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. 1995 Jul 3;14(13):3200–3205. doi: 10.1002/j.1460-2075.1995.tb07322.x

Rationally designed helix-turn-helix proteins and their conformational changes upon DNA binding.

P Percipalle 1, A Simoncsits 1, S Zakhariev 1, C Guarnaccia 1, R Sánchez 1, S Pongor 1
PMCID: PMC394381  PMID: 7621832

Abstract

Circular dichroism and electrophoretic mobility shift studies were performed to confirm that dimerized N-terminal domains of bacterial repressors containing helix-turn-helix motifs are capable of high-affinity and specific DNA recognition as opposed to the monomeric N-terminal domains. Specific, high-affinity DNA binding proteins were designed and produced in which two copies of the N-terminal 1-62 domain of the bacteriophage 434 repressor are connected either in a dyad-symmetric fashion, with a synthetic linker attached to the C-termini, or as direct sequence repeats. Both molecules bound to their presumptive cognate nearly as tightly as does the natural (full-length and non-covalently dimerized) 434 repressor, showing that covalent dimerization can be used to greatly enhance the binding activity of individual protein segments. Circular dichroism spectroscopy showed a pronounced increase in the alpha-helix content when these new proteins interacted with their cognate DNA and a similar, although 30% lower, increase was also seen upon their interaction with non-cognate DNA. These results imply that a gradual conformational change may occur when helix-turn-helix motifs bind to DNA, and that a scanning mechanism is just as plausible for this motif class as that which is proposed for the more flexible basic-leucine zipper and basic-helix-loop-helix motifs.

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Selected References

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

  1. Aggarwal A. K., Rodgers D. W., Drottar M., Ptashne M., Harrison S. C. Recognition of a DNA operator by the repressor of phage 434: a view at high resolution. Science. 1988 Nov 11;242(4880):899–907. doi: 10.1126/science.3187531. [DOI] [PubMed] [Google Scholar]
  2. Anderson J., Ptashne M., Harrison S. C. Cocrystals of the DNA-binding domain of phage 434 repressor and a synthetic phage 434 operator. Proc Natl Acad Sci U S A. 1984 Mar;81(5):1307–1311. doi: 10.1073/pnas.81.5.1307. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Anthony-Cahill S. J., Benfield P. A., Fairman R., Wasserman Z. R., Brenner S. L., Stafford W. F., 3rd, Altenbach C., Hubbell W. L., DeGrado W. F. Molecular characterization of helix-loop-helix peptides. Science. 1992 Feb 21;255(5047):979–983. doi: 10.1126/science.1312255. [DOI] [PubMed] [Google Scholar]
  4. Arrowsmith C. H., Pachter R., Altman R. B., Iyer S. B., Jardetzky O. Sequence-specific 1H NMR assignments and secondary structure in solution of Escherichia coli trp repressor. Biochemistry. 1990 Jul 10;29(27):6332–6341. doi: 10.1021/bi00479a002. [DOI] [PubMed] [Google Scholar]
  5. Berg O. G., von Hippel P. H. Selection of DNA binding sites by regulatory proteins. II. The binding specificity of cyclic AMP receptor protein to recognition sites. J Mol Biol. 1988 Apr 20;200(4):709–723. doi: 10.1016/0022-2836(88)90482-2. [DOI] [PubMed] [Google Scholar]
  6. Berg O. G., von Hippel P. H. Selection of DNA binding sites by regulatory proteins. Statistical-mechanical theory and application to operators and promoters. J Mol Biol. 1987 Feb 20;193(4):723–750. doi: 10.1016/0022-2836(87)90354-8. [DOI] [PubMed] [Google Scholar]
  7. Brent R., Ptashne M. A eukaryotic transcriptional activator bearing the DNA specificity of a prokaryotic repressor. Cell. 1985 Dec;43(3 Pt 2):729–736. doi: 10.1016/0092-8674(85)90246-6. [DOI] [PubMed] [Google Scholar]
  8. Castagnoli L., Vetriani C., Cesareni G. Linking an easily detectable phenotype to the folding of a common structural motif. Selection of rare turn mutations that prevent the folding of Rop. J Mol Biol. 1994 Apr 8;237(4):378–387. doi: 10.1006/jmbi.1994.1241. [DOI] [PubMed] [Google Scholar]
  9. Ellenberger T. E., Brandl C. J., Struhl K., Harrison S. C. The GCN4 basic region leucine zipper binds DNA as a dimer of uninterrupted alpha helices: crystal structure of the protein-DNA complex. Cell. 1992 Dec 24;71(7):1223–1237. doi: 10.1016/s0092-8674(05)80070-4. [DOI] [PubMed] [Google Scholar]
  10. Ferré-D'Amaré A. R., Prendergast G. C., Ziff E. B., Burley S. K. Recognition by Max of its cognate DNA through a dimeric b/HLH/Z domain. Nature. 1993 May 6;363(6424):38–45. doi: 10.1038/363038a0. [DOI] [PubMed] [Google Scholar]
  11. Harrison S. C., Aggarwal A. K. DNA recognition by proteins with the helix-turn-helix motif. Annu Rev Biochem. 1990;59:933–969. doi: 10.1146/annurev.bi.59.070190.004441. [DOI] [PubMed] [Google Scholar]
  12. Hu J. C., Newell N. E., Tidor B., Sauer R. T. Probing the roles of residues at the e and g positions of the GCN4 leucine zipper by combinatorial mutagenesis. Protein Sci. 1993 Jul;2(7):1072–1084. doi: 10.1002/pro.5560020701. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hu J. C., O'Shea E. K., Kim P. S., Sauer R. T. Sequence requirements for coiled-coils: analysis with lambda repressor-GCN4 leucine zipper fusions. Science. 1990 Dec 7;250(4986):1400–1403. doi: 10.1126/science.2147779. [DOI] [PubMed] [Google Scholar]
  14. Johnson N. P., Lindstrom J., Baase W. A., von Hippel P. H. Double-stranded DNA templates can induce alpha-helical conformation in peptides containing lysine and alanine: functional implications for leucine zipper and helix-loop-helix transcription factors. Proc Natl Acad Sci U S A. 1994 May 24;91(11):4840–4844. doi: 10.1073/pnas.91.11.4840. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kuziel W. A., Tucker P. W. Determination of vector: insert junctions in lambda gt10 cDNAs that do not recut with EcoRI. Nucleotide sequence of the lambda imm434 HindIII-EcoRI DNA fragment encoding part of the cI protein. Nucleic Acids Res. 1987 Apr 10;15(7):3181–3181. doi: 10.1093/nar/15.7.3181. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. König P., Richmond T. J. The X-ray structure of the GCN4-bZIP bound to ATF/CREB site DNA shows the complex depends on DNA flexibility. J Mol Biol. 1993 Sep 5;233(1):139–154. doi: 10.1006/jmbi.1993.1490. [DOI] [PubMed] [Google Scholar]
