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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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CarS1 structure and interactions with CarANt from NMR. (A) Superposition of the backbone traces for the 20 final NMR structures (left) and ribbon diagram of the average structure. (B) Cα-based overlay of the Abl tyrosine kinase SH3 domain (blue; PDB ID: 1JU5), R67-plasmid DHFR (yellow; PDB ID: 1VIE), or chloroplast FTR subunit B (green; PDB ID: 1DJ7) onto CarS1 (red). The respective DALI Z-score/rmsd (Å)/sequence identity (%)/number of superimposed residues are: Abl SH3 domain: 3.4/2.6/6/50; DHFR: 5/1.6/20/49; FTR B: 5.2/2.7/21/57. Below is a structure-based sequence alignment showing secondary structural elements and the RT, n-Src and distal (D) loops as denoted in SH3 domains. Residues are shaded black if identical in at least two sequences and gray if similar. (C) Portion of the 1H-13C HSQC spectrum of 13C, 15N-labeled H6CarS1 (0.24 mM) showing methyl crosspeaks perturbed by a 1.5-fold excess of unlabeled CarANt (red) compared to no CarANt added (black). Inset shows negligible perturbation of labeled Ala methyl crosspeaks for comparison. (D) Ribbon and electrostatic surface models of CarS1 showing residues that interact with CarANt from NMR data. Interacting side chains are depicted as magenta sticks with neighbouring acidic residues in red in the ribbon model.
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Figure 1: CarS1 structure and interactions with CarANt from NMR. (A) Superposition of the backbone traces for the 20 final NMR structures (left) and ribbon diagram of the average structure. (B) Cα-based overlay of the Abl tyrosine kinase SH3 domain (blue; PDB ID: 1JU5), R67-plasmid DHFR (yellow; PDB ID: 1VIE), or chloroplast FTR subunit B (green; PDB ID: 1DJ7) onto CarS1 (red). The respective DALI Z-score/rmsd (Å)/sequence identity (%)/number of superimposed residues are: Abl SH3 domain: 3.4/2.6/6/50; DHFR: 5/1.6/20/49; FTR B: 5.2/2.7/21/57. Below is a structure-based sequence alignment showing secondary structural elements and the RT, n-Src and distal (D) loops as denoted in SH3 domains. Residues are shaded black if identical in at least two sequences and gray if similar. (C) Portion of the 1H-13C HSQC spectrum of 13C, 15N-labeled H6CarS1 (0.24 mM) showing methyl crosspeaks perturbed by a 1.5-fold excess of unlabeled CarANt (red) compared to no CarANt added (black). Inset shows negligible perturbation of labeled Ala methyl crosspeaks for comparison. (D) Ribbon and electrostatic surface models of CarS1 showing residues that interact with CarANt from NMR data. Interacting side chains are depicted as magenta sticks with neighbouring acidic residues in red in the ribbon model.

Mentions: CarS1 is a functional, truncated form of CarS (lacking the last 25 residues) that, like CarS, forms a 1:1 complex with the CarA or CarH N-terminal domain (10,15,36). Given its excellent spectral dispersion and overall quality, NMR studies were carried out with CarS1. 1H, 15N and 13C resonances were assigned using standard triple resonance methods (20). The ensemble of the 20 lowest-energy structures calculated using experimental constraints (Supplementary Table S2) revealed a disordered nine-residue N-terminal segment, a relatively well-defined loop with two arms (residues 10–13 and 17–19) in extended conformations, a 310 helix (residues 37–39) and a five-stranded β-sheet (Figure 1A and Supplementary Figure S1). The β3–β4 hairpin is orthogonal to the β5–β1–β2 sheet, and long loops connect β1 to β2 and β2 to β3. Pairwise root mean square deviations (rmsd) for the superposition of the final structures, excluding the disordered, nine-residue N-terminal segment, were 0.8 ± 0.1 Å and 1.4 ± 0.3 Å for the backbone and heavy atoms, respectively (Supplementary Table S2). Steady-state, backbone 15N-{1H} heteronuclear NOEs of ≤0.65 for several non-P residues in the β1–β2 loop suggest flexibility with large amplitude motions on the subnanosecond timescale (Supplementary Figure S1C). Twenty-five residues mostly in strands β1 to β5 and involved in H-bonds showed the slowest amide 1H exchange with solvent (three to four orders of magnitude slower than random-coil values; Supplementary Figure S1A). The total solvent-accessible surface area (ASA) in native CarS1 is 6360 Å2, and its hydrophobic core is composed of 13 non-polar residues. Of the eight P in CarS1, only P59 is in the cis form and is stabilized by stacking interactions with Y58. With nine D and seven E but only two each of R, K and H, CarS1 is highly acidic (theoretical pI = 4.1). Basic residues lie on one face of the protein surface, while regions of high negative electrostatic potential are on two other adjoining faces (Supplementary Figure S1D). These latter are likely regions for interaction with CarANt, the highly basic and autonomously folded N-terminal DNA-binding domain of CarA.Figure 1.


