Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 2002 Jan 1;361(Pt 1):41–47. doi: 10.1042/0264-6021:3610041

The anti-toxin ParD of plasmid RK2 consists of two structurally distinct moieties and belongs to the ribbon-helix-helix family of DNA-binding proteins.

Monika Oberer 1, Klaus Zangger 1, Stefan Prytulla 1, Walter Keller 1
PMCID: PMC1222296  PMID: 11743881

Abstract

NMR and CD spectroscopy have been used to characterize, both structurally and dynamically, the 82-amino-acid ParD protein of the post-segregational killing module of the broad-host-range plasmid RP4/RK2. ParD occurs as a dimer in solution and exercises two different control functions; an autoregulatory function by binding to its own promoter P(parDE) and a plasmid-stabilizing function by inhibiting ParE toxicity in cells that express ParD and ParE. Analysis of the secondary structure based on the chemical-shift indices, sequential nuclear Overhauser enhancements (NOEs) and (3)J(Halpha-NH) scalar coupling constants showed that the N-terminal domain of ParD consists of a short beta-ribbon followed by three alpha-helices, demonstrating that ParD contains a ribbon-helix-helix fold, a DNA-binding motif found in a family of small prokaryotic repressors. (15)N longitudinal (T(1)) and transverse (T(2)) relaxation measurements and hetero nuclear NOEs showed that ParD is divided into two separate domains, a well-ordered N-terminal domain and a very flexible C-terminal domain. An increase in secondary structure was observed upon addition of trifluoroethanol, suggested to result from the formation of structured stretches in the C-terminal part of the protein. This is the first experimental evidence that the DNA-binding domain of ParD belongs to the ribbon-helix-helix fold family, and this structural motif is proposed to be present in functionally similar antidote proteins.

