Abstract
Interactions between the BCL-2 family proteins determine the cell's fate to live or die. How they interact with each other to regulate apoptosis remains as an unsettled central issue. So far, the antiapoptotic BCL-2 proteins are thought to interact with BAX weakly, but the physiological significance of this interaction has been vague. Herein, we show that recombinant BCL-2 and BCL-w interact potently with a BCL-2 homology (BH) 3 domain-containing peptide derived from BAX, exhibiting the dissociation constants of 15 and 23 nM, respectively. To clarify the basis for this strong interaction, we determined the three-dimensional structure of a complex of BCL-2 with a BAX peptide spanning its BH3 domain. It revealed that their interactions extended beyond the canonical BH3 domain and involved three nonconserved charged residues of BAX. A novel BAX variant, containing the alanine substitution of these three residues, had greatly impaired affinity for BCL-2 and BCL-w, but was otherwise indistinguishable from wild-type BAX. Critically, the apoptotic activity of the BAX variant could not be restrained by BCL-2 and BCL-w, pointing that the observed tight interactions are critical for regulating BAX activation. We also comprehensively quantified the binding affinities between the three BCL-2 subfamily proteins. Collectively, the data show that due to the high affinity of BAX for BCL-2, BCL-w and A1, and of BAK for BCL-XL, MCL-1 and A1, only a subset of BH3-only proteins, commonly including BIM, BID and PUMA, could be expected to free BAX or BAK from the antiapoptotic BCL-2 proteins to elicit apoptosis.
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References
Adams JM, Cory S . Life-or-death decisions by the Bcl-2 protein family. Trends Biochem Sci 2001; 26:61–66.
Opferman JT, Korsmeyer SJ . Apoptosis in the development and maintenance of the immune system. Nat Immunol 2003; 4:410–415.
Antignani A, Youle RJ . How do Bax and Bak lead to permeabilization of the outer mitochondrial membrane? Curr Opin Cell Biol 2006; 18:685–689.
Green DR, Kroemer G . The pathophysiology of mitochondrial cell death. Science 2004; 305:626–629.
Kim H, Rafiuddin-Shah M, Tu HC, et al. Hierarchical regulation of mitochondrion-dependent apoptosis by BCL-2 subfamilies. Nat Cell Biol 2006; 8:1348–1358.
Willis SN, Fletcher JI, Kaufmann T, et al. Apoptosis initiated when BH3 ligands engage multiple Bcl-2 homologs, not Bax or Bak. Science 2007; 315:856–859.
Sattler M, Liang H, Nettesheim D, et al. Structure of Bcl-xL-Bak peptide complex: recognition between regulators of apoptosis. Science 1997; 275:983–986.
Liu X, Dai S, Zhu Y, Marrack P, Kappler JW . The structure of a Bcl-xL/Bim fragment complex: implications for Bim function. Immunity 2003; 19:341–352.
Petros AM, Nettesheim DG, Wang Y, et al. Rationale for Bcl-xL/Bad peptide complex formation from structure, mutagenesis, and biophysical studies. Protein Sci 2000; 9:2528–2534.
Letai A, Bassik MC, Walensky LD, Sorcinelli MD, Weiler S, Korsmeyer SJ . Distinct BH3 domains either sensitize or activate mitochondrial apoptosis, serving as prototype cancer therapeutics. Cancer Cell 2002; 2:183–192.
Cartron PF, Gallenne T, Bougras G, et al. The first alpha helix of Bax plays a necessary role in its ligand-induced activation by the BH3-only proteins Bid and PUMA. Mol Cell 2004; 16:807–818.
Certo M, Del Gaizo Moore V, Nishino M, et al. Mitochondria primed by death signals determine cellular addiction to antiapoptotic BCL-2 family members. Cancer Cell 2006; 9:351–365.
Kim H, Tu HC, Ren D, et al. Stepwise activation of BAX and BAK by tBID, BIM, and PUMA initiates mitochondrial apoptosis. Mol Cell 2009; 36:487–499.
Adams JM, Cory S . Bcl-2-regulated apoptosis: mechanism and therapeutic potential. Curr Opin Immunol 2007; 19:488–496.
Uren RT, Dewson G, Chen L, et al. Mitochondrial permeabilization relies on BH3 ligands engaging multiple prosurvival Bcl-2 relatives, not Bak. J Cell Biol 2007; 177:277–287.
Fletcher JI, Meusburger S, Hawkins CJ, et al. Apoptosis is triggered when prosurvival Bcl-2 proteins cannot restrain Bax. Proc Natl Acad Sci USA 2008; 105:18081–18087.
Youle RJ, Strasser A . The BCL-2 protein family: opposing activities that mediate cell death. Nat Rev Mol Cell Biol 2008; 9:47–59.
Zhai D, Jin C, Huang Z, Satterthwait AC, Reed JC . Differential regulation of Bax and Bak by anti-apoptotic Bcl-2 family proteins Bcl-B and Mcl-1. J Biol Chem 2008; 283:9580–9586.
Liu D, Yang B, Cao R, Huang Z . A chemical strategy to promote helical peptide-protein interactions involved in apoptosis. Bioorg Med Chem Lett 2005; 15:4467–4469.
Chen L, Willis SN, Wei A, et al. Differential targeting of prosurvival Bcl-2 proteins by their BH3-only ligands allows complementary apoptotic function. Mol Cell 2005; 17:393–403.
Ku B, Woo JS, Liang C, et al. Structural and biochemical bases for the inhibition of autophagy and apoptosis by viral BCL-2 of murine gamma-herpesvirus 68. PLoS Pathog 2008; 4:e25.
Willis SN, Chen L, Dewson G, et al. Proapoptotic Bak is sequestered by Mcl-1 and Bcl-xL, but not Bcl-2, until displaced by BH3-only proteins. Genes Dev 2005; 19:1294–1305.
Hsu YT, Youle RJ . Bax in murine thymus is a soluble monomeric protein that displays differential detergent-induced conformations. J Biol Chem 1998; 273:10777–10783.
Simmons MJ, Fan G, Zong WX, Degenhardt K, White E, Gelinas C . Bfl-1/A1 functions, similar to Mcl-1, as a selective tBid and Bak antagonist. Oncogene 2008; 27:1421–1428.
Werner AB, de Vries E, Tait SW, Bontjer I, Borst J . Bcl-2 family member Bfl-1/A1 sequesters truncated bid to inhibit is collaboration with pro-apoptotic Bak or Bax. J Biol Chem 2002; 277:22781–22788.
Zhang H, Cowan-Jacob SW, 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; 275:11092–11099.
Suzuki M, Youle RJ, Tjandra N . Structure of Bax: coregulation of dimer formation and intracellular localization. Cell 2000; 103:645–654.
Hsu YT, Youle RJ . Nonionic detergents induce dimerization among members of the Bcl-2 family. J Biol Chem 1997; 272:13829–13834.
Oltvai ZN, Milliman CL, Korsmeyer SJ . Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death. Cell 1993; 74:609–619.
Zha H, Aime-Sempe C, Sato T, Reed JC . Proapoptotic protein Bax heterodimerizes with Bcl-2 and homodimerizes with Bax via a novel domain (BH3) distinct from BH1 and BH2. J Biol Chem 1996; 271:7440–7444.
Moldoveanu T, Liu Q, Tocilj A, Watson M, Shore G, Gehring K . The X-ray structure of a BAK homodimer reveals an inhibitory zinc binding site. Mol Cell 2006; 24:677–688.
Leber B, Lin J, Andrews DW . Embedded together: the life and death consequences of interaction of the Bcl-2 family with membranes. Apoptosis 2007; 12:897–911.
Cuconati A, Mukherjee C, Perez D, White E . DNA damage response and MCL-1 destruction initiate apoptosis in adenovirus-infected cells. Genes Dev 2003; 17:2922–2932.
Chittenden T, Flemington C, Houghton AB, et al. A conserved domain in Bak, distinct from BH1 and BH2, mediates cell death and protein binding functions. EMBO J 1995; 14:5589–5596.
Tan C, Dlugosz PJ, Peng J, et al. Auto-activation of the apoptosis protein Bax increases mitochondrial membrane permeability and is inhibited by Bcl-2. J Biol Chem 2006; 281:14764–14775.
Zhou H, Hou Q, Hansen JL, Hsu YT . Complete activation of Bax by a single site mutation. Oncogene 2007; 26:7092–7102.
Gavathiotis E, Suzuki M, Davis ML, et al. BAX activation is initiated at a novel interaction site. Nature 2008; 455:1076–1081.
Walensky LD, Pitter K, Morash J, et al. A stapled BID BH3 helix directly binds and activates BAX. Mol Cell 2006; 24:199–210.
Lovell JF, Billen LP, Bindner S, et al. Membrane binding by tBid initiates an ordered series of events culminating in membrane permeabilization by Bax. Cell 2008; 135:1074–1084.
Petros AM, Medek A, Nettesheim DG, et al. Solution structure of the antiapoptotic protein bcl-2. Proc Natl Acad Sci USA 2001; 98:3012–3017.
Smits C, Czabotar PE, Hinds MG, Day CL . Structural plasticity underpins promiscuous binding of the prosurvival protein A1. Structure 2008; 16:818–829.
Vagin A, Teplyakov A . MOLREP: an Automated Program for Molecular Replacement. J Appl Cryst 1997; 30:1022–1025.
Emsley P, Cowtan K . Coot: model-building tools for molecular graphics. Acta Crystallogr D Biol Crystallogr 2004; 60:2126–2132.
Brunger AT, Adams PD, Clore GM, et al. Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr D Biol Crystallogr 1998; 54:905–921.
Acknowledgements
This study made use of the beam line 4A at the Pohang Accelerator Laboratory in Korea. This work was supported by the GRL Program (K20815000001) from the National Research Foundation of Korea (B-HO and JUJ); the Brain Korea 21 Project (BK); US Public Health Service grants CA140964, AI083841; the Leukemia & Lymphoma Society of USA; and the Wright Foundation and the Baxter Foundation (CL).
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( Supplementary information is linked to the online version of the paper on the Cell Research website.)
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Supplementary information, Table S1
Data collection and structure refinement statistics (PDF 59 kb)
Supplementary information, Figure S1
Comparison of the structures of antiapoptotic BCL-2 proteins bound to a BH3 peptide (PDF 251 kb)
Supplementary information, Figure S2
The BH3 domain-containing segment in the structure of free BAX (PDF 146 kb)
Supplementary information, Figure S3
Phenotypes of BAX(AAA) (PDF 139 kb)
Supplementary information, Figure S4
Expression levels of proteins (PDF 108 kb)
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Ku, B., Liang, C., Jung, J. et al. Evidence that inhibition of BAX activation by BCL-2 involves its tight and preferential interaction with the BH3 domain of BAX. Cell Res 21, 627–641 (2011). https://doi.org/10.1038/cr.2010.149
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DOI: https://doi.org/10.1038/cr.2010.149
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