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Sequence-specific DNA binding determined by contacts outside the helix-turn-helix motif of the ParB homolog KorB

Nature Structural & Molecular Biology volume 11, pages 656–663 (2004)Cite this article

Abstract

The KorB protein of the broad-host-range plasmid RP4 acts as a multifunctional regulator of plasmid housekeeping genes, including those responsible for replication, maintenance and conjugation. Additionally, KorB functions as the ParB analog of the plasmid's partitioning system. The protein structure consists of eight helices, two of which belong to a predicted helix-turn-helix motif. Each half-site of the palindromic operator DNA binds one copy of the protein in the major groove. As confirmed by mutagenesis, recognition specificity is based mainly on two side chain interactions outside the helix-turn-helix motif with two bases next to the central base pair of the 13-base pair operator sequence. The surface of the KorB DNA-binding domain mirrors the overall acidity of KorB, whereas DNA binding occurs via a basic interaction surface. We present a model of KorB, including the structure of its dimerization domain, and discuss its interactions with the highly basic ParA homolog IncC.

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Figure 1: Sequence comparison of KorB proteins and secondary structure of RP4 KorB-O.
Figure 2: Organization of the KorB-O–OB complex.
Figure 3: Molecular structure of the KorB-O–operator complex.
Figure 4: Specific KorB-O–OB contacts.
Figure 5: KorB-O–DNA contacts in the crystal and verified by site-directed mutagenesis.
Figure 6: DNA binding by intact KorB.

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Protein Data Bank

References

  1. Møller-Jensen, J., Jensen, R.B. & Gerdes, K. Plasmid and chromosome segregation in prokaryotes. Trends Microbiol. 8, 313–320 (2000).

    Article  Google Scholar 

  2. Mori, H. et al. Purification and characterization of SopA protein and SopB protein essential for F-plasmid partitioning. J. Biol. Chem. 264, 15535–15541 (1989).

    CAS  PubMed  Google Scholar 

  3. Abeles, A.L., Reaves, L.D. & Austin, S.J. Protein-DNA interactions in regulation of P1 plasmid replication. J. Bacteriol. 171, 43–52 (1989).

    Article  CAS  Google Scholar 

  4. Davis, M.A. et al. The P1 ParA protein and its ATPase activity play a direct role in the segregation of plasmid copies to daughter cells. Mol. Microbiol. 21, 1029–1036 (1996).

    Article  CAS  Google Scholar 

  5. Motallebi-Veshareh, M., Rouch, D.A. & Thomas, C.M. A family of ATPases involved in active partitioning of diverse bacterial plasmids. Mol. Microbiol. 4, 1455–1463 (1990).

    Article  CAS  Google Scholar 

  6. Watanabe, E., Wachi, M., Yamasaki, M. & Nagai, K. ATPase activity of SopA, a protein essential for active partitioning of F-plasmid. Mol. Gen. Genet. 234, 346–352 (1992).

    Article  CAS  Google Scholar 

  7. Gerdes, K., Møller-Jensen, J. & Jensen, R.B. Plasmid and chromosome partitioning: surprises from phylogeny. Mol. Microbiol. 37, 455–466 (2000).

    Article  CAS  Google Scholar 

  8. van den Ent, F., Møller-Jensen, J., Amos, L.A., Gerdes, K. & Lowe, J. F-actin-like filaments formed by plasmid segregation protein ParM. EMBO J. 21, 6935–6943 (2002).

    Article  CAS  Google Scholar 

  9. Møller-Jensen, J., Jensen, R.B., Lowe, J. & Gerdes, K. Prokaryotic DNA segregation by an actin-like filament. EMBO J. 21, 3119–3127 (2002).

    Article  Google Scholar 

  10. Møller-Jensen, J. et al. Bacterial mitosis: ParM of plasmid R1 moves plasmid DNA by an actin-like insertional polymerization mechanism. Mol. Cell 12, 1477–1487 (2003).

    Article  Google Scholar 

  11. Gerdes, K., Møller-Jensen, J., Ebersbach, G., Kruse, T. & Nordström, K. Bacterial mitotic machineries. Cell 116, 359–366 (2004).

    Article  CAS  Google Scholar 

  12. Pansegrau, W. et al. Complete nucleotide sequence of Birmingham IncP-α plasmids. Compilation and comparative analysis. J. Mol. Biol. 239, 623–663 (1994).

    Article  CAS  Google Scholar 

  13. Adamczyk, M. & Jagura-Burdzy, G. Spread and survival of promiscuous IncP-1 plasmids. Acta Biochim. Pol. 50, 425–453 (2003).

    CAS  Google Scholar 

  14. Heinemann, J.A. & Sprague, G.F. Bacterial conjugative plasmids mobilize DNA transfer between bacteria and yeast. Nature 340, 205–209 (1989).

    Article  CAS  Google Scholar 

  15. Waters, V.L. Conjugation between bacterial and mammalian cells. Nat. Genet. 29, 375–376 (2001).

    Article  CAS  Google Scholar 

  16. Pansegrau, W. & Lanka, E. Conservation of a common 'backbone' in the genetic organization of the IncP plasmids RP4 and R751. Nucleic Acids Res. 15, 2385 (1987).

    Article  CAS  Google Scholar 

  17. Thorsted, P.A. et al. Complete sequence of the IncP beta plasmid R751: implications for evolution and organisation of the IncP backbone. J. Mol. Biol. 282, 969–990 (1998).

    Article  CAS  Google Scholar 

  18. Bignell, C. & Thomas, C.M. The bacterial ParA-ParB partitioning proteins. J. Biotechnol. 91, 1–34 (2001).

    Article  CAS  Google Scholar 

  19. Rosche, T.M., Siddique, A., Larsen, M.H. & Figurski, D.H. Incompatibility protein IncC and global regulator KorB interact in active partition of promiscuous plasmid RK2. J. Bacteriol. 182, 6014–6026 (2000).

    Article  CAS  Google Scholar 

  20. Siddique, A. & Figurski, D.H. The active partition gene incC of IncP plasmids is required for stable maintenance in a broad range of hosts. J. Bacteriol. 184, 1788–1793 (2002).

    Article  CAS  Google Scholar 

  21. Balzer, D., Ziegelin, G., Pansegrau, W., Kruft, V. & Lanka, E. KorB protein of promiscuous plasmid RP4 recognizes inverted sequence repetitions in regions essential for conjugative plasmid transfer. Nucleic Acids Res. 20, 1851–1858 (1992).

    Article  CAS  Google Scholar 

  22. Jagura-Burdzy, G. et al. IncC of broad-host-range plasmid RK2 modulates KorB transcriptional repressor activity in vivo and operator binding in vitro. J. Bacteriol. 181, 2807–2815 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Williams, D.R., Macartney, D.P. & Thomas, C.M. The partitioning activity of the RK2 central control region requires only incC, korB and KorB-binding site OB3 but other KorB-binding sites form destabilizing complexes in the absence of OB3. Microbiology 144, 3369–3378 (1998).

    Article  CAS  Google Scholar 

  24. Macartney, D.P., Williams, D.R., Stafford, T. & Thomas, C.M. Divergence and conservation of the partitioning and global regulation functions in the central control region of the IncP plasmids RK2 and R751. Microbiology 143, 2167–2177 (1997).

    Article  CAS  Google Scholar 

  25. Williams, D.R., Motallebi-Veshareh, M. & Thomas, C.M. Multifunctional repressor KorB can block transcription by preventing isomerization of RNA polymerase-promoter complexes. Nucleic Acids Res. 21, 1141–1148 (1993).

    Article  CAS  Google Scholar 

  26. Delbrück, H., Ziegelin, G., Lanka, E. & Heinemann, U. An Src homology 3-like domain is responsible for dimerization of the repressor protein KorB encoded by the promiscuous IncP plasmid RP4. J. Biol. Chem. 277, 4191–4198 (2002).

    Article  Google Scholar 

  27. Jagura-Burdzy, G. et al. Repression at a distance by the global regulator KorB of promiscuous IncP plasmids. Mol. Microbiol. 32, 519–532 (1999).

    Article  CAS  Google Scholar 

  28. Kostelidou, K. & Thomas, C.M. The hierarchy of KorB binding at its 12 binding sites on the broad-host-range plasmid RK2 and modulation of this binding by IncC1 protein. J. Mol. Biol. 295, 411–422 (2000).

    Article  CAS  Google Scholar 

  29. Bechhofer, D.H., Kornacki, J.A., Firshein, W. & Figurski, D.H. Gene control in broad host range plasmid RK2: expression, polypeptide product, and multiple regulatory functions of KorB. Proc. Natl. Acad. Sci. USA 83, 394–398 (1986).

    Article  CAS  Google Scholar 

  30. Theophilus, B.D.M. & Thomas, C.M. Nucleotide sequence of the transcriptional repressor gene KorB which plays a key role in regulation of the copy number of broad host range plasmid RK2. Nucleic Acids Res. 15, 7443–7450 (1987).

    Article  CAS  Google Scholar 

  31. Kornacki, J.A., Balderes, P.J. & Figurski, D.H. Nucleotide sequence of KorB, a replication control gene of broad host-range plasmid RK2. J. Mol. Biol. 198, 211–222 (1987).

    Article  CAS  Google Scholar 

  32. Dostál, L. et al. RP4 repressor protein KorB binds to the major groove of the operator DNA: a Raman study. Biochemistry 42, 14476–14482 (2003).

    Article  Google Scholar 

  33. Lavery, R. & Sklenar, H. Defining the structure of irregular nucleic acids: conventions and principles. J. Biomol. Struct. Dyn. 6, 655–667 (1989).

    Article  CAS  Google Scholar 

  34. Kostelidou, K., Jones, A.C. & Thomas, C.M. Conserved C-terminal region of global regulator KorA of broad-host-range plasmid RK2 is required for co-operativity between KorA and a second RK2 global regulator, KorB. J. Mol. Biol. 289, 211–221 (1999).

    Article  CAS  Google Scholar 

  35. Holm, L. & Sander, C. Protein structure comparison by alignment of distance matrices. J. Mol. Biol. 233, 123–138 (1993).

    Article  CAS  Google Scholar 

  36. Pabo, C.O. & Sauer, R.T. Protein-DNA recognition. Annu. Rev. Biochem. 53, 293–321 (1984).

    Article  CAS  Google Scholar 

  37. Brennan, R.G. & Matthews, B.W. Structural basis of DNA-protein recognition. Trends Biochem. Sci. 14, 286–290 (1989).

    Article  CAS  Google Scholar 

  38. Harrison, S.C. & Aggarwal, A.K. DNA recognition by proteins with the helix-turn-helix motif. Annu. Rev. Biochem. 59, 933–969 (1990).

    Article  CAS  Google Scholar 

  39. Lukaszewicz, M. et al. Functional dissection of the ParB homologue (KorB) from IncP-1 plasmid RK2. Nucleic Acids Res. 30, 1046–1055 (2002).

    Article  CAS  Google Scholar 

  40. Surtees, J.A. & Funnell, B.E. P1 ParB domain structure includes two independent multimerization domains. J. Bacteriol. 181, 5898–5908 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Kim, S.K. & Shim, J. Interaction between F plasmid partition proteins SopA and SopB. Biochem. Biophys. Res. Commun. 263, 113–117 (1999).

    Article  CAS  Google Scholar 

  42. Kabsch, W. Automatic processing of rotation diffraction data from crystals of initially unknown symmetry and cell constants. J. Appl. Crystallogr. 26, 795–800 (1993).

    Article  CAS  Google Scholar 

  43. Terwilliger, T.C. & Berendzen, J. Automated MAD and MIR structure solution. Acta Crystallogr. D 55, 849–861 (1999).

    Article  CAS  Google Scholar 

  44. Terwilliger, T.C. Maximum-likelihood density modification. Acta Crystallogr. D 56, 965–972 (2000).

    Article  CAS  Google Scholar 

  45. Jones, T.A., Zou, J.Y., Cowan, S.W. & Kjeldgaard, M. Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr. A 47, 110–119 (1991).

    Article  Google Scholar 

  46. Collaborative Computational Project, No. 4. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D 50, 760–763 (1994).

  47. Perrakis, A., Harkiolaki, M., Wilson, K.S. & Lamzin, V.S. ARP/wARP and molecular replacement. Acta Crystallogr. D 57, 1445–1450 (2001).

    Article  CAS  Google Scholar 

  48. Hooft, R.W.W., Vriend, G., Sander, C. & Abola, E.E. Errors in protein structures. Nature 381, 272 (1996).

    Article  CAS  Google Scholar 

  49. Laskowski, R.A., MacArthur, M.A., Moss, D.S. & Thornton, J.M. PROCHECK: a program to check the stereochemical quality of protein structures. J. Appl. Crystallogr. 26, 283–291 (1993).

    Article  CAS  Google Scholar 

  50. Koradi, R., Billeter, M. & Wüthrich, K. MOLMOL: a program for display and analysis of macromolecular structures. J. Mol. Graphics 14, 51–55 (1996).

    Article  CAS  Google Scholar 

  51. Nicholls, A., Sharp, K.A. & Honig, B. Protein folding and association: insights from the interfacial and thermodynamic properties of hydrocarbons. Proteins 11, 281–296 (1993).

    Article  Google Scholar 

  52. Diederichs, K. & Karplus, P.A. Improved R-factors for diffraction data analysis in macromolecular crystallography. Nat. Struct. Biol. 4, 269–275 (1997).

    Article  CAS  Google Scholar 

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Acknowledgements

We express our gratitude to C. Bernert for assistance with protein purification work to U. Müller and M. Fieber-Erdmann for helpful assistance at the ID14.2 beamline at BESSY, Berlin, to E.-C. Müller and A. Otto for protein sequencing and analyses by mass spectrometry, and to J.J. Müller and K. Fälber for useful discussions and help with crystallographic problems. E.L. and G.Z. thank H. Lehrach for generous support and M. Schlicht for expert assistance. This work was supported by the Deutsche Forschungsgemeinschaft (DFG) and the Fonds der Chemischen Industrie. D.K. acknowledges a DFG fellowship through GRK 80/2.3.

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Authors and Affiliations

  1. Max-Delbrück-Centrum für Molekulare Medizin, Robert-Rössle-Strasse 10, Berlin, D-13125, Germany

    Dheeraj Khare & Udo Heinemann

  2. Max-Planck-Institut fnr Molekulare Genetik, Ihnestrasse 73, Berlin, D-14195, Germany

    Günter Ziegelin & Erich Lanka

  3. Institut fnr Chemie Kristallographie, Freie UniversitSt, Takustrasse 6, Berlin, D-14195, Germany

    Udo Heinemann

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Correspondence to Udo Heinemann.

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Supplementary Fig. 1

Stereo view of the protein-DNA interface. (PDF 131 kb)

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Khare, D., Ziegelin, G., Lanka, E. et al. Sequence-specific DNA binding determined by contacts outside the helix-turn-helix motif of the ParB homolog KorB. Nat Struct Mol Biol 11, 656–663 (2004). https://doi.org/10.1038/nsmb773

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