Full Text

The Full Text of this article is available as a PDF (211.1 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Berman H. M., Westbrook J., Feng Z., Gilliland G., Bhat T. N., Weissig H., Shindyalov I. N., Bourne P. E. The Protein Data Bank. Nucleic Acids Res. 2000 Jan 1;28(1):235–242. doi: 10.1093/nar/28.1.235. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Buck M., Radford S. E., Dobson C. M. A partially folded state of hen egg white lysozyme in trifluoroethanol: structural characterization and implications for protein folding. Biochemistry. 1993 Jan 19;32(2):669–678. doi: 10.1021/bi00053a036. [DOI] [PubMed] [Google Scholar]
  3. Burgering M. J., Boelens R., Gilbert D. E., Breg J. N., Knight K. L., Sauer R. T., Kaptein R. Solution structure of dimeric Mnt repressor (1-76). Biochemistry. 1994 Dec 20;33(50):15036–15045. doi: 10.1021/bi00254a012. [DOI] [PubMed] [Google Scholar]
  4. Cooper T. F., Heinemann J. A. Postsegregational killing does not increase plasmid stability but acts to mediate the exclusion of competing plasmids. Proc Natl Acad Sci U S A. 2000 Nov 7;97(23):12643–12648. doi: 10.1073/pnas.220077897. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Dao-Thi M. H., Messens J., Wyns L., Backmann J. The thermodynamic stability of the proteins of the ccd plasmid addiction system. J Mol Biol. 2000 Jun 23;299(5):1373–1386. doi: 10.1006/jmbi.2000.3815. [DOI] [PubMed] [Google Scholar]
  6. Delaglio F., Grzesiek S., Vuister G. W., Zhu G., Pfeifer J., Bax A. NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J Biomol NMR. 1995 Nov;6(3):277–293. doi: 10.1007/BF00197809. [DOI] [PubMed] [Google Scholar]
  7. Eberl L., Givskov M., Schwab H. The divergent promoters mediating transcription of the par locus of plasmid RP4 are subject to autoregulation. Mol Microbiol. 1992 Jul;6(14):1969–1979. doi: 10.1111/j.1365-2958.1992.tb01370.x. [DOI] [PubMed] [Google Scholar]
  8. Engelberg-Kulka H., Glaser G. Addiction modules and programmed cell death and antideath in bacterial cultures. Annu Rev Microbiol. 1999;53:43–70. doi: 10.1146/annurev.micro.53.1.43. [DOI] [PubMed] [Google Scholar]
  9. Gast K., Zirwer D., Müller-Frohne M., Damaschun G. Trifluoroethanol-induced conformational transitions of proteins: insights gained from the differences between alpha-lactalbumin and ribonuclease A. Protein Sci. 1999 Mar;8(3):625–634. doi: 10.1110/ps.8.3.625. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Gazit E., Sauer R. T. Stability and DNA binding of the phd protein of the phage P1 plasmid addiction system. J Biol Chem. 1999 Jan 29;274(5):2652–2657. doi: 10.1074/jbc.274.5.2652. [DOI] [PubMed] [Google Scholar]
  11. Gazit E., Sauer R. T. The Doc toxin and Phd antidote proteins of the bacteriophage P1 plasmid addiction system form a heterotrimeric complex. J Biol Chem. 1999 Jun 11;274(24):16813–16818. doi: 10.1074/jbc.274.24.16813. [DOI] [PubMed] [Google Scholar]
  12. Gerdes K. Toxin-antitoxin modules may regulate synthesis of macromolecules during nutritional stress. J Bacteriol. 2000 Feb;182(3):561–572. doi: 10.1128/jb.182.3.561-572.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gomis-Rüth F. X., Solá M., Acebo P., Párraga A., Guasch A., Eritja R., González A., Espinosa M., del Solar G., Coll M. The structure of plasmid-encoded transcriptional repressor CopG unliganded and bound to its operator. EMBO J. 1998 Dec 15;17(24):7404–7415. doi: 10.1093/emboj/17.24.7404. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Grønlund H., Gerdes K. Toxin-antitoxin systems homologous with relBE of Escherichia coli plasmid P307 are ubiquitous in prokaryotes. J Mol Biol. 1999 Jan 29;285(4):1401–1415. doi: 10.1006/jmbi.1998.2416. [DOI] [PubMed] [Google Scholar]
  15. Heidelberg J. F., Eisen J. A., Nelson W. C., Clayton R. A., Gwinn M. L., Dodson R. J., Haft D. H., Hickey E. K., Peterson J. D., Umayam L. DNA sequence of both chromosomes of the cholera pathogen Vibrio cholerae. Nature. 2000 Aug 3;406(6795):477–483. doi: 10.1038/35020000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Holcík M., Iyer V. N. Conditionally lethal genes associated with bacterial plasmids. Microbiology. 1997 Nov;143(Pt 11):3403–3416. doi: 10.1099/00221287-143-11-3403. [DOI] [PubMed] [Google Scholar]
  17. Jasanoff A., Fersht A. R. Quantitative determination of helical propensities from trifluoroethanol titration curves. Biochemistry. 1994 Mar 1;33(8):2129–2135. doi: 10.1021/bi00174a020. [DOI] [PubMed] [Google Scholar]
  18. Jensen R. B., Gerdes K. Programmed cell death in bacteria: proteic plasmid stabilization systems. Mol Microbiol. 1995 Jul;17(2):205–210. doi: 10.1111/j.1365-2958.1995.mmi_17020205.x. [DOI] [PubMed] [Google Scholar]
  19. Jensen R. B., Grohmann E., Schwab H., Díaz-Orejas R., Gerdes K. Comparison of ccd of F, parDE of RP4, and parD of R1 using a novel conditional replication control system of plasmid R1. Mol Microbiol. 1995 Jul;17(2):211–220. doi: 10.1111/j.1365-2958.1995.mmi_17020211.x. [DOI] [PubMed] [Google Scholar]
  20. Johnson E. P., Strom A. R., Helinski D. R. Plasmid RK2 toxin protein ParE: purification and interaction with the ParD antitoxin protein. J Bacteriol. 1996 Mar;178(5):1420–1429. doi: 10.1128/jb.178.5.1420-1429.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Jones D. T. GenTHREADER: an efficient and reliable protein fold recognition method for genomic sequences. J Mol Biol. 1999 Apr 9;287(4):797–815. doi: 10.1006/jmbi.1999.2583. [DOI] [PubMed] [Google Scholar]
