Skip to main content
PMC home page
The EMBO Journal logoLink to The EMBO Journal
. 1998 Jan 15;17(2):535–543. doi: 10.1093/emboj/17.2.535

Determinants of protein-protein recognition by four helix bundles: changing the dimerization specificity of Tet repressor.

D Schnappinger 1, P Schubert 1, K Pfleiderer 1, W Hillen 1
PMCID: PMC1170403  PMID: 9430644

Abstract

Homo- and heterodimerization is essential for the activity of many proteins, particularly transcription factors. One widely distributed structural motif for protein recognition is the four helix bundle. To understand the molecular details determining specificity of subunit recognition in a dimer formed by a four helix bundle, we investigated Tet repressor (TetR) sequence variants TetR(B) and TetR(D), which do not form heterodimers. We used molecular modeling to identify residues with the potential to determine recognition of subunits. Directed mutagenesis of these residues in TetR(B) by the TetR(D) sequence resulted in chimeric TetR(B/D) repressors with new subunit recognition specificities. The single LS192 exchange in TetR(B/D)192 in the center of the helix bundle leads to a relaxed specificity since this variant dimerizes with TetR(B) and (D). To construct a variant with a new specificity it was not sufficient to mutate the contacting residue, F197, in the other subunit. Instead, it was necessary to exchange two more residues in the vicinity of F197 and S192. The resulting TetR(B/D)188, 192,193,197 forms dimers with TetR(D) but not with TetR(B), indicating that four amino acid exchanges are sufficient to change subunit recognition. These results establish that targeted alterations in the structural complementarity of protein-protein interaction surfaces can be used to construct new recognition specificities. However, it is not sufficient to adjust the complementary residues since the surrounding amino acids contribute essentially to protein-protein recognition.

Full Text

The Full Text of this article is available as a PDF (507.6 KB).

