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A bacterial antirepressor with SH3 domain topology mimics operator DNA in sequestering the repressor DNA recognition helix.

León E, Navarro-Avilés G, Santiveri CM, Flores-Flores C, Rico M, González C, Murillo FJ, Elías-Arnanz M, Jiménez MA, Padmanabhan S - Nucleic Acids Res. (2010)

Bottom Line: Direct targeting of critical DNA-binding elements of a repressor by its cognate antirepressor is an effective means to sequester the repressor and remove a transcription initiation block.CarA and CarH repress the carB operon in the dark.Our findings uncover an unprecedented use of the SH3 domain-like fold for protein-protein recognition whereby an antirepressor mimics operator DNA in sequestering the repressor DNA recognition helix to activate transcription.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Química-Física Rocasolano, Consejo Superior de Investigaciones Científicas (CSIC), Madrid, Spain.

ABSTRACT
Direct targeting of critical DNA-binding elements of a repressor by its cognate antirepressor is an effective means to sequester the repressor and remove a transcription initiation block. Structural descriptions for this, though often proposed for bacterial and phage repressor-antirepressor systems, are unavailable. Here, we describe the structural and functional basis of how the Myxococcus xanthus CarS antirepressor recognizes and neutralizes its cognate repressors to turn on a photo-inducible promoter. CarA and CarH repress the carB operon in the dark. CarS, produced in the light, physically interacts with the MerR-type winged-helix DNA-binding domain of these repressors leading to activation of carB. The NMR structure of CarS1, a functional CarS variant, reveals a five-stranded, antiparallel beta-sheet fold resembling SH3 domains, protein-protein interaction modules prevalent in eukaryotes but rare in prokaryotes. NMR studies and analysis of site-directed mutants in vivo and in vitro unveil a solvent-exposed hydrophobic pocket lined by acidic residues in CarS, where the CarA DNA recognition helix docks with high affinity in an atypical ligand-recognition mode for SH3 domains. Our findings uncover an unprecedented use of the SH3 domain-like fold for protein-protein recognition whereby an antirepressor mimics operator DNA in sequestering the repressor DNA recognition helix to activate transcription.

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Comparison of CarA α2 contacting CarS1 and DNA. (A) Top: CarS1-CarNt complex as ribbon models (left and centre) or with CarS1 in electrostatic surface representation (right). Charged side chains at the interface are shown as sticks and CarANt α2 is in purple. Bottom: CarANt in complex with the distorted pI half-site of operator DNA (10). CarANt is shown as in the models above. DNA is represented as a stick model with one strand in black, the other in gray, and phosphates as red spheres (left and centre), or by the electrostatic surface (right). (B) Close-up view of the CarANt α2-binding site in operator DNA (left) and in CarS1 (right). Charged side chains at the interface are displayed as sticks with α2 oriented as in the ribbon models on the left in (A).
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Figure 6: Comparison of CarA α2 contacting CarS1 and DNA. (A) Top: CarS1-CarNt complex as ribbon models (left and centre) or with CarS1 in electrostatic surface representation (right). Charged side chains at the interface are shown as sticks and CarANt α2 is in purple. Bottom: CarANt in complex with the distorted pI half-site of operator DNA (10). CarANt is shown as in the models above. DNA is represented as a stick model with one strand in black, the other in gray, and phosphates as red spheres (left and centre), or by the electrostatic surface (right). (B) Close-up view of the CarANt α2-binding site in operator DNA (left) and in CarS1 (right). Charged side chains at the interface are displayed as sticks with α2 oriented as in the ribbon models on the left in (A).

Mentions: Determinants necessary for tight binding to CarS1 as well as to operator DNA reside in the basic α2 (theoretical pI = 10.7). Hence, we compared α2 contacts with CarS1 versus those with DNA (Figure 6). CarANt conserves the winged-helix topology and most of the crucial DNA contacts of MerR-family proteins and, like the latter, operator-binding bends the DNA about the central dinucleotide (10). In our model of the complex, α2 occupies the major groove of this bent operator half-site, with its axis nearly perpendicular to that of DNA and burying 1150 Å2 of surface area at the interface. The operator and CarS1 surfaces that contact α2 are both polar and similar in size, and the relative locations and trajectories of negatively charged groups involved in the interactions are comparable (Figure 6). This is consistent with a critical role for electrostatic interactions in both cases, with complex formation being salt-sensitive and crucially dependent on specific charged residues. Thus, three out of the four R in α2 determine operator binding and their mutation relieves carB repression, while the fourth (R24′) has a marginal though observable effect (10). At least two of these (R24′ and R25′) are also critical in the binding to CarS1, as are specific D and E residues in the latter.Figure 6.


