Skip to main content
PMC home page
Protein Science : A Publication of the Protein Society logoLink to Protein Science : A Publication of the Protein Society
. 1999 Sep;8(9):1773–1779. doi: 10.1110/ps.8.9.1773

Change in oligomerization specificity of the p53 tetramerization domain by hydrophobic amino acid substitutions.

E S Stavridi 1, N H Chehab 1, L C Caruso 1, T D Halazonetis 1
PMCID: PMC2144399  PMID: 10493578

Abstract

The tumor suppressor function of the wild-type p53 protein is transdominantly inhibited by tumor-derived mutant p53 proteins. Such transdominant inhibition limits the prospects for gene therapy approaches that aim to introduce wild-type p53 into cancer cells. The molecular mechanism for transdominant inhibition involves sequestration of wild-type p53 subunits into inactive wild-type/mutant hetero-tetramers. Thus, p53 proteins, whose oligomerization specificity is altered so they cannot interact with tumor-derived mutant p53, would escape transdominant inhibition. Aided by the known three-dimensional structure of the p53 tetramerization domain and by trial and error we designed a novel domain with seven amino acid substitutions in the hydrophobic core. A full-length p53 protein bearing this novel domain formed homo-tetramers and had tumor suppressor function, but did not hetero-oligomerize with tumor-derived mutant p53 and resisted transdominant inhibition. Thus, hydrophobic core residues influence the oligomerization specificity of the p53 tetramerization domain.

Full Text

The Full Text of this article is available as a PDF (542.1 KB).

Selected References

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

  1. Baker S. J., Markowitz S., Fearon E. R., Willson J. K., Vogelstein B. Suppression of human colorectal carcinoma cell growth by wild-type p53. Science. 1990 Aug 24;249(4971):912–915. doi: 10.1126/science.2144057. [DOI] [PubMed] [Google Scholar]
  2. Bargonetti J., Manfredi J. J., Chen X., Marshak D. R., Prives C. A proteolytic fragment from the central region of p53 has marked sequence-specific DNA-binding activity when generated from wild-type but not from oncogenic mutant p53 protein. Genes Dev. 1993 Dec;7(12B):2565–2574. doi: 10.1101/gad.7.12b.2565. [DOI] [PubMed] [Google Scholar]
  3. Bargonetti J., Reynisdóttir I., Friedman P. N., Prives C. Site-specific binding of wild-type p53 to cellular DNA is inhibited by SV40 T antigen and mutant p53. Genes Dev. 1992 Oct;6(10):1886–1898. doi: 10.1101/gad.6.10.1886. [DOI] [PubMed] [Google Scholar]
  4. Clarke A. R., Purdie C. A., Harrison D. J., Morris R. G., Bird C. C., Hooper M. L., Wyllie A. H. Thymocyte apoptosis induced by p53-dependent and independent pathways. Nature. 1993 Apr 29;362(6423):849–852. doi: 10.1038/362849a0. [DOI] [PubMed] [Google Scholar]
  5. Clore G. M., Ernst J., Clubb R., Omichinski J. G., Kennedy W. M., Sakaguchi K., Appella E., Gronenborn A. M. Refined solution structure of the oligomerization domain of the tumour suppressor p53. Nat Struct Biol. 1995 Apr;2(4):321–333. doi: 10.1038/nsb0495-321. [DOI] [PubMed] [Google Scholar]
  6. Clore G. M., Omichinski J. G., Sakaguchi K., Zambrano N., Sakamoto H., Appella E., Gronenborn A. M. High-resolution structure of the oligomerization domain of p53 by multidimensional NMR. Science. 1994 Jul 15;265(5170):386–391. doi: 10.1126/science.8023159. [DOI] [PubMed] [Google Scholar]
  7. Conseiller E., Debussche L., Landais D., Venot C., Maratrat M., Sierra V., Tocque B., Bracco L. CTS1: a p53-derived chimeric tumor suppressor gene with enhanced in vitro apoptotic properties. J Clin Invest. 1998 Jan 1;101(1):120–127. doi: 10.1172/JCI1140. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Diller L., Kassel J., Nelson C. E., Gryka M. A., Litwak G., Gebhardt M., Bressac B., Ozturk M., Baker S. J., Vogelstein B. p53 functions as a cell cycle control protein in osteosarcomas. Mol Cell Biol. 1990 Nov;10(11):5772–5781. doi: 10.1128/mcb.10.11.5772. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Eliyahu D., Michalovitz D., Eliyahu S., Pinhasi-Kimhi O., Oren M. Wild-type p53 can inhibit oncogene-mediated focus formation. Proc Natl Acad Sci U S A. 1989 Nov;86(22):8763–8767. doi: 10.1073/pnas.86.22.8763. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Favrot M., Coll J. L., Louis N., Negoescu A. Cell death and cancer: replacement of apoptotic genes and inactivation of death suppressor genes in therapy. Gene Ther. 1998 Jun;5(6):728–739. doi: 10.1038/sj.gt.3300661. [DOI] [PubMed] [Google Scholar]
  11. Fields S., Jang S. K. Presence of a potent transcription activating sequence in the p53 protein. Science. 1990 Aug 31;249(4972):1046–1049. doi: 10.1126/science.2144363. [DOI] [PubMed] [Google Scholar]
  12. Finlay C. A., Hinds P. W., Levine A. J. The p53 proto-oncogene can act as a suppressor of transformation. Cell. 1989 Jun 30;57(7):1083–1093. doi: 10.1016/0092-8674(89)90045-7. [DOI] [PubMed] [Google Scholar]
  13. Hainaut P., Hall A., Milner J. Analysis of p53 quaternary structure in relation to sequence-specific DNA binding. Oncogene. 1994 Jan;9(1):299–303. [PubMed] [Google Scholar]
  14. Halazonetis T. D., Kandil A. N. Conformational shifts propagate from the oligomerization domain of p53 to its tetrameric DNA binding domain and restore DNA binding to select p53 mutants. EMBO J. 1993 Dec 15;12(13):5057–5064. doi: 10.1002/j.1460-2075.1993.tb06199.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hedrick J. L., Smith A. J. Size and charge isomer separation and estimation of molecular weights of proteins by disc gel electrophoresis. Arch Biochem Biophys. 1968 Jul;126(1):155–164. doi: 10.1016/0003-9861(68)90569-9. [DOI] [PubMed] [Google Scholar]
  16. Hollstein M., Sidransky D., Vogelstein B., Harris C. C. p53 mutations in human cancers. Science. 1991 Jul 5;253(5015):49–53. doi: 10.1126/science.1905840. [DOI] [PubMed] [Google Scholar]
  17. Jeffrey P. D., Gorina S., Pavletich N. P. Crystal structure of the tetramerization domain of the p53 tumor suppressor at 1.7 angstroms. Science. 1995 Mar 10;267(5203):1498–1502. doi: 10.1126/science.7878469. [DOI] [PubMed] [Google Scholar]
  18. Kern S. E., Pietenpol J. A., Thiagalingam S., Seymour A., Kinzler K. W., Vogelstein B. Oncogenic forms of p53 inhibit p53-regulated gene expression. Science. 1992 May 8;256(5058):827–830. doi: 10.1126/science.1589764. [DOI] [PubMed] [Google Scholar]
  19. Kohn W. D., Kay C. M., Hodges R. S. Orientation, positional, additivity, and oligomerization-state effects of interhelical ion pairs in alpha-helical coiled-coils. J Mol Biol. 1998 Nov 13;283(5):993–1012. doi: 10.1006/jmbi.1998.2125. [DOI] [PubMed] [Google Scholar]
  20. Kuerbitz S. J., Plunkett B. S., Walsh W. V., Kastan M. B. Wild-type p53 is a cell cycle checkpoint determinant following irradiation. Proc Natl Acad Sci U S A. 1992 Aug 15;89(16):7491–7495. doi: 10.1073/pnas.89.16.7491. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. 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]
  22. Lee W., Harvey T. S., Yin Y., Yau P., Litchfield D., Arrowsmith C. H. Solution structure of the tetrameric minimum transforming domain of p53. Nat Struct Biol. 1994 Dec;1(12):877–890. doi: 10.1038/nsb1294-877. [DOI] [PubMed] [Google Scholar]
  23. Levine A. J. p53, the cellular gatekeeper for growth and division. Cell. 1997 Feb 7;88(3):323–331. doi: 10.1016/s0092-8674(00)81871-1. [DOI] [PubMed] [Google Scholar]
  24. Lowe S. W., Schmitt E. M., Smith S. W., Osborne B. A., Jacks T. p53 is required for radiation-induced apoptosis in mouse thymocytes. Nature. 1993 Apr 29;362(6423):847–849. doi: 10.1038/362847a0. [DOI] [PubMed] [Google Scholar]
  25. Lumb K. J., Kim P. S. Measurement of interhelical electrostatic interactions in the GCN4 leucine zipper. Science. 1995 Apr 21;268(5209):436–439. doi: 10.1126/science.7716550. [DOI] [PubMed] [Google Scholar]
  26. Martinez J., Georgoff I., Martinez J., Levine A. J. Cellular localization and cell cycle regulation by a temperature-sensitive p53 protein. Genes Dev. 1991 Feb;5(2):151–159. doi: 10.1101/gad.5.2.151. [DOI] [PubMed] [Google Scholar]
  27. McCoy M., Stavridi E. S., Waterman J. L., Wieczorek A. M., Opella S. J., Halazonetis T. D. Hydrophobic side-chain size is a determinant of the three-dimensional structure of the p53 oligomerization domain. EMBO J. 1997 Oct 15;16(20):6230–6236. doi: 10.1093/emboj/16.20.6230. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Mercer W. E., Shields M. T., Amin M., Sauve G. J., Appella E., Romano J. W., Ullrich S. J. Negative growth regulation in a glioblastoma tumor cell line that conditionally expresses human wild-type p53. Proc Natl Acad Sci U S A. 1990 Aug;87(16):6166–6170. doi: 10.1073/pnas.87.16.6166. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Milner J., Medcalf E. A. Cotranslation of activated mutant p53 with wild type drives the wild-type p53 protein into the mutant conformation. Cell. 1991 May 31;65(5):765–774. doi: 10.1016/0092-8674(91)90384-b. [DOI] [PubMed] [Google Scholar]
  30. Nielsen L. L., Maneval D. C. P53 tumor suppressor gene therapy for cancer. Cancer Gene Ther. 1998 Jan-Feb;5(1):52–63. [PubMed] [Google Scholar]
  31. O'Shea E. K., Klemm J. D., Kim P. S., Alber T. X-ray structure of the GCN4 leucine zipper, a two-stranded, parallel coiled coil. Science. 1991 Oct 25;254(5031):539–544. doi: 10.1126/science.1948029. [DOI] [PubMed] [Google Scholar]
  32. O'Shea E. K., Rutkowski R., Kim P. S. Evidence that the leucine zipper is a coiled coil. Science. 1989 Jan 27;243(4890):538–542. doi: 10.1126/science.2911757. [DOI] [PubMed] [Google Scholar]
  33. O'Shea E. K., Rutkowski R., Kim P. S. Mechanism of specificity in the Fos-Jun oncoprotein heterodimer. Cell. 1992 Feb 21;68(4):699–708. doi: 10.1016/0092-8674(92)90145-3. [DOI] [PubMed] [Google Scholar]
  34. Pietenpol J. A., Tokino T., Thiagalingam S., el-Deiry W. S., Kinzler K. W., Vogelstein B. Sequence-specific transcriptional activation is essential for growth suppression by p53. Proc Natl Acad Sci U S A. 1994 Mar 15;91(6):1998–2002. doi: 10.1073/pnas.91.6.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Reed M., Wang Y., Mayr G., Anderson M. E., Schwedes J. F., Tegtmeyer P. p53 domains: suppression, transformation, and transactivation. Gene Expr. 1993;3(1):95–107. [PMC free article] [PubMed] [Google Scholar]
  36. Roth J. A., Nguyen D., Lawrence D. D., Kemp B. L., Carrasco C. H., Ferson D. Z., Hong W. K., Komaki R., Lee J. J., Nesbitt J. C. Retrovirus-mediated wild-type p53 gene transfer to tumors of patients with lung cancer. Nat Med. 1996 Sep;2(9):985–991. doi: 10.1038/nm0996-985. [DOI] [PubMed] [Google Scholar]
  37. Sakamoto H., Lewis M. S., Kodama H., Appella E., Sakaguchi K. Specific sequences from the carboxyl terminus of human p53 gene product form anti-parallel tetramers in solution. Proc Natl Acad Sci U S A. 1994 Sep 13;91(19):8974–8978. doi: 10.1073/pnas.91.19.8974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Shaulian E., Zauberman A., Ginsberg D., Oren M. Identification of a minimal transforming domain of p53: negative dominance through abrogation of sequence-specific DNA binding. Mol Cell Biol. 1992 Dec;12(12):5581–5592. doi: 10.1128/mcb.12.12.5581. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Shaw P., Bovey R., Tardy S., Sahli R., Sordat B., Costa J. Induction of apoptosis by wild-type p53 in a human colon tumor-derived cell line. Proc Natl Acad Sci U S A. 1992 May 15;89(10):4495–4499. doi: 10.1073/pnas.89.10.4495. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Soussi T., May P. Structural aspects of the p53 protein in relation to gene evolution: a second look. J Mol Biol. 1996 Aug 2;260(5):623–637. doi: 10.1006/jmbi.1996.0425. [DOI] [PubMed] [Google Scholar]
  41. Unger T., Mietz J. A., Scheffner M., Yee C. L., Howley P. M. Functional domains of wild-type and mutant p53 proteins involved in transcriptional regulation, transdominant inhibition, and transformation suppression. Mol Cell Biol. 1993 Sep;13(9):5186–5194. doi: 10.1128/mcb.13.9.5186. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Wang P., Reed M., Wang Y., Mayr G., Stenger J. E., Anderson M. E., Schwedes J. F., Tegtmeyer P. p53 domains: structure, oligomerization, and transformation. Mol Cell Biol. 1994 Aug;14(8):5182–5191. doi: 10.1128/mcb.14.8.5182. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Waterman J. L., Shenk J. L., Halazonetis T. D. The dihedral symmetry of the p53 tetramerization domain mandates a conformational switch upon DNA binding. EMBO J. 1995 Feb 1;14(3):512–519. doi: 10.1002/j.1460-2075.1995.tb07027.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Waterman M. J., Stavridi E. S., Waterman J. L., Halazonetis T. D. ATM-dependent activation of p53 involves dephosphorylation and association with 14-3-3 proteins. Nat Genet. 1998 Jun;19(2):175–178. doi: 10.1038/542. [DOI] [PubMed] [Google Scholar]
  45. Waterman M. J., Waterman J. L., Halazonetis T. D. An engineered four-stranded coiled coil substitutes for the tetramerization domain of wild-type p53 and alleviates transdominant inhibition by tumor-derived p53 mutants. Cancer Res. 1996 Jan 1;56(1):158–163. [PubMed] [Google Scholar]
  46. Wieczorek A. M., Waterman J. L., Waterman M. J., Halazonetis T. D. Structure-based rescue of common tumor-derived p53 mutants. Nat Med. 1996 Oct;2(10):1143–1146. doi: 10.1038/nm1096-1143. [DOI] [PubMed] [Google Scholar]
  47. Yonish-Rouach E., Resnitzky D., Lotem J., Sachs L., Kimchi A., Oren M. Wild-type p53 induces apoptosis of myeloid leukaemic cells that is inhibited by interleukin-6. Nature. 1991 Jul 25;352(6333):345–347. doi: 10.1038/352345a0. [DOI] [PubMed] [Google Scholar]
  48. Zeng X., Herndon A. M., Hu J. C. Buried asparagines determine the dimerization specificities of leucine zipper mutants. Proc Natl Acad Sci U S A. 1997 Apr 15;94(8):3673–3678. doi: 10.1073/pnas.94.8.3673. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Zeng X., Zhu H., Lashuel H. A., Hu J. C. Oligomerization properties of GCN4 leucine zipper e and g position mutants. Protein Sci. 1997 Oct;6(10):2218–2226. doi: 10.1002/pro.5560061016. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Protein Science : A Publication of the Protein Society are provided here courtesy of The Protein Society

RESOURCES