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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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Gentamicin interrupts IcaR and DNA interaction. (A) The effects of antibiotics to the IcaR binding to ica operator in EMSA experiment. Only two aminoglycosides are capable of interfering the DNA binding, whereas gentamicin inhibited the binding to a significantly greater extent than streptomycin. The other four antibiotics kanamycin, tetracycline, ampicillin and chloramphenicol did not show significant effect on the IcaR–DNA binding. (B) Possible effect of gentamicin on the IcaR binding to ica operator. A negatively charged cavity was observed in each IcaR monomer, near the dimer interface. Binding of gentamicin to this cavity may result in conformation changes that lead to a larger separation of the N-terminal domains. (C) A schematic diagram of the putative antibiotics-binding cavity. The cavity is located at the dimer interface and surrounded by helices α5, α6, α7 and α8 of one monomer as well as α8′ and α9′ of the counter monomer. The α8–α9 loop has different conformations in the two crystal forms (Figure 1D), suggesting some functional flexibility.
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Figure 6: Gentamicin interrupts IcaR and DNA interaction. (A) The effects of antibiotics to the IcaR binding to ica operator in EMSA experiment. Only two aminoglycosides are capable of interfering the DNA binding, whereas gentamicin inhibited the binding to a significantly greater extent than streptomycin. The other four antibiotics kanamycin, tetracycline, ampicillin and chloramphenicol did not show significant effect on the IcaR–DNA binding. (B) Possible effect of gentamicin on the IcaR binding to ica operator. A negatively charged cavity was observed in each IcaR monomer, near the dimer interface. Binding of gentamicin to this cavity may result in conformation changes that lead to a larger separation of the N-terminal domains. (C) A schematic diagram of the putative antibiotics-binding cavity. The cavity is located at the dimer interface and surrounded by helices α5, α6, α7 and α8 of one monomer as well as α8′ and α9′ of the counter monomer. The α8–α9 loop has different conformations in the two crystal forms (Figure 1D), suggesting some functional flexibility.

Mentions: As shown in Figure 6A, only two of the six antibiotics were effective: streptomycin and gentamicin, both belonging to the aminoglycoside class. Gentamicin appeared to inhibit the DNA binding to a significantly greater extent than streptomycin, partially due to the higher molar concentration of gentamicin. Comparison of their chemical structures suggests that gentamicin is more positively charged and less polar than the other antibiotics. Identical results were obtained with the inclusion of spermine, a polycation polyamine, in the EMSA (data not shown), which ruled out nonspecific effect of the positive charges. Furthermore, in order to investigate interactions of IcaR and DNA with the antibiotics, a series of ITC experiments were carried out for gentamicin, streptomycin and kanamycin. As shown in Table 2, gentamicin and streptomycin both bound to IcaR, although not with great strength. The stoichiometry showed further similarity of IcaR to QacR (16). It suggests that only one drug molecule is bound by each dimer, despite the existence of two binding pockets. In contrast, binding to IcaR was not observed for kanamycin, which on the other hand interacted with DNA (see also Figure S4 in the Supplementary Material). Although we cannot exclude the possibility of binding to DNA rather than the repressor, it is also likely that the two aminoglycoside antibiotics interacted with IcaR in a similar way as the drugs with TetR and QacR.Figure 6.


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)

Gentamicin interrupts IcaR and DNA interaction. (A) The effects of antibiotics to the IcaR binding to ica operator in EMSA experiment. Only two aminoglycosides are capable of interfering the DNA binding, whereas gentamicin inhibited the binding to a significantly greater extent than streptomycin. The other four antibiotics kanamycin, tetracycline, ampicillin and chloramphenicol did not show significant effect on the IcaR–DNA binding. (B) Possible effect of gentamicin on the IcaR binding to ica operator. A negatively charged cavity was observed in each IcaR monomer, near the dimer interface. Binding of gentamicin to this cavity may result in conformation changes that lead to a larger separation of the N-terminal domains. (C) A schematic diagram of the putative antibiotics-binding cavity. The cavity is located at the dimer interface and surrounded by helices α5, α6, α7 and α8 of one monomer as well as α8′ and α9′ of the counter monomer. The α8–α9 loop has different conformations in the two crystal forms (Figure 1D), suggesting some functional flexibility.
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Related In: Results  -  Collection

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Figure 6: Gentamicin interrupts IcaR and DNA interaction. (A) The effects of antibiotics to the IcaR binding to ica operator in EMSA experiment. Only two aminoglycosides are capable of interfering the DNA binding, whereas gentamicin inhibited the binding to a significantly greater extent than streptomycin. The other four antibiotics kanamycin, tetracycline, ampicillin and chloramphenicol did not show significant effect on the IcaR–DNA binding. (B) Possible effect of gentamicin on the IcaR binding to ica operator. A negatively charged cavity was observed in each IcaR monomer, near the dimer interface. Binding of gentamicin to this cavity may result in conformation changes that lead to a larger separation of the N-terminal domains. (C) A schematic diagram of the putative antibiotics-binding cavity. The cavity is located at the dimer interface and surrounded by helices α5, α6, α7 and α8 of one monomer as well as α8′ and α9′ of the counter monomer. The α8–α9 loop has different conformations in the two crystal forms (Figure 1D), suggesting some functional flexibility.
Mentions: As shown in Figure 6A, only two of the six antibiotics were effective: streptomycin and gentamicin, both belonging to the aminoglycoside class. Gentamicin appeared to inhibit the DNA binding to a significantly greater extent than streptomycin, partially due to the higher molar concentration of gentamicin. Comparison of their chemical structures suggests that gentamicin is more positively charged and less polar than the other antibiotics. Identical results were obtained with the inclusion of spermine, a polycation polyamine, in the EMSA (data not shown), which ruled out nonspecific effect of the positive charges. Furthermore, in order to investigate interactions of IcaR and DNA with the antibiotics, a series of ITC experiments were carried out for gentamicin, streptomycin and kanamycin. As shown in Table 2, gentamicin and streptomycin both bound to IcaR, although not with great strength. The stoichiometry showed further similarity of IcaR to QacR (16). It suggests that only one drug molecule is bound by each dimer, despite the existence of two binding pockets. In contrast, binding to IcaR was not observed for kanamycin, which on the other hand interacted with DNA (see also Figure S4 in the Supplementary Material). Although we cannot exclude the possibility of binding to DNA rather than the repressor, it is also likely that the two aminoglycoside antibiotics interacted with IcaR in a similar way as the drugs with TetR and QacR.Figure 6.

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