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Crystal structure of KorA bound to operator DNA: insight into repressor cooperation in RP4 gene regulation.

König B, Müller JJ, Lanka E, Heinemann U - Nucleic Acids Res. (2009)

Bottom Line: As confirmed by mutagenesis, recognition specificity is based on two KorA side chains forming hydrogen bonds to four bases within each operator half-site.KorA has a unique dimerization module shared by the RP4 proteins TrbA and KlcB.We propose that these proteins cooperate with the global RP4 repressor KorB in a similar manner via this dimerization module and thus regulate RP4 inheritance.

View Article: PubMed Central - PubMed

Affiliation: Max-Delbrück-Center for Molecular Medicine, Berlin, Germany.

ABSTRACT
KorA is a global repressor in RP4 which regulates cooperatively the expression of plasmid genes whose products are involved in replication, conjugative transfer and stable inheritance. The structure of KorA bound to an 18-bp DNA duplex that contains the symmetric operator sequence and incorporates 5-bromo-deoxyuridine nucleosides has been determined by multiple-wavelength anomalous diffraction phasing at 1.96-A resolution. KorA is present as a symmetric dimer and contacts DNA via a helix-turn-helix motif. Each half-site of the symmetric operator DNA binds one copy of the protein in the major groove. As confirmed by mutagenesis, recognition specificity is based on two KorA side chains forming hydrogen bonds to four bases within each operator half-site. KorA has a unique dimerization module shared by the RP4 proteins TrbA and KlcB. We propose that these proteins cooperate with the global RP4 repressor KorB in a similar manner via this dimerization module and thus regulate RP4 inheritance.

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KorA binds its operator binding site. (A) OA* with the central 12-bp consensus operator sequence in red and green. (In OA the BrU bases are replaced by T.) The symmetric operator has two half-sites, each of which binds one KorA monomer in identical geometry. Two different types of protein–DNA interactions are shown in the two half-sites. In the top half-site (green), only direct protein–DNA contacts are shown, whereas water-mediated interactions are shown in the bottom half-site. Both types of interactions occur simultaneously in both half-sites of the operator DNA. Amino acids circled in red are from the HTH motif of KorA. Direct hydrogen-bonded contacts, involving protein side chains and DNA, are indicated by black solid arrows, those between protein backbone and DNA by black dotted arrows. Contacts mediated by water molecules are marked by blue arrows, those between protein side chains and DNA by blue solid lines, and those between protein backbone and DNA by blue dotted lines. (B) Electrostatic surface potential of KorA; positive charge of the protein in blue and negative charge in red. The DNA is bound to the KorA dimer in two orientations with duplexes EF (gray) and GH (green). (C) Two-fold disorder of KorA-bound OA* DNA. Left, base pair A2-BrU17 with electron density before allowing for 2-fold disorder of the KorA-bound DNA. Right, A2-BrU17 base pairs of the superimposed DNA duplexes EF (red) and GH (blue) after refinement. The 2Fo–Fc density (gray) is contoured at 1.0 σ, the Fo–Fc difference density (green) is contoured at 3.0 σ.
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Figure 4: KorA binds its operator binding site. (A) OA* with the central 12-bp consensus operator sequence in red and green. (In OA the BrU bases are replaced by T.) The symmetric operator has two half-sites, each of which binds one KorA monomer in identical geometry. Two different types of protein–DNA interactions are shown in the two half-sites. In the top half-site (green), only direct protein–DNA contacts are shown, whereas water-mediated interactions are shown in the bottom half-site. Both types of interactions occur simultaneously in both half-sites of the operator DNA. Amino acids circled in red are from the HTH motif of KorA. Direct hydrogen-bonded contacts, involving protein side chains and DNA, are indicated by black solid arrows, those between protein backbone and DNA by black dotted arrows. Contacts mediated by water molecules are marked by blue arrows, those between protein side chains and DNA by blue solid lines, and those between protein backbone and DNA by blue dotted lines. (B) Electrostatic surface potential of KorA; positive charge of the protein in blue and negative charge in red. The DNA is bound to the KorA dimer in two orientations with duplexes EF (gray) and GH (green). (C) Two-fold disorder of KorA-bound OA* DNA. Left, base pair A2-BrU17 with electron density before allowing for 2-fold disorder of the KorA-bound DNA. Right, A2-BrU17 base pairs of the superimposed DNA duplexes EF (red) and GH (blue) after refinement. The 2Fo–Fc density (gray) is contoured at 1.0 σ, the Fo–Fc difference density (green) is contoured at 3.0 σ.

Mentions: The structure of the complex containing KorA (1–101) and the 18-bp OA* oligomer, in which three thymine residues were replaced by 5-bromodeoxyuracil (Figure 4A), was solved by multiple-wavelength anomalous diffraction (MAD). The program HKL2MAP (22) located six heavy atom sites, differing in relative occupancy and anomalous contribution. The number and position of the bromine sites was the first indication of a 2-fold disorder of the OA* duplex.


