations versus [DNA]/[protein] ratio using equation given by Morton et al. (25). Titration data were analyzed assuming that the observed chemical-shift perturbation is a weighted average between the two extreme values corresponding to the free (Δδ = 0) and the bound state (Δδ = Δδmax) so that:where [Op]0 and [I]0 are the total molar concentrations of DNA operator and protein. Statistical analysis using Monte-Carlo simulations were used to evaluate the uncertainty of the fitted parameters.Figure 2.


Conformational and thermodynamic changes of the repressor/DNA operator complex upon monomerization shed new light on regulation mechanisms of bacterial resistance against beta-lactam antibiotics.

Boudet J, Duval V, Van Melckebeke H, Blackledge M, Amoroso A, Joris B, Simorre JP - Nucleic Acids Res. (2007)

Determination of dissociation constants using chemical shift titration obtained by NMR. Weighted sum of the 1H,15N chemical shift variation measured for the protein on 15N-HSQC is plotted as a function of the DNA/protein ratio for the different complexes. (A) B. licheniformis BlaI-NTD with the B. licheniformis 1/2-operator of the blaP gene and (B) with the S. aureus 1/2-operator of the mecAgene, (C) S. aureus MecI-NTD with the 1/2-operator of the mecA gene and (D) with the B. licheniformis 1/2-operator of the blaP gene. By fitting the curves, the different dissociation constants Kd were obtained: Kd1 [BLBlaI-NTD][1/2OP1blaP] = 190 ± 50 μM, Kd2 [BLBlaI-NTD][1/2OPmecA] = 170 ± 50 μM, Kd3 [SAMecI-NTD][1/2OP1blaP] = 860 ± 80 μM and Kd4 [SAMecI-NTD][1/2OPmecA] = 160 ± 60 μM.
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Figure 2: Determination of dissociation constants using chemical shift titration obtained by NMR. Weighted sum of the 1H,15N chemical shift variation measured for the protein on 15N-HSQC is plotted as a function of the DNA/protein ratio for the different complexes. (A) B. licheniformis BlaI-NTD with the B. licheniformis 1/2-operator of the blaP gene and (B) with the S. aureus 1/2-operator of the mecAgene, (C) S. aureus MecI-NTD with the 1/2-operator of the mecA gene and (D) with the B. licheniformis 1/2-operator of the blaP gene. By fitting the curves, the different dissociation constants Kd were obtained: Kd1 [BLBlaI-NTD][1/2OP1blaP] = 190 ± 50 μM, Kd2 [BLBlaI-NTD][1/2OPmecA] = 170 ± 50 μM, Kd3 [SAMecI-NTD][1/2OP1blaP] = 860 ± 80 μM and Kd4 [SAMecI-NTD][1/2OPmecA] = 160 ± 60 μM.
Mentions: Titration of 15N-labeled BLBlaI or SAMecI truncated repressor with progressive amounts of unlabeled DNA half-dyads was performed by monitoring changes in 1H-15N HSQC spectra. Significant chemical-shift changes for correlation peaks in the 1H-15N HSQC spectra were observed when the 1/2OP1blaP and the 1/2OPmecA were added to protein samples. [DNA]/[Protein] molar ratios were varied from 0 to 10 for [BLBlaI-NTD]/[1/2OP1blaP], [BLBlaI-NTD]/[1/2OPmecA], [SAMecI-NTD]/[1/2OPmecA] and from 0 to 50 for [SAMecI-NTD]/[1/2OP1blaP]. The chemical-shift changes observed upon complexes formation reached a plateau over 7 [DNA]/[protein] molar ratios for [BLBlaI-NTD]/[1/2OP1blaP], [BLBlaI-NTD]/[1/2OPmecA], [SAMecI-NTD]/[1/2OPmecA] and a plateau over 30 for [SAMecI-NTD]/[1/2OP1blaP] (Figure 2). These results confirm the formation of stable intermolecular interactions with saturating DNA quantities. At any point during the titration, specific single correlation peaks were detected, suggesting that the truncated monomeric repressors/semi-operators complexes are in fast exchange regarding the NMR time scale. Only well-resolved correlation peaks with a chemical-shift cut-off equal or superior to 0.03 ppm were used in the four experiments. The affinity constants were calculated from a non-linear fit of the significant chemical-shift variations versus [DNA]/[protein] ratio using equation given by Morton et al. (25). Titration data were analyzed assuming that the observed chemical-shift perturbation is a weighted average between the two extreme values corresponding to the free (Δδ = 0) and the bound state (Δδ = Δδmax) so that:where [Op]0 and [I]0 are the total molar concentrations of DNA operator and protein. Statistical analysis using Monte-Carlo simulations were used to evaluate the uncertainty of the fitted parameters.Figure 2.

Bottom Line: To understand the loss of the high DNA affinity of the truncated repressor, we have determined the different dissociation constants of the system and solved the solution structure of the B. licheniformis monomeric repressor complexed to the semi-operating sequence OP1 of blaP (1/2OP1blaP) by using a de novo docking approach based on inter-molecular nuclear Overhauser effects and chemical-shift differences measured on each macromolecular partner.Although the N-terminal domain of the repressor is not subject to internal structural rearrangements upon DNA binding, the molecules adopt a tertiary conformation different from the crystallographic operator-repressor dimer complex, leading to a 30 degrees rotation of the monomer with respect to a central axis extended across the DNA.These results open new insights for the repression and induction mechanisms of bacterial resistance to beta-lactams.

View Article: PubMed Central - PubMed

Affiliation: Institut de Biologie Structurale Jean-Pierre Ebel CEA-CNRS-UJF, 41 Avenue Jules Horowitz, 38027 Grenoble Cedex 1, France.

ABSTRACT
In absence of beta-lactam antibiotics, BlaI and MecI homodimeric repressors negatively control the expression of genes involved in beta-lactam resistance in Bacillus licheniformis and in Staphylococcus aureus. Subsequently to beta-lactam presence, BlaI/MecI is inactivated by a single-point proteolysis that separates its N-terminal DNA-binding domain to its C-terminal domain responsible for its dimerization. Concomitantly to this proteolysis, the truncated repressor acquires a low affinity for its DNA target that explains the expression of the structural gene for resistance. To understand the loss of the high DNA affinity of the truncated repressor, we have determined the different dissociation constants of the system and solved the solution structure of the B. licheniformis monomeric repressor complexed to the semi-operating sequence OP1 of blaP (1/2OP1blaP) by using a de novo docking approach based on inter-molecular nuclear Overhauser effects and chemical-shift differences measured on each macromolecular partner. Although the N-terminal domain of the repressor is not subject to internal structural rearrangements upon DNA binding, the molecules adopt a tertiary conformation different from the crystallographic operator-repressor dimer complex, leading to a 30 degrees rotation of the monomer with respect to a central axis extended across the DNA. These results open new insights for the repression and induction mechanisms of bacterial resistance to beta-lactams.

Show MeSH
Related in: MedlinePlus