  17. Neri D., Billeter M., Wüthrich K. Determination of the nuclear magnetic resonance solution structure of the DNA-binding domain (residues 1 to 69) of the 434 repressor and comparison with the X-ray crystal structure. J Mol Biol. 1992 Feb 5;223(3):743–767. doi: 10.1016/0022-2836(92)90987-u. [DOI] [PubMed] [Google Scholar]
  18. O'Neil K. T., Hoess R. H., DeGrado W. F. Design of DNA-binding peptides based on the leucine zipper motif. Science. 1990 Aug 17;249(4970):774–778. doi: 10.1126/science.2389143. [DOI] [PubMed] [Google Scholar]
  19. O'Neil K. T., Shuman J. D., Ampe C., DeGrado W. F. DNA-induced increase in the alpha-helical content of C/EBP and GCN4. Biochemistry. 1991 Sep 17;30(37):9030–9034. doi: 10.1021/bi00101a017. [DOI] [PubMed] [Google Scholar]
  20. Percipalle P., Saletti R., Pongor S., Foti S., Tossi A., Fisichella S. Structural characterization of synthetic model peptides of the DNA-binding cI434 repressor by electrospray ionization and fast atom bombardment mass spectrometry. Biol Mass Spectrom. 1994 Dec;23(12):727–733. doi: 10.1002/bms.1200231203. [DOI] [PubMed] [Google Scholar]
  21. Pu W. T., Struhl K. Dimerization of leucine zippers analyzed by random selection. Nucleic Acids Res. 1993 Sep 11;21(18):4348–4355. doi: 10.1093/nar/21.18.4348. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Saudek V., Pasley H. S., Gibson T., Gausepohl H., Frank R., Pastore A. Solution structure of the basic region from the transcriptional activator GCN4. Biochemistry. 1991 Feb 5;30(5):1310–1317. doi: 10.1021/bi00219a022. [DOI] [PubMed] [Google Scholar]
  23. Schmidt-Dörr T., Oertel-Buchheit P., Pernelle C., Bracco L., Schnarr M., Granger-Schnarr M. Construction, purification, and characterization of a hybrid protein comprising the DNA binding domain of the LexA repressor and the Jun leucine zipper: a circular dichroism and mutagenesis study. Biochemistry. 1991 Oct 8;30(40):9657–9664. doi: 10.1021/bi00104a013. [DOI] [PubMed] [Google Scholar]
  24. Schoepfer R. The pRSET family of T7 promoter expression vectors for Escherichia coli. Gene. 1993 Feb 14;124(1):83–85. doi: 10.1016/0378-1119(93)90764-t. [DOI] [PubMed] [Google Scholar]
  25. Simoncsits A., Bristulf J., Tjörnhammar M. L., Cserzö M., Pongor S., Rybakina E., Gatti S., Bartfai T. Deletion mutants of human interleukin 1 beta with significantly reduced agonist properties: search for the agonist/antagonist switch in ligands to the interleukin 1 receptors. Cytokine. 1994 Mar;6(2):206–214. doi: 10.1016/1043-4666(94)90043-4. [DOI] [PubMed] [Google Scholar]
  26. Spolar R. S., Record M. T., Jr Coupling of local folding to site-specific binding of proteins to DNA. Science. 1994 Feb 11;263(5148):777–784. doi: 10.1126/science.8303294. [DOI] [PubMed] [Google Scholar]
  27. Studier F. W., Rosenberg A. H., Dunn J. J., Dubendorff J. W. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 1990;185:60–89. doi: 10.1016/0076-6879(90)85008-c. [DOI] [PubMed] [Google Scholar]
  28. Talanian R. V., McKnight C. J., Kim P. S. Sequence-specific DNA binding by a short peptide dimer. Science. 1990 Aug 17;249(4970):769–771. doi: 10.1126/science.2389142. [DOI] [PubMed] [Google Scholar]
  29. Tsao D. H., Gruschus J. M., Wang L. H., Nirenberg M., Ferretti J. A. Elongation of helix III of the NK-2 homeodomain upon binding to DNA: a secondary structure study by NMR. Biochemistry. 1994 Dec 20;33(50):15053–15060. doi: 10.1021/bi00254a014. [DOI] [PubMed] [Google Scholar]
  30. Vinson C. R., Sigler P. B., McKnight S. L. Scissors-grip model for DNA recognition by a family of leucine zipper proteins. Science. 1989 Nov 17;246(4932):911–916. doi: 10.1126/science.2683088. [DOI] [PubMed] [Google Scholar]
  31. Weiss M. A., Ellenberger T., Wobbe C. R., Lee J. P., Harrison S. C., Struhl K. Folding transition in the DNA-binding domain of GCN4 on specific binding to DNA. Nature. 1990 Oct 11;347(6293):575–578. doi: 10.1038/347575a0. [DOI] [PubMed] [Google Scholar]
  32. Wu C. S., Ikeda K., Yang J. T. Ordered conformation of polypeptides and proteins in acidic dodecyl sulfate solution. Biochemistry. 1981 Feb 3;20(3):566–570. doi: 10.1021/bi00506a019. [DOI] [PubMed] [Google Scholar]
  33. Zhang H., Zhao D., Revington M., Lee W., Jia X., Arrowsmith C., Jardetzky O. The solution structures of the trp repressor-operator DNA complex. J Mol Biol. 1994 May 13;238(4):592–614. doi: 10.1006/jmbi.1994.1317. [DOI] [PubMed] [Google Scholar]
  34. von Hippel P. H., Berg O. G. On the specificity of DNA-protein interactions. Proc Natl Acad Sci U S A. 1986 Mar;83(6):1608–1612. doi: 10.1073/pnas.83.6.1608. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. von Hippel P. H. Protein-DNA recognition: new perspectives and underlying themes. Science. 1994 Feb 11;263(5148):769–770. doi: 10.1126/science.8303292. [DOI] [PubMed] [Google Scholar]

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