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)

CarS1 structure and interactions with CarANt from NMR. (A) Superposition of the backbone traces for the 20 final NMR structures (left) and ribbon diagram of the average structure. (B) Cα-based overlay of the Abl tyrosine kinase SH3 domain (blue; PDB ID: 1JU5), R67-plasmid DHFR (yellow; PDB ID: 1VIE), or chloroplast FTR subunit B (green; PDB ID: 1DJ7) onto CarS1 (red). The respective DALI Z-score/rmsd (Å)/sequence identity (%)/number of superimposed residues are: Abl SH3 domain: 3.4/2.6/6/50; DHFR: 5/1.6/20/49; FTR B: 5.2/2.7/21/57. Below is a structure-based sequence alignment showing secondary structural elements and the RT, n-Src and distal (D) loops as denoted in SH3 domains. Residues are shaded black if identical in at least two sequences and gray if similar. (C) Portion of the 1H-13C HSQC spectrum of 13C, 15N-labeled H6CarS1 (0.24 mM) showing methyl crosspeaks perturbed by a 1.5-fold excess of unlabeled CarANt (red) compared to no CarANt added (black). Inset shows negligible perturbation of labeled Ala methyl crosspeaks for comparison. (D) Ribbon and electrostatic surface models of CarS1 showing residues that interact with CarANt from NMR data. Interacting side chains are depicted as magenta sticks with neighbouring acidic residues in red in the ribbon model.
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Figure 1: CarS1 structure and interactions with CarANt from NMR. (A) Superposition of the backbone traces for the 20 final NMR structures (left) and ribbon diagram of the average structure. (B) Cα-based overlay of the Abl tyrosine kinase SH3 domain (blue; PDB ID: 1JU5), R67-plasmid DHFR (yellow; PDB ID: 1VIE), or chloroplast FTR subunit B (green; PDB ID: 1DJ7) onto CarS1 (red). The respective DALI Z-score/rmsd (Å)/sequence identity (%)/number of superimposed residues are: Abl SH3 domain: 3.4/2.6/6/50; DHFR: 5/1.6/20/49; FTR B: 5.2/2.7/21/57. Below is a structure-based sequence alignment showing secondary structural elements and the RT, n-Src and distal (D) loops as denoted in SH3 domains. Residues are shaded black if identical in at least two sequences and gray if similar. (C) Portion of the 1H-13C HSQC spectrum of 13C, 15N-labeled H6CarS1 (0.24 mM) showing methyl crosspeaks perturbed by a 1.5-fold excess of unlabeled CarANt (red) compared to no CarANt added (black). Inset shows negligible perturbation of labeled Ala methyl crosspeaks for comparison. (D) Ribbon and electrostatic surface models of CarS1 showing residues that interact with CarANt from NMR data. Interacting side chains are depicted as magenta sticks with neighbouring acidic residues in red in the ribbon model.
Mentions: CarS1 is a functional, truncated form of CarS (lacking the last 25 residues) that, like CarS, forms a 1:1 complex with the CarA or CarH N-terminal domain (10,15,36). Given its excellent spectral dispersion and overall quality, NMR studies were carried out with CarS1. 1H, 15N and 13C resonances were assigned using standard triple resonance methods (20). The ensemble of the 20 lowest-energy structures calculated using experimental constraints (Supplementary Table S2) revealed a disordered nine-residue N-terminal segment, a relatively well-defined loop with two arms (residues 10–13 and 17–19) in extended conformations, a 310 helix (residues 37–39) and a five-stranded β-sheet (Figure 1A and Supplementary Figure S1). The β3–β4 hairpin is orthogonal to the β5–β1–β2 sheet, and long loops connect β1 to β2 and β2 to β3. Pairwise root mean square deviations (rmsd) for the superposition of the final structures, excluding the disordered, nine-residue N-terminal segment, were 0.8 ± 0.1 Å and 1.4 ± 0.3 Å for the backbone and heavy atoms, respectively (Supplementary Table S2). Steady-state, backbone 15N-{1H} heteronuclear NOEs of ≤0.65 for several non-P residues in the β1–β2 loop suggest flexibility with large amplitude motions on the subnanosecond timescale (Supplementary Figure S1C). Twenty-five residues mostly in strands β1 to β5 and involved in H-bonds showed the slowest amide 1H exchange with solvent (three to four orders of magnitude slower than random-coil values; Supplementary Figure S1A). The total solvent-accessible surface area (ASA) in native CarS1 is 6360 Å2, and its hydrophobic core is composed of 13 non-polar residues. Of the eight P in CarS1, only P59 is in the cis form and is stabilized by stacking interactions with Y58. With nine D and seven E but only two each of R, K and H, CarS1 is highly acidic (theoretical pI = 4.1). Basic residues lie on one face of the protein surface, while regions of high negative electrostatic potential are on two other adjoining faces (Supplementary Figure S1D). These latter are likely regions for interaction with CarANt, the highly basic and autonomously folded N-terminal DNA-binding domain of CarA.Figure 1.

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