  22. Jones D. T. Protein secondary structure prediction based on position-specific scoring matrices. J Mol Biol. 1999 Sep 17;292(2):195–202. doi: 10.1006/jmbi.1999.3091. [DOI] [PubMed] [Google Scholar]
  23. Le Dantec C., Winter N., Gicquel B., Vincent V., Picardeau M. Genomic sequence and transcriptional analysis of a 23-kilobase mycobacterial linear plasmid: evidence for horizontal transfer and identification of plasmid maintenance systems. J Bacteriol. 2001 Apr;183(7):2157–2164. doi: 10.1128/JB.183.7.2157-2164.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Magnuson R., Lehnherr H., Mukhopadhyay G., Yarmolinsky M. B. Autoregulation of the plasmid addiction operon of bacteriophage P1. J Biol Chem. 1996 Aug 2;271(31):18705–18710. doi: 10.1074/jbc.271.31.18705. [DOI] [PubMed] [Google Scholar]
  25. Masuda Y., Miyakawa K., Nishimura Y., Ohtsubo E. chpA and chpB, Escherichia coli chromosomal homologs of the pem locus responsible for stable maintenance of plasmid R100. J Bacteriol. 1993 Nov;175(21):6850–6856. doi: 10.1128/jb.175.21.6850-6856.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Nooren I. M., Rietveld A. W., Melacini G., Sauer R. T., Kaptein R., Boelens R. The solution structure and dynamics of an Arc repressor mutant reveal premelting conformational changes related to DNA binding. Biochemistry. 1999 May 11;38(19):6035–6042. doi: 10.1021/bi982677t. [DOI] [PubMed] [Google Scholar]
  27. Oberer M., Lindner H., Glatter O., Kratky C., Keller W. Thermodynamic properties and DNA binding of the ParD protein from the broad host-range plasmid RK2/RP4 killing system. Biol Chem. 1999 Dec;380(12):1413–1420. doi: 10.1515/BC.1999.181. [DOI] [PubMed] [Google Scholar]
  28. Phillips S. E. The beta-ribbon DNA recognition motif. Annu Rev Biophys Biomol Struct. 1994;23:671–701. doi: 10.1146/annurev.bb.23.060194.003323. [DOI] [PubMed] [Google Scholar]
  29. Raumann B. E., Rould M. A., Pabo C. O., Sauer R. T. DNA recognition by beta-sheets in the Arc repressor-operator crystal structure. Nature. 1994 Feb 24;367(6465):754–757. doi: 10.1038/367754a0. [DOI] [PubMed] [Google Scholar]
  30. Rawlings D. E. Proteic toxin-antitoxin, bacterial plasmid addiction systems and their evolution with special reference to the pas system of pTF-FC2. FEMS Microbiol Lett. 1999 Jul 15;176(2):269–277. doi: 10.1111/j.1574-6968.1999.tb13672.x. [DOI] [PubMed] [Google Scholar]
  31. Roberts R. C., Spangler C., Helinski D. R. Characteristics and significance of DNA binding activity of plasmid stabilization protein ParD from the broad host-range plasmid RK2. J Biol Chem. 1993 Dec 25;268(36):27109–27117. [PubMed] [Google Scholar]
  32. Ruiz-Echevarría M. J., de Torrontegui G., Giménez-Gallego G., Díaz-Orejas R. Structural and functional comparison between the stability systems ParD of plasmid R1 and Ccd of plasmid F. Mol Gen Genet. 1991 Mar;225(3):355–362. doi: 10.1007/BF00261674. [DOI] [PubMed] [Google Scholar]
  33. Salmon M. A., Van Melderen L., Bernard P., Couturier M. The antidote and autoregulatory functions of the F plasmid CcdA protein: a genetic and biochemical survey. Mol Gen Genet. 1994 Sep 1;244(5):530–538. doi: 10.1007/BF00583904. [DOI] [PubMed] [Google Scholar]
  34. Santos Sierra S., Giraldo R., Díaz Orejas R. Functional interactions between chpB and parD, two homologous conditional killer systems found in the Escherichia coli chromosome and in plasmid R1. FEMS Microbiol Lett. 1998 Nov 1;168(1):51–58. doi: 10.1111/j.1574-6968.1998.tb13254.x. [DOI] [PubMed] [Google Scholar]
  35. Simpson A. J., Reinach F. C., Arruda P., Abreu F. A., Acencio M., Alvarenga R., Alves L. M., Araya J. E., Baia G. S., Baptista C. S. The genome sequence of the plant pathogen Xylella fastidiosa. The Xylella fastidiosa Consortium of the Organization for Nucleotide Sequencing and Analysis. Nature. 2000 Jul 13;406(6792):151–159. doi: 10.1038/35018003. [DOI] [PubMed] [Google Scholar]
  36. Smith A. S., Rawlings D. E. The poison-antidote stability system of the broad-host-range Thiobacillus ferrooxidans plasmid pTF-FC2. Mol Microbiol. 1997 Dec;26(5):961–970. doi: 10.1046/j.1365-2958.1997.6332000.x. [DOI] [PubMed] [Google Scholar]
  37. Somers W. S., Phillips S. E. Crystal structure of the met repressor-operator complex at 2.8 A resolution reveals DNA recognition by beta-strands. Nature. 1992 Oct 1;359(6394):387–393. doi: 10.1038/359387a0. [DOI] [PubMed] [Google Scholar]
  38. Thompson J. D., Higgins D. G., Gibson T. J. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 1994 Nov 11;22(22):4673–4680. doi: 10.1093/nar/22.22.4673. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Van Melderen L., Thi M. H., Lecchi P., Gottesman S., Couturier M., Maurizi M. R. ATP-dependent degradation of CcdA by Lon protease. Effects of secondary structure and heterologous subunit interactions. J Biol Chem. 1996 Nov 1;271(44):27730–27738. doi: 10.1074/jbc.271.44.27730. [DOI] [PubMed] [Google Scholar]
  40. Wishart D. S., Sykes B. D. The 13C chemical-shift index: a simple method for the identification of protein secondary structure using 13C chemical-shift data. J Biomol NMR. 1994 Mar;4(2):171–180. doi: 10.1007/BF00175245. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES