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. 2002 Nov 15;368(Pt 1):213–221. doi: 10.1042/BJ20020836

Evidence for crucial electrostatic interactions between Bcl-2 homology domains BH3 and BH4 in the anti-apoptotic Nr-13 protein.

Philippe Lalle 1, Abdel Aouacheria 1, Agnès Dumont-Miscopein 1, Martin Jambon 1, Séverine Venet 1, Hélène Bobichon 1, Pierre Colas 1, Gilbert Deléage 1, Christophe Geourjon 1, Germain Gillet 1
PMCID: PMC1222957  PMID: 12133006

Abstract

Nr-13 is an anti-apoptotic member of the Bcl-2 family previously shown to interact with Bax. The biological significance of this interaction was explored both in yeast and vertebrate cells and revealed that Nr-13 is able to counteract the pro-apoptotic activity of Bax. The Bax-interacting domain has been identified and corresponds to alpha-helices 5 and 6 in Nr-13. Site-directed mutagenesis has revealed that the N-terminal region of Nr-13 is essential for activity and corresponds to a genuine Bcl-2 homology domain (BH4). The modelling of Nr-13, based on its similarity with other Bcl-2 family proteins and energy minimization, suggests the possibility of electrostatic interactions between the two N-terminal-conserved domains BH4 and BH3. Disruption of these interactions severely affects Nr-13 anti-apoptotic activity. Together our results suggest that electrostatic interactions between BH4 and BH3 domains play a role in the control of activity of Nr-13 and a subset of Bcl-2 family members.

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Selected References

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

  1. Adams J. M., Cory S. Life-or-death decisions by the Bcl-2 protein family. Trends Biochem Sci. 2001 Jan;26(1):61–66. doi: 10.1016/s0968-0004(00)01740-0. [DOI] [PubMed] [Google Scholar]
  2. Adams J. M., Cory S. The Bcl-2 protein family: arbiters of cell survival. Science. 1998 Aug 28;281(5381):1322–1326. doi: 10.1126/science.281.5381.1322. [DOI] [PubMed] [Google Scholar]
  3. Aouacheria A., Arnaud E., Venet S., Lalle P., Gouy M., Rigal D., Gillet G. Nrh, a human homologue of Nr-13 associates with Bcl-Xs and is an inhibitor of apoptosis. Oncogene. 2001 Sep 13;20(41):5846–5855. doi: 10.1038/sj.onc.1204740. [DOI] [PubMed] [Google Scholar]
  4. Blagosklonny M. V. Unwinding the loop of Bcl-2 phosphorylation. Leukemia. 2001 Jun;15(6):869–874. doi: 10.1038/sj.leu.2402134. [DOI] [PubMed] [Google Scholar]
  5. Chen Y. H., Yang J. T., Chau K. H. Determination of the helix and beta form of proteins in aqueous solution by circular dichroism. Biochemistry. 1974 Jul 30;13(16):3350–3359. doi: 10.1021/bi00713a027. [DOI] [PubMed] [Google Scholar]
  6. Combet Christophe, Jambon Martin, Deléage Gilbert, Geourjon Christophe. Geno3D: automatic comparative molecular modelling of protein. Bioinformatics. 2002 Jan;18(1):213–214. doi: 10.1093/bioinformatics/18.1.213. [DOI] [PubMed] [Google Scholar]
  7. Evan G., Littlewood T. A matter of life and cell death. Science. 1998 Aug 28;281(5381):1317–1322. doi: 10.1126/science.281.5381.1317. [DOI] [PubMed] [Google Scholar]
  8. Finley R. L., Jr, Brent R. Interaction mating reveals binary and ternary connections between Drosophila cell cycle regulators. Proc Natl Acad Sci U S A. 1994 Dec 20;91(26):12980–12984. doi: 10.1073/pnas.91.26.12980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Geourjon C., Deléage G. SOPMA: significant improvements in protein secondary structure prediction by consensus prediction from multiple alignments. Comput Appl Biosci. 1995 Dec;11(6):681–684. doi: 10.1093/bioinformatics/11.6.681. [DOI] [PubMed] [Google Scholar]
  10. Gietz R. D., Schiestl R. H., Willems A. R., Woods R. A. Studies on the transformation of intact yeast cells by the LiAc/SS-DNA/PEG procedure. Yeast. 1995 Apr 15;11(4):355–360. doi: 10.1002/yea.320110408. [DOI] [PubMed] [Google Scholar]
  11. Gillet G., Guerin M., Trembleau A., Brun G. A Bcl-2-related gene is activated in avian cells transformed by the Rous sarcoma virus. EMBO J. 1995 Apr 3;14(7):1372–1381. doi: 10.1002/j.1460-2075.1995.tb07123.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gillet G., Michel D., Crisanti P., Guérin M., Herault Y., Pessac B., Calothy G., Brun G., Volovitch M. Serum factors and v-src control two complementary mitogenic pathways in quail neuroretinal cells in culture. Oncogene. 1993 Mar;8(3):565–574. [PubMed] [Google Scholar]
  13. Greenhalf W., Stephan C., Chaudhuri B. Role of mitochondria and C-terminal membrane anchor of Bcl-2 in Bax induced growth arrest and mortality in Saccharomyces cerevisiae. FEBS Lett. 1996 Feb 12;380(1-2):169–175. doi: 10.1016/0014-5793(96)00044-0. [DOI] [PubMed] [Google Scholar]
  14. Gross A., McDonnell J. M., Korsmeyer S. J. BCL-2 family members and the mitochondria in apoptosis. Genes Dev. 1999 Aug 1;13(15):1899–1911. doi: 10.1101/gad.13.15.1899. [DOI] [PubMed] [Google Scholar]
  15. Hanada M., Aimé-Sempé C., Sato T., Reed J. C. Structure-function analysis of Bcl-2 protein. Identification of conserved domains important for homodimerization with Bcl-2 and heterodimerization with Bax. J Biol Chem. 1995 May 19;270(20):11962–11969. doi: 10.1074/jbc.270.20.11962. [DOI] [PubMed] [Google Scholar]
  16. Hirotani M., Zhang Y., Fujita N., Naito M., Tsuruo T. NH2-terminal BH4 domain of Bcl-2 is functional for heterodimerization with Bax and inhibition of apoptosis. J Biol Chem. 1999 Jul 16;274(29):20415–20420. doi: 10.1074/jbc.274.29.20415. [DOI] [PubMed] [Google Scholar]
  17. Huang D. C., Adams J. M., Cory S. The conserved N-terminal BH4 domain of Bcl-2 homologues is essential for inhibition of apoptosis and interaction with CED-4. EMBO J. 1998 Feb 16;17(4):1029–1039. doi: 10.1093/emboj/17.4.1029. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kabsch W., Sander C. How good are predictions of protein secondary structure? FEBS Lett. 1983 May 8;155(2):179–182. doi: 10.1016/0014-5793(82)80597-8. [DOI] [PubMed] [Google Scholar]
  19. Ke N., Godzik A., Reed J. C. Bcl-B, a novel Bcl-2 family member that differentially binds and regulates Bax and Bak. J Biol Chem. 2001 Feb 21;276(16):12481–12484. doi: 10.1074/jbc.C000871200. [DOI] [PubMed] [Google Scholar]
  20. King R. D., Sternberg M. J. Identification and application of the concepts important for accurate and reliable protein secondary structure prediction. Protein Sci. 1996 Nov;5(11):2298–2310. doi: 10.1002/pro.5560051116. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Lee L. C., Hunter J. J., Mujeeb A., Turck C., Parslow T. G. Evidence for alpha-helical conformation of an essential N-terminal region in the human Bcl2 protein. J Biol Chem. 1996 Sep 20;271(38):23284–23288. doi: 10.1074/jbc.271.38.23284. [DOI] [PubMed] [Google Scholar]
  22. Lee R. M., Gillet G., Burnside J., Thomas S. J., Neiman P. Role of Nr13 in regulation of programmed cell death in the bursa of Fabricius. Genes Dev. 1999 Mar 15;13(6):718–728. doi: 10.1101/gad.13.6.718. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Lee R., Chen J., Matthews C. P., McDougall J. K., Neiman P. E. Characterization of NR13-related human cell death regulator, Boo/Diva, in normal and cancer tissues. Biochim Biophys Acta. 2001 Sep 21;1520(3):187–194. doi: 10.1016/s0167-4781(01)00268-8. [DOI] [PubMed] [Google Scholar]
  24. Ligr M., Madeo F., Fröhlich E., Hilt W., Fröhlich K. U., Wolf D. H. Mammalian Bax triggers apoptotic changes in yeast. FEBS Lett. 1998 Oct 30;438(1-2):61–65. doi: 10.1016/s0014-5793(98)01227-7. [DOI] [PubMed] [Google Scholar]
  25. Mangeney M., Schmitt J. R., Leverrier Y., Thomas J., Marvel J., Brun G., Gillet G. The product of the v-src-inducible gene nr-13 is a potent anti-apoptotic factor. Oncogene. 1996 Oct 3;13(7):1441–1446. [PubMed] [Google Scholar]
  26. McDonnell J. M., Fushman D., Milliman C. L., Korsmeyer S. J., Cowburn D. Solution structure of the proapoptotic molecule BID: a structural basis for apoptotic agonists and antagonists. Cell. 1999 Mar 5;96(5):625–634. doi: 10.1016/s0092-8674(00)80573-5. [DOI] [PubMed] [Google Scholar]
  27. Minn A. J., Kettlun C. S., Liang H., Kelekar A., Vander Heiden M. G., Chang B. S., Fesik S. W., Fill M., Thompson C. B. Bcl-xL regulates apoptosis by heterodimerization-dependent and -independent mechanisms. EMBO J. 1999 Feb 1;18(3):632–643. doi: 10.1093/emboj/18.3.632. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Moradi-Améli Mahnaz, Lorca Thierry, Ficheux Damien, di Pietro Attilio, Gillet Germain. Interaction between the antiapoptotic protein Nr-13 and cytochrome c. Antagonistic effect of the BH3 domain of Bax. Biochemistry. 2002 Jul 9;41(27):8540–8550. doi: 10.1021/bi0110286. [DOI] [PubMed] [Google Scholar]
  29. Muchmore S. W., Sattler M., Liang H., Meadows R. P., Harlan J. E., Yoon H. S., Nettesheim D., Chang B. S., Thompson C. B., Wong S. L. X-ray and NMR structure of human Bcl-xL, an inhibitor of programmed cell death. Nature. 1996 May 23;381(6580):335–341. doi: 10.1038/381335a0. [DOI] [PubMed] [Google Scholar]
  30. Nechushtan A., Smith C. L., Hsu Y. T., Youle R. J. Conformation of the Bax C-terminus regulates subcellular location and cell death. EMBO J. 1999 May 4;18(9):2330–2341. doi: 10.1093/emboj/18.9.2330. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Nechushtan A., Smith C. L., Lamensdorf I., Yoon S. H., Youle R. J. Bax and Bak coalesce into novel mitochondria-associated clusters during apoptosis. J Cell Biol. 2001 Jun 11;153(6):1265–1276. doi: 10.1083/jcb.153.6.1265. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Oltvai Z. N., Milliman C. L., Korsmeyer S. J. Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death. Cell. 1993 Aug 27;74(4):609–619. doi: 10.1016/0092-8674(93)90509-o. [DOI] [PubMed] [Google Scholar]
  33. Priault M., Camougrand N., Chaudhuri B., Manon S. Role of the C-terminal domain of Bax and Bcl-XL in their localization and function in yeast cells. FEBS Lett. 1999 Jan 25;443(2):225–228. doi: 10.1016/s0014-5793(98)01661-5. [DOI] [PubMed] [Google Scholar]
  34. Reed J. C. Double identity for proteins of the Bcl-2 family. Nature. 1997 Jun 19;387(6635):773–776. doi: 10.1038/42867. [DOI] [PubMed] [Google Scholar]
  35. Rost B., Sander C. Prediction of protein secondary structure at better than 70% accuracy. J Mol Biol. 1993 Jul 20;232(2):584–599. doi: 10.1006/jmbi.1993.1413. [DOI] [PubMed] [Google Scholar]
  36. Sato T., Hanada M., Bodrug S., Irie S., Iwama N., Boise L. H., Thompson C. B., Golemis E., Fong L., Wang H. G. Interactions among members of the Bcl-2 protein family analyzed with a yeast two-hybrid system. Proc Natl Acad Sci U S A. 1994 Sep 27;91(20):9238–9242. doi: 10.1073/pnas.91.20.9238. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Schendel S. L., Xie Z., Montal M. O., Matsuyama S., Montal M., Reed J. C. Channel formation by antiapoptotic protein Bcl-2. Proc Natl Acad Sci U S A. 1997 May 13;94(10):5113–5118. doi: 10.1073/pnas.94.10.5113. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Shibasaki F., Kondo E., Akagi T., McKeon F. Suppression of signalling through transcription factor NF-AT by interactions between calcineurin and Bcl-2. Nature. 1997 Apr 17;386(6626):728–731. doi: 10.1038/386728a0. [DOI] [PubMed] [Google Scholar]
  39. Shimizu S., Konishi A., Kodama T., Tsujimoto Y. BH4 domain of antiapoptotic Bcl-2 family members closes voltage-dependent anion channel and inhibits apoptotic mitochondrial changes and cell death. Proc Natl Acad Sci U S A. 2000 Mar 28;97(7):3100–3105. doi: 10.1073/pnas.97.7.3100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Shitashige M., Toi M., Yano T., Shibata M., Matsuo Y., Shibasaki F. Dissociation of Bax from a Bcl-2/Bax heterodimer triggered by phosphorylation of serine 70 of Bcl-2. J Biochem. 2001 Dec;130(6):741–748. doi: 10.1093/oxfordjournals.jbchem.a003044. [DOI] [PubMed] [Google Scholar]
  41. Simonen M., Keller H., Heim J. The BH3 domain of Bax is sufficient for interaction of Bax with itself and with other family members and it is required for induction of apoptosis. Eur J Biochem. 1997 Oct 1;249(1):85–91. doi: 10.1111/j.1432-1033.1997.t01-1-00085.x. [DOI] [PubMed] [Google Scholar]
  42. Suzuki M., Youle R. J., Tjandra N. Structure of Bax: coregulation of dimer formation and intracellular localization. Cell. 2000 Nov 10;103(4):645–654. doi: 10.1016/s0092-8674(00)00167-7. [DOI] [PubMed] [Google Scholar]
  43. Tao W., Kurschner C., Morgan J. I. Modulation of cell death in yeast by the Bcl-2 family of proteins. J Biol Chem. 1997 Jun 13;272(24):15547–15552. doi: 10.1074/jbc.272.24.15547. [DOI] [PubMed] [Google Scholar]
  44. 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]
  45. Wang K., Gross A., Waksman G., Korsmeyer S. J. Mutagenesis of the BH3 domain of BAX identifies residues critical for dimerization and killing. Mol Cell Biol. 1998 Oct;18(10):6083–6089. doi: 10.1128/mcb.18.10.6083. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Wang K., Yin X. M., Chao D. T., Milliman C. L., Korsmeyer S. J. BID: a novel BH3 domain-only death agonist. Genes Dev. 1996 Nov 15;10(22):2859–2869. doi: 10.1101/gad.10.22.2859. [DOI] [PubMed] [Google Scholar]
  47. Wei M. C., Zong W. X., Cheng E. H., Lindsten T., Panoutsakopoulou V., Ross A. J., Roth K. A., MacGregor G. R., Thompson C. B., Korsmeyer S. J. Proapoptotic BAX and BAK: a requisite gateway to mitochondrial dysfunction and death. Science. 2001 Apr 27;292(5517):727–730. doi: 10.1126/science.1059108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Zakharov S. D., Lindeberg M., Griko Y., Salamon Z., Tollin G., Prendergast F. G., Cramer W. A. Membrane-bound state of the colicin E1 channel domain as an extended two-dimensional helical array. Proc Natl Acad Sci U S A. 1998 Apr 14;95(8):4282–4287. doi: 10.1073/pnas.95.8.4282. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Zhang H., Cowan-Jacob S. W., Simonen M., Greenhalf W., Heim J., Meyhack B. Structural basis of BFL-1 for its interaction with BAX and its anti-apoptotic action in mammalian and yeast cells. J Biol Chem. 2000 Apr 14;275(15):11092–11099. doi: 10.1074/jbc.275.15.11092. [DOI] [PubMed] [Google Scholar]
  50. Zhang H., Holzgreve W., De Geyter C. Bcl2-L-10, a novel anti-apoptotic member of the Bcl-2 family, blocks apoptosis in the mitochondria death pathway but not in the death receptor pathway. Hum Mol Genet. 2001 Oct 1;10(21):2329–2339. doi: 10.1093/hmg/10.21.2329. [DOI] [PubMed] [Google Scholar]

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