Selected References

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

  1. Altschmied L., Baumeister R., Pfleiderer K., Hillen W. A threonine to alanine exchange at position 40 of Tet repressor alters the recognition of the sixth base pair of tet operator from GC to AT. EMBO J. 1988 Dec 1;7(12):4011–4017. doi: 10.1002/j.1460-2075.1988.tb03290.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Backes H., Berens C., Helbl V., Walter S., Schmid F. X., Hillen W. Combinations of the alpha-helix-turn-alpha-helix motif of TetR with respective residues from LacI or 434Cro: DNA recognition, inducer binding, and urea-dependent denaturation. Biochemistry. 1997 May 6;36(18):5311–5322. doi: 10.1021/bi961527k. [DOI] [PubMed] [Google Scholar]
  3. Berens C., Altschmied L., Hillen W. The role of the N terminus in Tet repressor for tet operator binding determined by a mutational analysis. J Biol Chem. 1992 Jan 25;267(3):1945–1952. [PubMed] [Google Scholar]
  4. Berens C., Pfleiderer K., Helbl V., Hillen W. Deletion mutagenesis of Tn 10 Tet repressor--localization of regions important for dimerization and inducibility in vivo. Mol Microbiol. 1995 Nov;18(3):437–448. doi: 10.1111/j.1365-2958.1995.mmi_18030437.x. [DOI] [PubMed] [Google Scholar]
  5. Berens C., Schnappinger D., Hillen W. The role of the variable region in Tet repressor for inducibility by tetracycline. J Biol Chem. 1997 Mar 14;272(11):6936–6942. doi: 10.1074/jbc.272.11.6936. [DOI] [PubMed] [Google Scholar]
  6. Davies D. R., Cohen G. H. Interactions of protein antigens with antibodies. Proc Natl Acad Sci U S A. 1996 Jan 9;93(1):7–12. doi: 10.1073/pnas.93.1.7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Garrell J., Modolell J. The Drosophila extramacrochaetae locus, an antagonist of proneural genes that, like these genes, encodes a helix-loop-helix protein. Cell. 1990 Apr 6;61(1):39–48. doi: 10.1016/0092-8674(90)90213-x. [DOI] [PubMed] [Google Scholar]
  8. Gordon C. L., King J. Temperature-sensitive mutations in the phage P22 coat protein which interfere with polypeptide chain folding. J Biol Chem. 1993 May 5;268(13):9358–9368. [PubMed] [Google Scholar]
  9. Hai T., Curran T. Cross-family dimerization of transcription factors Fos/Jun and ATF/CREB alters DNA binding specificity. Proc Natl Acad Sci U S A. 1991 May 1;88(9):3720–3724. doi: 10.1073/pnas.88.9.3720. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Herskowitz I. Functional inactivation of genes by dominant negative mutations. Nature. 1987 Sep 17;329(6136):219–222. doi: 10.1038/329219a0. [DOI] [PubMed] [Google Scholar]
  11. Hillen W., Berens C. Mechanisms underlying expression of Tn10 encoded tetracycline resistance. Annu Rev Microbiol. 1994;48:345–369. doi: 10.1146/annurev.mi.48.100194.002021. [DOI] [PubMed] [Google Scholar]
  12. Hinrichs W., Kisker C., Düvel M., Müller A., Tovar K., Hillen W., Saenger W. Structure of the Tet repressor-tetracycline complex and regulation of antibiotic resistance. Science. 1994 Apr 15;264(5157):418–420. doi: 10.1126/science.8153629. [DOI] [PubMed] [Google Scholar]
  13. Hsu H. L., Wadman I., Tsan J. T., Baer R. Positive and negative transcriptional control by the TAL1 helix-loop-helix protein. Proc Natl Acad Sci U S A. 1994 Jun 21;91(13):5947–5951. doi: 10.1073/pnas.91.13.5947. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Jones S., Thornton J. M. Principles of protein-protein interactions. Proc Natl Acad Sci U S A. 1996 Jan 9;93(1):13–20. doi: 10.1073/pnas.93.1.13. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kallipolitis B. H., Nørregaard-Madsen M., Valentin-Hansen P. Protein-protein communication: structural model of the repression complex formed by CytR and the global regulator CRP. Cell. 1997 Jun 27;89(7):1101–1109. doi: 10.1016/s0092-8674(00)80297-4. [DOI] [PubMed] [Google Scholar]
  16. Kouzarides T., Ziff E. The role of the leucine zipper in the fos-jun interaction. Nature. 1988 Dec 15;336(6200):646–651. doi: 10.1038/336646a0. [DOI] [PubMed] [Google Scholar]
  17. Landschulz W. H., Johnson P. F., McKnight S. L. The leucine zipper: a hypothetical structure common to a new class of DNA binding proteins. Science. 1988 Jun 24;240(4860):1759–1764. doi: 10.1126/science.3289117. [DOI] [PubMed] [Google Scholar]
  18. Landt O., Grunert H. P., Hahn U. A general method for rapid site-directed mutagenesis using the polymerase chain reaction. Gene. 1990 Nov 30;96(1):125–128. doi: 10.1016/0378-1119(90)90351-q. [DOI] [PubMed] [Google Scholar]
  19. Lavigne P., Kondejewski L. H., Houston M. E., Jr, Sönnichsen F. D., Lix B., Skyes B. D., Hodges R. S., Kay C. M. Preferential heterodimeric parallel coiled-coil formation by synthetic Max and c-Myc leucine zippers: a description of putative electrostatic interactions responsible for the specificity of heterodimerization. J Mol Biol. 1995 Dec 1;254(3):505–520. doi: 10.1006/jmbi.1995.0634. [DOI] [PubMed] [Google Scholar]
  20. Lin S. L., Tsai C. J., Nussinov R. A study of four-helix bundles: investigating protein folding via similar architectural motifs in protein cores and in subunit interfaces. J Mol Biol. 1995 Apr 21;248(1):151–161. doi: 10.1006/jmbi.1995.0208. [DOI] [PubMed] [Google Scholar]
  21. Lupas A. Coiled coils: new structures and new functions. Trends Biochem Sci. 1996 Oct;21(10):375–382. [PubMed] [Google Scholar]
  22. Müller G., Hecht B., Helbl V., Hinrichs W., Saenger W., Hillen W. Characterization of non-inducible Tet repressor mutants suggests conformational changes necessary for induction. Nat Struct Biol. 1995 Aug;2(8):693–703. doi: 10.1038/nsb0895-693. [DOI] [PubMed] [Google Scholar]
  23. Nakabeppu Y., Nathans D. A naturally occurring truncated form of FosB that inhibits Fos/Jun transcriptional activity. Cell. 1991 Feb 22;64(4):751–759. doi: 10.1016/0092-8674(91)90504-r. [DOI] [PubMed] [Google Scholar]
  24. Neet K. E., Timm D. E. Conformational stability of dimeric proteins: quantitative studies by equilibrium denaturation. Protein Sci. 1994 Dec;3(12):2167–2174. doi: 10.1002/pro.5560031202. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Nossal N. G. Protein-protein interactions at a DNA replication fork: bacteriophage T4 as a model. FASEB J. 1992 Feb 1;6(3):871–878. doi: 10.1096/fasebj.6.3.1310946. [DOI] [PubMed] [Google Scholar]
  26. Pabo C. O., Sauer R. T. Transcription factors: structural families and principles of DNA recognition. Annu Rev Biochem. 1992;61:1053–1095. doi: 10.1146/annurev.bi.61.070192.005201. [DOI] [PubMed] [Google Scholar]
  27. Ptashne M., Gann A. Transcriptional activation by recruitment. Nature. 1997 Apr 10;386(6625):569–577. doi: 10.1038/386569a0. [DOI] [PubMed] [Google Scholar]
  28. Sancar A. Mechanisms of DNA excision repair. Science. 1994 Dec 23;266(5193):1954–1956. doi: 10.1126/science.7801120. [DOI] [PubMed] [Google Scholar]
  29. Schreiber G., Fersht A. R. Energetics of protein-protein interactions: analysis of the barnase-barstar interface by single mutations and double mutant cycles. J Mol Biol. 1995 Apr 28;248(2):478–486. doi: 10.1016/s0022-2836(95)80064-6. [DOI] [PubMed] [Google Scholar]
  30. Smith L. D., Bertrand K. P. Mutations in the Tn10 tet repressor that interfere with induction. Location of the tetracycline-binding domain. J Mol Biol. 1988 Oct 20;203(4):949–959. doi: 10.1016/0022-2836(88)90120-9. [DOI] [PubMed] [Google Scholar]
  31. Vinson C. R., Hai T., Boyd S. M. Dimerization specificity of the leucine zipper-containing bZIP motif on DNA binding: prediction and rational design. Genes Dev. 1993 Jun;7(6):1047–1058. doi: 10.1101/gad.7.6.1047. [DOI] [PubMed] [Google Scholar]
  32. Wells J. A. Binding in the growth hormone receptor complex. Proc Natl Acad Sci U S A. 1996 Jan 9;93(1):1–6. doi: 10.1073/pnas.93.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Wissmann A., Wray L. V., Jr, Somaggio U., Baumeister R., Geissendörfer M., Hillen W. Selection for Tn10 tet repressor binding to tet operator in Escherichia coli: isolation of temperature-sensitive mutants and combinatorial mutagenesis in the DNA binding motif. Genetics. 1991 Jun;128(2):225–232. doi: 10.1093/genetics/128.2.225. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Yu M. H., King J. Single amino acid substitutions influencing the folding pathway of the phage P22 tail spike endorhamnosidase. Proc Natl Acad Sci U S A. 1984 Nov;81(21):6584–6588. doi: 10.1073/pnas.81.21.6584. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Zechel C., Shen X. Q., Chen J. Y., Chen Z. P., Chambon P., Gronemeyer H. The dimerization interfaces formed between the DNA binding domains of RXR, RAR and TR determine the binding specificity and polarity of the full-length receptors to direct repeats. EMBO J. 1994 Mar 15;13(6):1425–1433. doi: 10.1002/j.1460-2075.1994.tb06396.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES