Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

BAX activation is initiated at a novel interaction site

Nature volume 455, pages 1076–1081 (2008)Cite this article

Abstract

BAX is a pro-apoptotic protein of the BCL-2 family that is stationed in the cytosol until activated by a diversity of stress stimuli to induce cell death. Anti-apoptotic proteins such as BCL-2 counteract BAX-mediated cell death. Although an interaction site that confers survival functionality has been defined for anti-apoptotic proteins, an activation site has not been identified for BAX, rendering its explicit trigger mechanism unknown. We previously developed stabilized α-helix of BCL-2 domains (SAHBs) that directly initiate BAX-mediated mitochondrial apoptosis. Here we demonstrate by NMR analysis that BIM SAHB binds BAX at an interaction site that is distinct from the canonical binding groove characterized for anti-apoptotic proteins. The specificity of the human BIM-SAHB–BAX interaction is highlighted by point mutagenesis that disrupts functional activity, confirming that BAX activation is initiated at this novel structural location. Thus, we have now defined a BAX interaction site for direct activation, establishing a new target for therapeutic modulation of apoptosis.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Learn more

Prices may be subject to local taxes which are calculated during checkout

Figure 1: NMR analysis of BAX on BIM SAHB titration.
Figure 2: Orientation of BIM SAHB at the BAX binding site.
Figure 3: BIM SAHB directly initiates BAX activation in vitro.
Figure 4: Sequence specificity of BIM-SAHB-induced BAX activation.
Figure 5: Mutagenesis of the BAX interaction site impairs activation and BAX-mediated apoptosis.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

The structural coordinates have been submitted to the Protein Data Bank under accession number 2K7W.

References

  1. Danial, N. N. & Korsmeyer, S. J. Cell death: critical control points. Cell 116, 205–219 (2004)

    Article  CAS  Google Scholar 

  2. Leber, B., Lin, J. & Andrews, D. W. Embedded together: the life and death consequences of interaction of the Bcl-2 family with membranes. Apoptosis 12, 897–911 (2007)

    Article  CAS  Google Scholar 

  3. Youle, R. J. & Strasser, A. The BCL-2 protein family: opposing activities that mediate cell death. Nature Rev. Mol. Cell Biol. 9, 47–59 (2008)

    Article  CAS  Google Scholar 

  4. Petros, A. M., Olejniczak, E. T. & Fesik, S. W. Structural biology of the Bcl-2 family of proteins. Biochim. Biophys. Acta 1644, 83–94 (2004)

    Article  CAS  Google Scholar 

  5. Huang, D. C. & Strasser, A. BH3-only proteins—essential initiators of apoptotic cell death. Cell 103, 839–842 (2000)

    Article  CAS  Google Scholar 

  6. Walensky, L. D. BCL-2 in the crosshairs: tipping the balance of life and death. Cell Death Differ. 13, 1339–1350 (2006)

    Article  CAS  Google Scholar 

  7. Cheng, E. H. et al. BCL-2, BCL-X(L) sequester BH3 domain-only molecules preventing BAX- and BAK-mediated mitochondrial apoptosis. Mol. Cell 8, 705–711 (2001)

    Article  CAS  Google Scholar 

  8. Wei, M. C. et al. Proapoptotic BAX and BAK: a requisite gateway to mitochondrial dysfunction and death. Science 292, 727–730 (2001)

    Article  CAS  ADS  Google Scholar 

  9. Li, P. et al. Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell 91, 479–489 (1997)

    Article  CAS  Google Scholar 

  10. Sattler, M. et al. Structure of Bcl-xL–Bak peptide complex: recognition between regulators of apoptosis. Science 275, 983–986 (1997)

    Article  CAS  Google Scholar 

  11. Certo, M. et al. Mitochondria primed by death signals determine cellular addiction to antiapoptotic BCL-2 family members. Cancer Cell 9, 351–365 (2006)

    Article  CAS  Google Scholar 

  12. Chen, L. et al. Differential targeting of prosurvival Bcl-2 proteins by their BH3-only ligands allows complementary apoptotic function. Mol. Cell 17, 393–403 (2005)

    Article  CAS  Google Scholar 

  13. Kim, H. et al. Hierarchical regulation of mitochondrion-dependent apoptosis by BCL-2 subfamilies. Nature Cell Biol. 8, 1348–1358 (2006)

    Article  CAS  Google Scholar 

  14. Oltval, Z. N., Milliman, C. L. & Korsmeyer, S. J. Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death. Cell 74, 609–619 (1993)

    Article  Google Scholar 

  15. Willis, S. N. et al. Proapoptotic Bak is sequestered by Mcl-1 and Bcl-xL, but not Bcl-2, until displaced by BH3-only proteins. Genes Dev. 19, 1294–1305 (2005)

    Article  CAS  Google Scholar 

  16. Zhai, D., Jin, C., Huang, Z., Satterthwait, A. C. & Reed, J. C. Differential regulation of Bax and Bak by anti-apoptotic Bcl-2 family proteins Bcl-B and Mcl-1. J. Biol. Chem. 283, 9580–9586 (2008)

    Article  CAS  Google Scholar 

  17. Oltersdorf, T. et al. An inhibitor of Bcl-2 family proteins induces regression of solid tumours. Nature 435, 677–681 (2005)

    Article  CAS  ADS  Google Scholar 

  18. Tse, C. et al. ABT-263: a potent and orally bioavailable Bcl-2 family inhibitor. Cancer Res. 68, 3421–3428 (2008)

    Article  CAS  Google Scholar 

  19. Willis, S. N. et al. Apoptosis initiated when BH3 ligands engage multiple Bcl-2 homologs, not Bax or Bak. Science 315, 856–859 (2007)

    Article  CAS  ADS  Google Scholar 

  20. Annis, M. G. et al. Bax forms multispanning monomers that oligomerize to permeabilize membranes during apoptosis. EMBO J. 24, 2096–2103 (2005)

    Article  CAS  MathSciNet  Google Scholar 

  21. Goping, I. S. et al. Regulated targeting of BAX to mitochondria. J. Cell Biol. 143, 207–215 (1998)

    Article  CAS  Google Scholar 

  22. Gross, A., Jockel, J., Wei, M. C. & Korsmeyer, S. J. Enforced dimerization of BAX results in its translocation, mitochondrial dysfunction and apoptosis. EMBO J. 17, 3878–3885 (1998)

    Article  CAS  Google Scholar 

  23. Wolter, K. G. et al. Movement of Bax from the cytosol to mitochondria during apoptosis. J. Cell Biol. 139, 1281–1292 (1997)

    Article  CAS  Google Scholar 

  24. Chipuk, J. E. et al. Direct activation of Bax by p53 mediates mitochondrial membrane permeabilization and apoptosis. Science 303, 1010–1014 (2004)

    Article  CAS  ADS  Google Scholar 

  25. Nie, C. et al. Cysteine 62 of Bax is critical for its conformational activation and its proapoptotic activity in response to H2O2-induced apoptosis. J. Biol. Chem. 283, 15359–15369 (2008)

    Article  CAS  Google Scholar 

  26. Pagliari, L. J. et al. The multidomain proapoptotic molecules Bax and Bak are directly activated by heat. Proc. Natl Acad. Sci. USA 102, 17975–17980 (2005)

    Article  CAS  ADS  Google Scholar 

  27. Cartron, P. F. et al. The first α helix of Bax plays a necessary role in its ligand-induced activation by the BH3-only proteins Bid and PUMA. Mol. Cell 16, 807–818 (2004)

    Article  CAS  Google Scholar 

  28. Harada, H., Quearry, B., Ruiz-Vela, A. & Korsmeyer, S. J. Survival factor-induced extracellular signal-regulated kinase phosphorylates BIM, inhibiting its association with BAX and proapoptotic activity. Proc. Natl Acad. Sci. USA 101, 15313–15317 (2004)

    Article  CAS  ADS  Google Scholar 

  29. Kuwana, T. et al. BH3 domains of BH3-only proteins differentially regulate Bax-mediated mitochondrial membrane permeabilization both directly and indirectly. Mol. Cell 17, 525–535 (2005)

    Article  CAS  Google Scholar 

  30. Kuwana, T. et al. Bid, Bax, and lipids cooperate to form supramolecular openings in the outer mitochondrial membrane. Cell 111, 331–342 (2002)

    Article  CAS  Google Scholar 

  31. Letai, A. et al. Distinct BH3 domains either sensitize or activate mitochondrial apoptosis, serving as prototype cancer therapeutics. Cancer Cell 2, 183–192 (2002)

    Article  CAS  Google Scholar 

  32. Marani, M., Tenev, T., Hancock, D., Downward, J. & Lemoine, N. R. Identification of novel isoforms of the BH3 domain protein Bim which directly activate Bax to trigger apoptosis. Mol. Cell. Biol. 22, 3577–3589 (2002)

    Article  CAS  Google Scholar 

  33. Walensky, L. D. et al. A stapled BID BH3 helix directly binds and activates BAX. Mol. Cell 24, 199–210 (2006)

    Article  CAS  Google Scholar 

  34. Wang, K., Yin, X. M., Chao, D. T., Milliman, C. L. & Korsmeyer, S. J. BID: a novel BH3 domain-only death agonist. Genes Dev. 10, 2859–2869 (1996)

    Article  CAS  Google Scholar 

  35. Suzuki, M., Youle, R. J. & Tjandra, N. Structure of Bax: coregulation of dimer formation and intracellular localization. Cell 103, 645–654 (2000)

    Article  CAS  Google Scholar 

  36. Battiste, J. L. & Wagner, G. Utilization of site-directed spin labeling and high-resolution heteronuclear nuclear magnetic resonance for global fold determination of large proteins with limited nuclear Overhauser effect data. Biochemistry 39, 5355–5365 (2000)

    Article  CAS  Google Scholar 

  37. Hsu, Y. T. & Youle, R. J. Nonionic detergents induce dimerization among members of the Bcl-2 family. J. Biol. Chem. 272, 13829–13834 (1997)

    Article  CAS  Google Scholar 

  38. Villunger, A. et al. p53- and drug-induced apoptotic responses mediated by BH3-only proteins Puma and Noxa. Science 302, 1036–1038 (2003)

    Article  CAS  ADS  Google Scholar 

  39. Chen, D. & Zhou, Q. Caspase cleavage of BimEL triggers a positive feedback amplification of apoptotic signaling. Proc. Natl Acad. Sci. USA 101, 1235–1240 (2004)

    Article  CAS  ADS  Google Scholar 

  40. Hsu, Y. T., Wolter, K. G. & Youle, R. J. Cytosol-to-membrane redistribution of Bax and Bcl-XL during apoptosis. Proc. Natl Acad. Sci. USA 94, 3668–3672 (1997)

    Article  CAS  ADS  Google Scholar 

  41. Muchmore, S. W. et al. X-ray and NMR structure of human Bcl-xL, an inhibitor of programmed cell death. Nature 381, 335–341 (1996)

    Article  CAS  ADS  Google Scholar 

  42. Cartron, P. F. et al. Involvement of the N-terminus of Bax in its intracellular localization and function. FEBS Lett. 512, 95–100 (2002)

    Article  CAS  Google Scholar 

  43. 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. 18, 2330–2341 (1999)

    Article  CAS  Google Scholar 

  44. Schinzel, A. et al. Conformational control of Bax localization and apoptotic activity by Pro168. J. Cell Biol. 164, 1021–1032 (2004)

    Article  CAS  Google Scholar 

  45. Dlugosz, P. J. et al. Bcl-2 changes conformation to inhibit Bax oligomerization. EMBO J. 25, 2287–2296 (2006)

    Article  CAS  Google Scholar 

  46. Bird, G. H., Bernal, F., Pitter, K. & Walensky, L. D. Synthesis and biophysical characterization of stabilized α-helices of BCL-2 domains. Methods Enzymol. 446, 369–386 (2008)

    Article  CAS  Google Scholar 

  47. Walensky, L. D. et al. Activation of apoptosis in vivo by a hydrocarbon-stapled BH3 helix. Science 305, 1466–1470 (2004)

    Article  CAS  ADS  Google Scholar 

  48. Pitter, K., Bernal, F., Labelle, J. & Walensky, L. D. Dissection of the BCL-2 family signaling network with stabilized α-helices of BCL-2 domains. Methods Enzymol. 446, 387–408 (2008)

    Article  CAS  Google Scholar 

  49. Grzesiek, S. & Bax, A. The importance of not saturating water in protein NMR. Application to sensitivity enhancement and NOE measurements. J. Am. Chem. Soc. 115, 12593–12594 (1993)

    Article  CAS  Google Scholar 

  50. DeLano, W. L. The PyMOL Molecular Graphics System. <http://www.pymol.org> (DeLano Scientific, 2002)

Download references

Acknowledgements

We thank E. Smith for editorial and graphics assistance, W. Beavers for amino acid analyses, A. Perry for technical assistance, C. Turner and A. Bielecki of the MIT/Harvard Center for Magnetic Resonance for NMR technical advice, and R. Youle for feedback on the manuscript. We acknowledge the indelible contributions of the late S. J. Korsmeyer, who inspired this work. L.D.W. is supported by National Cancer Institute (NCI) grant 5P01CA92625, a Burroughs Wellcome Fund Career Award in the Biomedical Sciences, a Culpeper Scholarship in Medical Science from the Goldman Philanthropic Partnerships, an American Society of Hematology Junior Faculty Scholar Award, and a grant from the William Lawrence Children’s Foundation. This research was also supported by NCI grant 5RO1CA50239. N.T. is supported by the Intramural Research Program of the National Heart, Lung and Blood Institute, NIH. E.H.-Y.C. is supported by the Searle Scholars Program and NCI grant 5RO1CA125562.

Author Contributions G.H.B. and L.D.W. designed, synthesized and characterized the SAHBs for structural and biological studies. M.S. and N.T. performed the NMR analysis of BAX and BIM SAHB, and E.G. and L.D.W. conducted the PRE NMR analysis of BAX using MTSL-derivatized SAHBs and performed the structure calculations. E.G., M.L.D. and K.P. executed the in vitro BAX activation studies. H.-C.T., H.K. and E.H.-Y.C. generated the BAX-reconstituted DKO MEFs and analysed their response to staurosporine, and S.G.K. examined the cellular response to BIM SAHB treatment.

Author information

Author notes

  1. Evripidis Gavathiotis and Motoshi Suzuki: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Pediatric Oncology and the Program in Cancer Chemical Biology, Dana-Farber Cancer Institute, 44 Binney Street, Boston, Massachusetts 02115, USA,

    Evripidis Gavathiotis, Marguerite L. Davis, Kenneth Pitter, Gregory H. Bird, Samuel G. Katz & Loren D. Walensky

  2. Division of Hematology/Oncology, Children’s Hospital Boston, 300 Longwood Avenue, Boston, Massachusetts 02115, USA,

    Evripidis Gavathiotis, Marguerite L. Davis, Kenneth Pitter, Gregory H. Bird, Samuel G. Katz & Loren D. Walensky

  3. Department of Pediatrics, Harvard Medical School, Boston, Massachusetts 02115, USA,

    Evripidis Gavathiotis, Marguerite L. Davis, Kenneth Pitter, Gregory H. Bird, Samuel G. Katz & Loren D. Walensky

  4. Laboratory of Molecular Biophysics, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892, USA ,

    Motoshi Suzuki & Nico Tjandra

  5. Departments of Internal Medicine and Pathology and Immunology, Washington University School of Medicine, Saint Louis, Missouri 63110, USA,

    Ho-Chou Tu, Hyungjin Kim & Emily H.-Y. Cheng

Authors
  1. Evripidis Gavathiotis
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Motoshi Suzuki
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Marguerite L. Davis
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Kenneth Pitter
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Gregory H. Bird
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Samuel G. Katz
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Ho-Chou Tu
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Hyungjin Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Emily H.-Y. Cheng
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Nico Tjandra
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Loren D. Walensky
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Nico Tjandra or Loren D. Walensky.

Ethics declarations

Competing interests

L.D.W. is a scientific advisory board member and consultant for Aileron Therapeutics, Inc.

Supplementary information

Supplementary Information

This file contains Supplementary Figures S1-S10 with Legends, Supplementary Table 1, Supplementary Methods and Supplementary Notes (PDF 858 kb)

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Gavathiotis, E., Suzuki, M., Davis, M. et al. BAX activation is initiated at a novel interaction site. Nature 455, 1076–1081 (2008). https://doi.org/10.1038/nature07396

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature07396

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing