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A BAK subdomain that binds mitochondrial lipids selectively and releases cytochrome C

Abstract

How BAK and BAX induce mitochondrial outer membrane (MOM) permeabilization (MOMP) during apoptosis is incompletely understood. Here we have used molecular dynamics simulations, surface plasmon resonance, and assays for membrane permeabilization in vitro and in vivo to assess the structure and function of selected BAK subdomains and their derivatives. Results of these studies demonstrate that BAK helical regions α5 and α6 bind the MOM lipid cardiolipin. While individual peptides corresponding to these helical regions lack the full biological activity of BAK, tandem peptides corresponding to α4–α5, α5–α6, or α6–α7/8 can localize exogenous proteins to mitochondria, permeabilize liposomes composed of MOM lipids, and cause MOMP in the absence of the remainder of the BAK protein. Importantly, the ability of these tandem helices to induce MOMP under cell-free conditions is diminished by mutations that disrupt the U-shaped helix-turn-helix structure of the tandem peptides or decrease their lipid binding. Likewise, BAK-induced apoptosis in intact cells is diminished by CLS1 gene interruption, which decreases mitochondrial cardiolipin content, or by BAK mutations that disrupt the U-shaped tandem peptide structure or diminish lipid binding. Collectively, these results suggest that BAK structural rearrangements during apoptosis might mobilize helices involved in specific protein-lipid interactions that are critical for MOMP.

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Fig. 1: Activation-associated binding of BAKΔTM to the MOM requires movement of multiple helices.
Fig. 2: BAK helical fragments other than α9 target mitochondria.
Fig. 3: The first or second most populated conformations of tandem helix peptides and selected variants.
Fig. 4: BAK tandem helices directly permeabilize the MOM and induce cell death.
Fig. 5: Preferential binding of BAK peptides to MOM lipids is essential for MOMP.
Fig. 6: BAK peptide alterations modulate lipid binding and cytochrome c release.
Fig. 7: Effect of α9 or proline insertion on BAK tandem peptide-induced membrane permeabilization and cytotoxicity.
Fig. 8: Effect of helix shortening on MOMP and cell killing.

Data availability

The authors declare that all data supporting the findings of this study are available within the article and its Supplementary Information files.

References

  1. Adams JM, Cory S. The BCL-2 arbiters of apoptosis and their growing role as cancer targets. Cell Death Differ. 2018;25:27–36.

    Article  CAS  PubMed  Google Scholar 

  2. Kale J, Osterlund EJ, Andrews DW. BCL-2 family proteins: changing partners in the dance towards death. Cell Death Differ. 2018;25:65–80.

    Article  CAS  PubMed  Google Scholar 

  3. Merino D, Kelly GL, Lessene G, Wei AH, Roberts AW, Strasser A. BH3-mimetic drugs: blazing the trail for new cancer medicines. Cancer Cell. 2018;34:879–91.

    Article  CAS  PubMed  Google Scholar 

  4. Jeng PS, Inoue-Yamauchi A, Hsieh JJ, Cheng EH. BH3-dependent and independent activation of BAX and BAK in mitochondrial apoptosis. Curr Opin Physiol. 2018;3:71–81.

    Article  PubMed  PubMed Central  Google Scholar 

  5. Antonsson B, Montessuit S, Sanchez B, Martinou JC. Bax is present as a high molecular weight oligomer/complex in the mitochondrial membrane of apoptotic cells. J Biol Chem. 2001;276:11615–23.

    Article  CAS  PubMed  Google Scholar 

  6. Llambi F, Moldoveanu T, Tait SW, Bouchier-Hayes L, Temirov J, McCormick LL, et al. A unified model of mammalian BCL-2 protein family interactions at the mitochondria. Mol Cell. 2011;44:517–31.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Kim H, Tu HC, Ren D, Takeuchi O, Jeffers JR, Zambetti GP, et al. Stepwise activation of BAX and BAK by tBID, BIM, and PUMA initiates mitochondrial apoptosis. Mol Cell. 2009;36:487–99.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Dai H, Smith A, Meng XW, Schneider PA, Pang Y-P, Kaufmann SH. Transient binding of an activator BH3 domain to the Bak BH3-binding groove initiates Bak oligomerization. J Cell Biol. 2011;194:39–48.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Chen HC, Kanai M, Inoue-Yamauchi A, Tu HC, Huang Y, Ren D, et al. An interconnected hierarchical model of cell death regulation by the BCL-2 family. Nat Cell Biol. 2015;17:1270–81.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Leshchiner ES, Braun CR, Bird GH, Walensky LD. Direct activation of full-length proapoptotic BAK. Proc Natl Acad Sci USA. 2013;110:E986–995.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Huang K, O’Neill KL, Li J, Zhou W, Han N, Pang X, et al. BH3-only proteins target BCL-xL/MCL-1, not BAX/BAK, to initiate apoptosis. Cell Res. 2019;29:942–52.

  12. Jiang X, Wang X. Cytochrome C-mediated apoptosis. Annu Rev Biochem. 2004;73:87–106.

    Article  CAS  PubMed  Google Scholar 

  13. Ekert PG, Vaux DL. The mitochondrial death squad—hardened killers or innocent bystanders? Curr Opin Cell Biol. 2005;17:626–30.

    Article  CAS  PubMed  Google Scholar 

  14. Strasser A, Cory S, Adams JM. Deciphering the rules of programmed cell death to improve therapy of cancer and other diseases. EMBO J. 2011;30:3667–83.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Birkinshaw RW, Czabotar PE. The BCL-2 family of proteins and mitochondrial outer membrane permeabilisation. Semin Cell Dev Biol. 2017;72:152–62.

    Article  CAS  PubMed  Google Scholar 

  16. Hauseman ZJ, Harvey EP, Newman CE, Wales TE, Bucci JC, Mintseris J, et al. Homogeneous oligomers of pro-apoptotic BAX reveal structural determinants of mitochondrial membrane permeabilization. Mol Cell. 2020;79:68–83.e67.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. 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;24:677–88.

    Article  CAS  PubMed  Google Scholar 

  18. Suzuki M, Youle RJ, Tjandra N. Structure of Bax: coregulation of dimer formation and intracellular localization. Cell. 2000;103:645–54.

    Article  CAS  PubMed  Google Scholar 

  19. Gavathiotis E, Reyna DE, Davis ML, Bird GH, Walensky LD. BH3-triggered structural reorganization drives the activation of proapoptotic BAX. Mol Cell. 2010;40:481–92.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Du H, Wolf J, Schafer B, Moldoveanu T, Chipuk JE, Kuwana T. BH3 domains other than Bim and Bid can directly activate Bax/Bak. J Biol Chem. 2011;286:491–501.

    Article  CAS  PubMed  Google Scholar 

  21. Oh KJ, Singh P, Lee K, Foss K, Lee S, Park M, et al. Conformational changes in BAK, a pore-forming proapoptotic Bcl-2 family member, upon membrane insertion and direct evidence for the existence of BH3-BH3 contact interface in BAK homo-oligomers. J Biol Chem. 2010;285:28924–37.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Birkinshaw RW, Iyer S, Lio D, Luo CS, Brouwer JM, Miller MS, et al. Structure of detergent-activated BAK dimers derived from the inert monomer. Mol Cell. 2021;81:2123–34.e2125

    Article  CAS  PubMed  Google Scholar 

  23. Antonsson B, Conti F, Ciavatta A, Montessuit S, Lewis S, Martinou I, et al. Inhibition of Bax channel-forming activity by Bcl-2. Science. 1997;277:370–2.

    Article  CAS  PubMed  Google Scholar 

  24. Schendel SL, Xie Z, Montal MO, Matsuyama S, Montal M, Reed JC. Channel formation by antiapoptotic protein Bcl-2. Proc Natl Acad Sci USA. 1997;94:5113–8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Westphal D, Dewson G, Menard M, Frederick P, Iyer S, Bartolo R, et al. Apoptotic pore formation is associated with in-plane insertion of Bak or Bax central helices into the mitochondrial outer membrane. Proc Natl Acad Sci USA. 2014;111:E4076–85.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Bleicken S, Jeschke G, Stegmueller C, Salvador-Gallego R, Garcia-Saez AJ, Bordignon E. Structural model of active Bax at the membrane. Mol Cell. 2014;56:496–505.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Salvador-Gallego R, Mund M, Cosentino K, Schneider J, Unsay J, Schraermeyer U, et al. Bax assembly into rings and arcs in apoptotic mitochondria is linked to membrane pores. EMBO J. 2016;35:389–401.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Uren RT, O’Hely M, Iyer S, Bartolo R, Shi MX, Brouwer JM, et al. Disordered clusters of Bak dimers rupture mitochondria during apoptosis. Elife 2017;6:e19944.

  29. Brouwer JM, Westphal D, Dewson G, Robin AY, Uren RT, Bartolo R, et al. Bak core and latch domains separate during activation, and freed core domains form symmetric homodimers. Mol Cell. 2014;55:938–46.

    Article  CAS  PubMed  Google Scholar 

  30. Cowan AD, Smith NA, Sandow JJ, Kapp EA, Rustam YH, Murphy JM, et al. BAK core dimers bind lipids and can be bridged by them. Nat Struct Mol Biol. 2020;27:1024–31.

    Article  CAS  PubMed  Google Scholar 

  31. Dewson G, Kratina T, Sim HW, Puthalakath H, Adams JM, Colman PM, et al. To trigger apoptosis, Bak exposes its BH3 domain and homodimerizes via BH3:groove interactions. Mol Cell. 2008;30:369–80.

    Article  CAS  PubMed  Google Scholar 

  32. Simbeni R, Pon L, Zinser E, Paltauf F, Daum G. Mitochondrial membrane contact sites of yeast. Characterization of lipid components and possible involvement in intramitochondrial translocation of phospholipids. J Biol Chem. 1991;266:10047–9.

    Article  CAS  PubMed  Google Scholar 

  33. Lutter M, Fang M, Luo X, Nishijima M, Xie X, Wang X. Cardiolipin provides specificity for targeting of tBid to mitochondria. Nat Cell Biol. 2000;2:754–61.

    Article  CAS  PubMed  Google Scholar 

  34. Raemy E, Montessuit S, Pierredon S, van Kampen AH, Vaz FM, Martinou JC. Cardiolipin or MTCH2 can serve as tBID receptors during apoptosis. Cell Death Differ. 2016;23:1165–74.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Dai H, Ding H, Meng XW, Peterson KL, Schneider PA, Karp JE, et al. Constitutive BAK activation as a determinant of drug sensitivity in malignant lymphohematopoietic cells. Genes Dev. 2015;29:2140–52.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Dewson G, Kratina T, Czabotar P, Day CL, Adams JM, Kluck RM. Bak activation for apoptosis involves oligomerization of dimers via their alpha6 helices. Mol Cell. 2009;36:696–703.

    Article  CAS  PubMed  Google Scholar 

  37. Zacharias DA, Violin JD, Newton AC, Tsien RY. Partitioning of lipid-modified monomeric GFPs into membrane microdomains of live cells. Science. 2002;296:913–6.

    Article  CAS  PubMed  Google Scholar 

  38. Gohil VM, Greenberg ML. Mitochondrial membrane biogenesis: phospholipids and proteins go hand in hand. J Cell Biol. 2009;184:469–72.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Horvath SE, Daum G. Lipids of mitochondria. Prog Lipid Res. 2013;52:590–614.

    Article  CAS  PubMed  Google Scholar 

  40. Fuertes G, Garcia-Saez AJ, Esteban-Martin S, Gimenez D, Sanchez-Munoz OL, Schwille P, et al. Pores formed by Baxalpha5 relax to a smaller size and keep at equilibrium. Biophys J. 2010;99:2917–25.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Garcia-Saez AJ, Coraiola M, Serra MD, Mingarro I, Muller P, Salgado J. Peptides corresponding to helices 5 and 6 of Bax can independently form large lipid pores. FEBS J. 2006;273:971–81.

    Article  CAS  PubMed  Google Scholar 

  42. Qian S, Wang W, Yang L, Huang HW. Structure of transmembrane pore induced by Bax-derived peptide: evidence for lipidic pores. Proc Natl Acad Sci USA. 2008;105:17379–83.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Krebs JJ, Hauser H, Carafoli E. Asymmetric distribution of phospholipids in the inner membrane of beef heart mitochondria. J Biol Chem. 1979;254:5308–16.

    Article  CAS  PubMed  Google Scholar 

  44. Hovius R, Lambrechts H, Nicolay K, de Kruijff B. Improved methods to isolate and subfractionate rat liver mitochondria. Lipid composition of the inner and outer membrane. Biochim Biophys Acta. 1990;1021:217–26.

    Article  CAS  PubMed  Google Scholar 

  45. de Kroon AI, Dolis D, Mayer A, Lill R, de Kruijff B. Phospholipid composition of highly purified mitochondrial outer membranes of rat liver and Neurospora crassa. Is cardiolipin present in the mitochondrial outer membrane? Biochim Biophys Acta. 1997;1325:108–16.

    Article  PubMed  Google Scholar 

  46. Schug ZT, Gottlieb E. Cardiolipin acts as a mitochondrial signalling platform to launch apoptosis. Biochim Biophys Acta. 2009;1788:2022–31.

    Article  CAS  PubMed  Google Scholar 

  47. Gonzalvez F, Pariselli F, Dupaigne P, Budihardjo I, Lutter M, Antonsson B, et al. tBid interaction with cardiolipin primarily orchestrates mitochondrial dysfunctions and subsequently activates Bax and Bak. Cell Death Differ. 2005;12:614–26.

    Article  CAS  PubMed  Google Scholar 

  48. Lucken-Ardjomande S, Montessuit S, Martinou JC. Contributions to Bax insertion and oligomerization of lipids of the mitochondrial outer membrane. Cell Death Differ. 2008;15:929–37.

    Article  CAS  PubMed  Google Scholar 

  49. Gonzalvez F, Schug ZT, Houtkooper RH, MacKenzie ED, Brooks DG, Wanders RJ, et al. Cardiolipin provides an essential activating platform for caspase-8 on mitochondria. J Cell Biol. 2008;183:681–96.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Sandow JJ, Tan IK, Huang AS, Masaldan S, Bernardini JP, Wardak AZ, et al. Dynamic reconfiguration of pro-apoptotic BAK on membranes. EMBO J. 2021;40:e107237.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. George NM, Evans JJ, Luo X. A three-helix homo-oligomerization domain containing BH3 and BH1 is responsible for the apoptotic activity of Bax. Genes Dev. 2007;21:1937–48.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Dai H, Ding H, Peterson KL, Meng XW, Schneider PA, Knorr KLB, et al. Measurement of BH3-only protein tolerance. Cell Death Differ. 2018;25:282–93.

    Article  CAS  PubMed  Google Scholar 

  53. Dai H, Meng XW, Lee S-H, Schneider PA, Kaufmann SH. Context-dependent Bcl-2/Bak interactions regulate lymphoid cell apoptosis. J Biol Chem. 2009;284:18311–22.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Wang C, Youle RJ. Predominant requirement of Bax for apoptosis in HCT116 cells is determined by Mcl-1’s inhibitory effect on Bak. Oncogene. 2012;31:3177–89.

    Article  CAS  PubMed  Google Scholar 

  55. Ye K, Meng WX, Sun H, Wu B, Chen M, Pang YP, et al. Characterization of an alternative BAK-binding site for BH3 peptides. Nat Commun. 2020;11:3301.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Lee SH, Meng XW, Flatten KS, Loegering DA, Kaufmann SH. Phosphatidylserine exposure during apoptosis reflects bidirectional trafficking between plasma membrane and cytoplasm. Cell Death Differ. 2013;20:64–76.

    Article  CAS  PubMed  Google Scholar 

  57. Goping IS, Gross A, Lavoie JN, Nguyen M, Jemmerson R, Roth K, et al. Regulated targeting of BAX to mitochondria. J Cell Biol. 1998;143:207–15.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Biacore T200 software handbook. GE Healthcare Bio-Sciences AB. Uppsala, Sweden, 2010. p. 165–168.

  59. Scheffe H. The analysis of variance. New York: John Wiley & Sons, Inc.; 1999.

  60. Ferrer PE, Frederick P, Gulbis JM, Dewson G, Kluck RM. Translocation of a Bak C-terminus mutant from cytosol to mitochondria to mediate cytochrome C release: implications for Bak and Bax apoptotic function. PLoS One. 2012;7:e31510.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank David Toft, Richard Youle, Qian Liu, and Kalle Gehring for gifts of reagents; Eric Roush (Cytiva) for advice regarding surface plasmon resonance analysis; Gregory Gores, Roderick Brown, and Husheng Ding for helpful discussions; and the two anonymous reviewers for insightful suggestions. We also acknowledge the computing resources provided by the University of Minnesota Supercomputing Institute and the Mayo Clinic high-performance computing facility at the University of Illinois Urbana-Champaign National Center for Supercomputing Applications.

Funding

This work was supported in part by grants from the National Cancer Institute (R01 CA166741, R01 CA225996 and P30 CA015083).

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Designed study: HD and SHK Conducted experiments: HD, KLP, KSF, XWM, AV, Y-PP. Analyzed data: HD, CC, MR-A, Y-PP, SHK. Wrote manuscript: HD, SHK. Edited and approved manuscript: HD, KLP, KSF, XWM, KLP, AV, CC, MR-A, Y-PP, SHK.

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Correspondence to Haiming Dai or Scott H. Kaufmann.

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Dai, H., Peterson, K.L., Flatten, K.S. et al. A BAK subdomain that binds mitochondrial lipids selectively and releases cytochrome C. Cell Death Differ (2022). https://doi.org/10.1038/s41418-022-01083-z

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