A bacterial antirepressor with SH3 domain topology mimics operator DNA in sequestering the repressor DNA recognition helix.

León E, Navarro-Avilés G, Santiveri CM, Flores-Flores C, Rico M, González C, Murillo FJ, Elías-Arnanz M, Jiménez MA, Padmanabhan S - Nucleic Acids Res. (2010)

Comparison of CarA α2 contacting CarS1 and DNA. (A) Top: CarS1-CarNt complex as ribbon models (left and centre) or with CarS1 in electrostatic surface representation (right). Charged side chains at the interface are shown as sticks and CarANt α2 is in purple. Bottom: CarANt in complex with the distorted pI half-site of operator DNA (10). CarANt is shown as in the models above. DNA is represented as a stick model with one strand in black, the other in gray, and phosphates as red spheres (left and centre), or by the electrostatic surface (right). (B) Close-up view of the CarANt α2-binding site in operator DNA (left) and in CarS1 (right). Charged side chains at the interface are displayed as sticks with α2 oriented as in the ribbon models on the left in (A).
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Related In: Results  -  Collection

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Figure 6: Comparison of CarA α2 contacting CarS1 and DNA. (A) Top: CarS1-CarNt complex as ribbon models (left and centre) or with CarS1 in electrostatic surface representation (right). Charged side chains at the interface are shown as sticks and CarANt α2 is in purple. Bottom: CarANt in complex with the distorted pI half-site of operator DNA (10). CarANt is shown as in the models above. DNA is represented as a stick model with one strand in black, the other in gray, and phosphates as red spheres (left and centre), or by the electrostatic surface (right). (B) Close-up view of the CarANt α2-binding site in operator DNA (left) and in CarS1 (right). Charged side chains at the interface are displayed as sticks with α2 oriented as in the ribbon models on the left in (A).
Mentions: Determinants necessary for tight binding to CarS1 as well as to operator DNA reside in the basic α2 (theoretical pI = 10.7). Hence, we compared α2 contacts with CarS1 versus those with DNA (Figure 6). CarANt conserves the winged-helix topology and most of the crucial DNA contacts of MerR-family proteins and, like the latter, operator-binding bends the DNA about the central dinucleotide (10). In our model of the complex, α2 occupies the major groove of this bent operator half-site, with its axis nearly perpendicular to that of DNA and burying 1150 Å2 of surface area at the interface. The operator and CarS1 surfaces that contact α2 are both polar and similar in size, and the relative locations and trajectories of negatively charged groups involved in the interactions are comparable (Figure 6). This is consistent with a critical role for electrostatic interactions in both cases, with complex formation being salt-sensitive and crucially dependent on specific charged residues. Thus, three out of the four R in α2 determine operator binding and their mutation relieves carB repression, while the fourth (R24′) has a marginal though observable effect (10). At least two of these (R24′ and R25′) are also critical in the binding to CarS1, as are specific D and E residues in the latter.Figure 6.

Bottom Line: Direct targeting of critical DNA-binding elements of a repressor by its cognate antirepressor is an effective means to sequester the repressor and remove a transcription initiation block.CarA and CarH repress the carB operon in the dark.Our findings uncover an unprecedented use of the SH3 domain-like fold for protein-protein recognition whereby an antirepressor mimics operator DNA in sequestering the repressor DNA recognition helix to activate transcription.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Química-Física Rocasolano, Consejo Superior de Investigaciones Científicas (CSIC), Madrid, Spain.

ABSTRACT
Direct targeting of critical DNA-binding elements of a repressor by its cognate antirepressor is an effective means to sequester the repressor and remove a transcription initiation block. Structural descriptions for this, though often proposed for bacterial and phage repressor-antirepressor systems, are unavailable. Here, we describe the structural and functional basis of how the Myxococcus xanthus CarS antirepressor recognizes and neutralizes its cognate repressors to turn on a photo-inducible promoter. CarA and CarH repress the carB operon in the dark. CarS, produced in the light, physically interacts with the MerR-type winged-helix DNA-binding domain of these repressors leading to activation of carB. The NMR structure of CarS1, a functional CarS variant, reveals a five-stranded, antiparallel beta-sheet fold resembling SH3 domains, protein-protein interaction modules prevalent in eukaryotes but rare in prokaryotes. NMR studies and analysis of site-directed mutants in vivo and in vitro unveil a solvent-exposed hydrophobic pocket lined by acidic residues in CarS, where the CarA DNA recognition helix docks with high affinity in an atypical ligand-recognition mode for SH3 domains. Our findings uncover an unprecedented use of the SH3 domain-like fold for protein-protein recognition whereby an antirepressor mimics operator DNA in sequestering the repressor DNA recognition helix to activate transcription.

Show MeSH
Related in: MedlinePlus