Crystal structure of KorA bound to operator DNA: insight into repressor cooperation in RP4 gene regulation.

König B, Müller JJ, Lanka E, Heinemann U - Nucleic Acids Res. (2009)

KorA binds its operator binding site. (A) OA* with the central 12-bp consensus operator sequence in red and green. (In OA the BrU bases are replaced by T.) The symmetric operator has two half-sites, each of which binds one KorA monomer in identical geometry. Two different types of protein–DNA interactions are shown in the two half-sites. In the top half-site (green), only direct protein–DNA contacts are shown, whereas water-mediated interactions are shown in the bottom half-site. Both types of interactions occur simultaneously in both half-sites of the operator DNA. Amino acids circled in red are from the HTH motif of KorA. Direct hydrogen-bonded contacts, involving protein side chains and DNA, are indicated by black solid arrows, those between protein backbone and DNA by black dotted arrows. Contacts mediated by water molecules are marked by blue arrows, those between protein side chains and DNA by blue solid lines, and those between protein backbone and DNA by blue dotted lines. (B) Electrostatic surface potential of KorA; positive charge of the protein in blue and negative charge in red. The DNA is bound to the KorA dimer in two orientations with duplexes EF (gray) and GH (green). (C) Two-fold disorder of KorA-bound OA* DNA. Left, base pair A2-BrU17 with electron density before allowing for 2-fold disorder of the KorA-bound DNA. Right, A2-BrU17 base pairs of the superimposed DNA duplexes EF (red) and GH (blue) after refinement. The 2Fo–Fc density (gray) is contoured at 1.0 σ, the Fo–Fc difference density (green) is contoured at 3.0 σ.
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Figure 4: KorA binds its operator binding site. (A) OA* with the central 12-bp consensus operator sequence in red and green. (In OA the BrU bases are replaced by T.) The symmetric operator has two half-sites, each of which binds one KorA monomer in identical geometry. Two different types of protein–DNA interactions are shown in the two half-sites. In the top half-site (green), only direct protein–DNA contacts are shown, whereas water-mediated interactions are shown in the bottom half-site. Both types of interactions occur simultaneously in both half-sites of the operator DNA. Amino acids circled in red are from the HTH motif of KorA. Direct hydrogen-bonded contacts, involving protein side chains and DNA, are indicated by black solid arrows, those between protein backbone and DNA by black dotted arrows. Contacts mediated by water molecules are marked by blue arrows, those between protein side chains and DNA by blue solid lines, and those between protein backbone and DNA by blue dotted lines. (B) Electrostatic surface potential of KorA; positive charge of the protein in blue and negative charge in red. The DNA is bound to the KorA dimer in two orientations with duplexes EF (gray) and GH (green). (C) Two-fold disorder of KorA-bound OA* DNA. Left, base pair A2-BrU17 with electron density before allowing for 2-fold disorder of the KorA-bound DNA. Right, A2-BrU17 base pairs of the superimposed DNA duplexes EF (red) and GH (blue) after refinement. The 2Fo–Fc density (gray) is contoured at 1.0 σ, the Fo–Fc difference density (green) is contoured at 3.0 σ.
Mentions: The structure of the complex containing KorA (1–101) and the 18-bp OA* oligomer, in which three thymine residues were replaced by 5-bromodeoxyuracil (Figure 4A), was solved by multiple-wavelength anomalous diffraction (MAD). The program HKL2MAP (22) located six heavy atom sites, differing in relative occupancy and anomalous contribution. The number and position of the bromine sites was the first indication of a 2-fold disorder of the OA* duplex.

Bottom Line: As confirmed by mutagenesis, recognition specificity is based on two KorA side chains forming hydrogen bonds to four bases within each operator half-site.KorA has a unique dimerization module shared by the RP4 proteins TrbA and KlcB.We propose that these proteins cooperate with the global RP4 repressor KorB in a similar manner via this dimerization module and thus regulate RP4 inheritance.

View Article: PubMed Central - PubMed

Affiliation: Max-Delbrück-Center for Molecular Medicine, Berlin, Germany.

ABSTRACT
KorA is a global repressor in RP4 which regulates cooperatively the expression of plasmid genes whose products are involved in replication, conjugative transfer and stable inheritance. The structure of KorA bound to an 18-bp DNA duplex that contains the symmetric operator sequence and incorporates 5-bromo-deoxyuridine nucleosides has been determined by multiple-wavelength anomalous diffraction phasing at 1.96-A resolution. KorA is present as a symmetric dimer and contacts DNA via a helix-turn-helix motif. Each half-site of the symmetric operator DNA binds one copy of the protein in the major groove. As confirmed by mutagenesis, recognition specificity is based on two KorA side chains forming hydrogen bonds to four bases within each operator half-site. KorA has a unique dimerization module shared by the RP4 proteins TrbA and KlcB. We propose that these proteins cooperate with the global RP4 repressor KorB in a similar manner via this dimerization module and thus regulate RP4 inheritance.

Show MeSH