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Crystal structure of IcaR, a repressor of the TetR family implicated in biofilm formation in Staphylococcus epidermidis.

Jeng WY, Ko TP, Liu CI, Guo RT, Liu CL, Shr HL, Wang AH - Nucleic Acids Res. (2008)

Bottom Line: The C-terminal domain shows a complementary surface charge distribution about the dyad axis, ideal for efficient and specific dimer formation.Computer modeling based on the known DNA-complex structure of QacR and site-specific mutagenesis experiments showed that direct protein-DNA interactions are mostly conserved, but with slight variations for recognizing the different sequences.By interfering with the binding of IcaR to DNA, aminoglycoside gentamicin and other antibiotics may activate the icaADBC genes and elicit biofilm production in S. epidermidis, and likely S. aureus, as a defense mechanism.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biological Chemistry, Taipei, Taiwan.

ABSTRACT
Expression of the gene cluster icaADBC is necessary for biofilm production in Staphylococcus epidermidis. The ica operon is negatively controlled by the repressor IcaR. Here, the crystal structure of IcaR was determined and the refined structure revealed a homodimer comprising entirely alpha-helices, typical of the tetracycline repressor protein family for gene regulations. The N-terminal domain contains a conserved helix-turn-helix DNA-binding motif with some conformational variations, indicating flexibility in this region. The C-terminal domain shows a complementary surface charge distribution about the dyad axis, ideal for efficient and specific dimer formation. The results of the electrophoretic mobility shift assay and isothermal titration calorimetry suggested that a 28 bp core segment of the ica operator is implicated in the cooperative binding of two IcaR dimers on opposite sides of the duplex DNA. Computer modeling based on the known DNA-complex structure of QacR and site-specific mutagenesis experiments showed that direct protein-DNA interactions are mostly conserved, but with slight variations for recognizing the different sequences. By interfering with the binding of IcaR to DNA, aminoglycoside gentamicin and other antibiotics may activate the icaADBC genes and elicit biofilm production in S. epidermidis, and likely S. aureus, as a defense mechanism.

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IcaR–DNA binding determined by ITC. Representative plots from an ITC experiment are shown with raw data in the upper panel and curve fit in the lower panel. The IcaR protein was titrated into the reaction cell containing the 28E dsDNA. Note that the first addition was 3/8 the volume of the other additions. The first two data points were not used in the fitting. Thermodynamic values obtained from the curve fit are: ΔS = 84.3 cal/mol K, ΔH = 13.8 ± 0.2 kcal/mol, KB = 2.0 ± 0.8 × 108 M–1, N = 2.18 ± 0.01. N is the stoichiometry of bound IcaR dimer per dsDNA.
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Figure 3: IcaR–DNA binding determined by ITC. Representative plots from an ITC experiment are shown with raw data in the upper panel and curve fit in the lower panel. The IcaR protein was titrated into the reaction cell containing the 28E dsDNA. Note that the first addition was 3/8 the volume of the other additions. The first two data points were not used in the fitting. Thermodynamic values obtained from the curve fit are: ΔS = 84.3 cal/mol K, ΔH = 13.8 ± 0.2 kcal/mol, KB = 2.0 ± 0.8 × 108 M–1, N = 2.18 ± 0.01. N is the stoichiometry of bound IcaR dimer per dsDNA.

Mentions: In Figure 2D, the EMSA experiments demonstrated that by incubating the double-stranded 28E DNA with the IcaR monomers at twice the molar equivalent, i.e. with a ratio of 1:2, the mobility of some fraction of the DNA segments was reduced while a significant fraction of the DNA remained unaffected. When the molar ratio was 1:4, all but a small fraction of the DNA was reduced in the gel-shift mobility. These results suggest that IcaR binds to the ica operator by a ratio of 1:4 in a highly cooperative manner. In other words, each operator DNA segment either binds simultaneously to two IcaR homodimers, or it does not bind. Cooperative binding was also observed in the ITC experiments. Here, the stoichiometry was unambiguously determined as two IcaR dimers to one DNA operator (see Figure 3 for details). Similar observations of cooperativity have been reported for QacR, which also binds to DNA with a 1:4 molar ratio, and EthR, which has a ratio of 1:8 (17,20). However, further increase of the molar ratio of dsDNA IcaR to 1:8 did not change the DNA mobility any further (Figure 2D). The length of the 28 bp ica operator DNA core segment also excludes the possibility of binding more than a pair of IcaR dimers. As revealed in the crystal structure of QacR–DNA complex, the two dimeric repressors bind to the DNA on opposite sides, and with overlapping regions of the encompassed base pairs. The cooperativity of the binding is mediated by the protein–DNA–protein interactions, rather than direct interactions between the repressor molecules. We believe that the same DNA-binding mechanism of QacR applies to IcaR as well.Figure 3.


Crystal structure of IcaR, a repressor of the TetR family implicated in biofilm formation in Staphylococcus epidermidis.

Jeng WY, Ko TP, Liu CI, Guo RT, Liu CL, Shr HL, Wang AH - Nucleic Acids Res. (2008)

IcaR–DNA binding determined by ITC. Representative plots from an ITC experiment are shown with raw data in the upper panel and curve fit in the lower panel. The IcaR protein was titrated into the reaction cell containing the 28E dsDNA. Note that the first addition was 3/8 the volume of the other additions. The first two data points were not used in the fitting. Thermodynamic values obtained from the curve fit are: ΔS = 84.3 cal/mol K, ΔH = 13.8 ± 0.2 kcal/mol, KB = 2.0 ± 0.8 × 108 M–1, N = 2.18 ± 0.01. N is the stoichiometry of bound IcaR dimer per dsDNA.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 3: IcaR–DNA binding determined by ITC. Representative plots from an ITC experiment are shown with raw data in the upper panel and curve fit in the lower panel. The IcaR protein was titrated into the reaction cell containing the 28E dsDNA. Note that the first addition was 3/8 the volume of the other additions. The first two data points were not used in the fitting. Thermodynamic values obtained from the curve fit are: ΔS = 84.3 cal/mol K, ΔH = 13.8 ± 0.2 kcal/mol, KB = 2.0 ± 0.8 × 108 M–1, N = 2.18 ± 0.01. N is the stoichiometry of bound IcaR dimer per dsDNA.
Mentions: In Figure 2D, the EMSA experiments demonstrated that by incubating the double-stranded 28E DNA with the IcaR monomers at twice the molar equivalent, i.e. with a ratio of 1:2, the mobility of some fraction of the DNA segments was reduced while a significant fraction of the DNA remained unaffected. When the molar ratio was 1:4, all but a small fraction of the DNA was reduced in the gel-shift mobility. These results suggest that IcaR binds to the ica operator by a ratio of 1:4 in a highly cooperative manner. In other words, each operator DNA segment either binds simultaneously to two IcaR homodimers, or it does not bind. Cooperative binding was also observed in the ITC experiments. Here, the stoichiometry was unambiguously determined as two IcaR dimers to one DNA operator (see Figure 3 for details). Similar observations of cooperativity have been reported for QacR, which also binds to DNA with a 1:4 molar ratio, and EthR, which has a ratio of 1:8 (17,20). However, further increase of the molar ratio of dsDNA IcaR to 1:8 did not change the DNA mobility any further (Figure 2D). The length of the 28 bp ica operator DNA core segment also excludes the possibility of binding more than a pair of IcaR dimers. As revealed in the crystal structure of QacR–DNA complex, the two dimeric repressors bind to the DNA on opposite sides, and with overlapping regions of the encompassed base pairs. The cooperativity of the binding is mediated by the protein–DNA–protein interactions, rather than direct interactions between the repressor molecules. We believe that the same DNA-binding mechanism of QacR applies to IcaR as well.Figure 3.

Bottom Line: The C-terminal domain shows a complementary surface charge distribution about the dyad axis, ideal for efficient and specific dimer formation.Computer modeling based on the known DNA-complex structure of QacR and site-specific mutagenesis experiments showed that direct protein-DNA interactions are mostly conserved, but with slight variations for recognizing the different sequences.By interfering with the binding of IcaR to DNA, aminoglycoside gentamicin and other antibiotics may activate the icaADBC genes and elicit biofilm production in S. epidermidis, and likely S. aureus, as a defense mechanism.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biological Chemistry, Taipei, Taiwan.

ABSTRACT
Expression of the gene cluster icaADBC is necessary for biofilm production in Staphylococcus epidermidis. The ica operon is negatively controlled by the repressor IcaR. Here, the crystal structure of IcaR was determined and the refined structure revealed a homodimer comprising entirely alpha-helices, typical of the tetracycline repressor protein family for gene regulations. The N-terminal domain contains a conserved helix-turn-helix DNA-binding motif with some conformational variations, indicating flexibility in this region. The C-terminal domain shows a complementary surface charge distribution about the dyad axis, ideal for efficient and specific dimer formation. The results of the electrophoretic mobility shift assay and isothermal titration calorimetry suggested that a 28 bp core segment of the ica operator is implicated in the cooperative binding of two IcaR dimers on opposite sides of the duplex DNA. Computer modeling based on the known DNA-complex structure of QacR and site-specific mutagenesis experiments showed that direct protein-DNA interactions are mostly conserved, but with slight variations for recognizing the different sequences. By interfering with the binding of IcaR to DNA, aminoglycoside gentamicin and other antibiotics may activate the icaADBC genes and elicit biofilm production in S. epidermidis, and likely S. aureus, as a defense mechanism.

Show MeSH
Related in: MedlinePlus