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.

Structure of the BAK-activating antibody 7D10 bound to BAK reveals an unexpected role for the α1-α2 loop in BAK activation

Cell Death & Differentiation volume 29, pages 1757–1768 (2022)Cite this article

Subjects

Abstract

Pro-apoptotic BAK and BAX are activated by BH3-only proteins to permeabilise the outer mitochondrial membrane. The antibody 7D10 also activates BAK on mitochondria and its epitope has previously been mapped to BAK residues in the loop connecting helices α1 and α2 of BAK. A crystal structure of the complex between the Fv fragment of 7D10 and the BAK mutant L100A suggests a possible mechanism of activation involving the α1-α2 loop residue M60. M60 mutants of BAK have reduced stability and elevated sensitivity to activation by BID, illustrating that M60, through its contacts with residues in helices α1, α5 and α6, is a linchpin stabilising the inert, monomeric structure of BAK. Our data demonstrate that BAK’s α1-α2 loop is not a passive covalent connector between secondary structure elements, but a direct restraint on BAK’s activation.

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

Access options

Rent or buy this article

Get just this article for as long as you need it

$39.95

Learn more

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

Fig. 1: Characterisation of the complex between antibody 7D10 and both BAK and BAKL100A.
Fig. 2: Structure of the complex between antibody 7D10 and BAKL100A.
Fig. 3: Differences between apo- and 7D10-bound structures of BAK.
Fig. 4: Molecular dynamics.
Fig. 5: Stability and activity profiles of ΒΑΚΔΝ22ΔC25Δcys and M60 mutants.
Fig. 6: M60 mutants are less stable on mitochondria and more sensitive to activation.
Fig. 7: Schematic of BAK activation by the 7D10 antibody.

Data availability

PDB entry 7LK4 is presently on hold at https://www.ebi.ac.uk/pdbe/entry/pdb/7lk4.

References

  1. Czabotar PE, Lessene G, Strasser A, Adams JM. Control of apoptosis by the BCL-2 protein family: implications for physiology and therapy. Nat Rev Mol Cell Biol. 2014;15:49–63.

    Article  CAS  PubMed  Google Scholar 

  2. Wei MC, Zong WX, Cheng EH, Lindsten T, Panoutsakopoulou V, Ross AJ, et al. Proapoptotic BAX and BAK: a requisite gateway to mitochondrial dysfunction and death. Science. 2001;292:727–30.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Hsu YT, Youle RJ. Bax in murine thymus is a soluble monomeric protein that displays differential detergent-induced conformations. J Biol Chem. 1998;273:10777–83.

    Article  CAS  PubMed  Google Scholar 

  4. Dewson G, Kluck RM. Mechanisms by which Bak and Bax permeabilise mitochondria during apoptosis. J Cell Sci. 2009;122:2801–8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. 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 

  6. Czabotar PE, Westphal D, Dewson G, Ma S, Hockings C, Fairlie WD, et al. Bax Crystal Structures Reveal How BH3 Domains Activate Bax and Nucleate Its Oligomerization to Induce Apoptosis. Cell. 2013;152:519–31.

    Article  CAS  PubMed  Google Scholar 

  7. 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 

  8. Dewson G, Ma S, Frederick P, Hockings C, Tan I, Kratina T, et al. Bax dimerizes via a symmetric BH3:groove interface during apoptosis. Cell Death Differ. 2012;19:661–70.

    Article  CAS  PubMed  Google Scholar 

  9. Chen L, Willis SN, Wei A, Smith BJ, Fletcher JI, Hinds MG, et al. Differential targeting of prosurvival Bcl-2 proteins by their BH3-only ligands allows complementary apoptotic function. Mol Cell. 2005;17:393–403.

    Article  CAS  PubMed  Google Scholar 

  10. 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 

  11. Iyer S, Anwari K, Alsop AE, Yuen WS, Huang DC, Carroll J, et al. Identification of an activation site in Bak and mitochondrial Bax triggered by antibodies. Nat Commun. 2016;7:11734.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. 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 

  13. Robin AY, Iyer S, Birkinshaw RW, Sandow J, Wardak A, Luo CS, et al. Ensemble Properties of Bax Determine Its Function. Structure. 2018;26:1346–59 e1345.

    Article  CAS  PubMed  Google Scholar 

  14. Kvansakul M, Yang H, Fairlie WD, Czabotar PE, Fischer SF, Perugini MA, et al. Vaccinia virus anti-apoptotic F1L is a novel Bcl-2-like domain-swapped dimer that binds a highly selective subset of BH3-containing death ligands. Cell Death Differ. 2008;15:1564–71.

    Article  CAS  PubMed  Google Scholar 

  15. 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 

  16. Brouwer JM, Lan P, Cowan AD, Bernardini JP, Birkinshaw RW, van Delft MF, et al. Conversion of Bim-BH3 from Activator to Inhibitor of Bak through Structure-Based Design. Mol Cell. 2017;68:659–72 e659.

    Article  CAS  PubMed  Google Scholar 

  17. Chikh GG, Li WM, Schutze-Redelmeier MP, Meunier JC, Bally MB. Attaching histidine-tagged peptides and proteins to lipid-based carriers through use of metal-ion-chelating lipids. Biochim Biophys Acta. 2002;1567:204–12.

    Article  CAS  PubMed  Google Scholar 

  18. Lee EF, Dewson G, Smith BJ, Evangelista M, Pettikiriarachchi A, Dogovski C, et al. Crystal structure of a BCL-W domain-swapped dimer: implications for the function of BCL-2 family proteins. Structure. 2011;19:1467–76.

    Article  CAS  PubMed  Google Scholar 

  19. Lawrence MC, Colman PM. Shape Complementarity at Protein-Protein Interfaces. J Mol Biol. 1993;234:946–50.

    Article  CAS  PubMed  Google Scholar 

  20. Epa VC, Colman PM. Shape and electrostatic complementarity at viral antigen-antibody complexes. Curr Top Microbiol Immunol. 2001;260:45–53.

    CAS  PubMed  Google Scholar 

  21. Alsop AE, Fennell SC, Bartolo RC, Tan IK, Dewson G, Kluck RM. Dissociation of Bak alpha1 helix from the core and latch domains is required for apoptosis. Nat Commun. 2015;6:6841.

    Article  CAS  PubMed  Google Scholar 

  22. Wang H, Takemoto C, Akasaka R, Uchikubo-Kamo T, Kishishita S, Murayama K, et al. Novel dimerization mode of the human Bcl-2 family protein Bak, a mitochondrial apoptosis regulator. J Struct Biol. 2009;166:32–37.

    Article  CAS  PubMed  Google Scholar 

  23. Berman HM, Westbrook J, Feng Z, Gilliland G, Bhat TN, Weissig H, et al. The Protein Data Bank. Nucleic Acids Res. 2000;28:235–42.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Tandon H, de Brevern AG, Srinivasan N. Transient association between proteins elicits alteration of dynamics at sites far away from interfaces. Structure. 2021;29:371–84 e373.

    Article  CAS  PubMed  Google Scholar 

  25. Pires DE, Ascher DB, Blundell TL. DUET: a server for predicting effects of mutations on protein stability using an integrated computational approach. Nucleic Acids Res. 2014;42:W314–319.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. 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.

    Article  CAS  PubMed  Google Scholar 

  27. 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 

  28. Gavathiotis E, Suzuki M, Davis ML, Pitter K, Bird GH, Katz SG, et al. BAX activation is initiated at a novel interaction site. Nature. 2008;455:1076–81.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Dengler MA, Gibson L, Adams JM. BAX mitochondrial integration is regulated allosterically by its alpha1-alpha2 loop. Cell Death Differ. 2021;28:3270–81.

    Article  CAS  PubMed  Google Scholar 

  30. Dengler MA, Robin AY, Gibson L, Li MX, Sandow JJ, Iyer S, et al. BAX Activation: Mutations Near Its Proposed Non-canonical BH3 Binding Site Reveal Allosteric Changes Controlling Mitochondrial Association. Cell Rep. 2019;27:359–73 e356.

    Article  CAS  PubMed  Google Scholar 

  31. Cooper A, Dryden DTF. Allostery without Conformational Change - a Plausible Model. Eur Biophys J Biophy. 1984;11:103–9.

    Article  CAS  Google Scholar 

  32. 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 

  33. McCoy AJ, Grosse-Kunstleve RW, Adams PD, Winn MD, Storoni LC, Read RJ. Phaser crystallographic software. J Appl Crystallogr. 2007;40:658–74.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Emsley P, Lohkamp B, Scott WG, Cowtan K. Features and development of Coot. Acta Crystallogr D Biol Crystallogr. 2010;66:486–501.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Adams PD, Afonine PV, Bunkoczi G, Chen VB, Davis IW, Echols N, et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr D Biol Crystallogr. 2010;66:213–21.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Webb B, Sali A. Comparative Protein Structure Modeling Using Modeller. Curr Protoc Bioinformatics. 2016;54:5.6.1-5.6.37.

  37. Abraham MJ, Murtola T, Schulz R, Pall S, Smith JC, Hess B, et al. GROMACS: high performance molecular simulations through multi-level parallelism from laptops to supercomputers. SoftwareX. 2015;1-2:6.

    Article  Google Scholar 

  38. Huang J, Rauscher S, Nawrocki G, Ran T, Feig M, de Groot BL, et al. CHARMM36m: an improved force field for folded and intrinsically disordered proteins. Nat Methods. 2017;14:71–3.

    Article  CAS  PubMed  Google Scholar 

  39. Bussi G, Donadio D, Parrinello M. Canonical sampling through velocity rescaling. J Chem Phys. 2007;126:014101.

    Article  PubMed  CAS  Google Scholar 

  40. Parrinello M, Rahman A. Canonical sampling through velocity rescaling. J Appl Phys. 1981;52:9.

    Article  Google Scholar 

  41. Essmann U, Perera L, Berkowitz M, Darden T, Lee H, Pedersen LG. A smooth particle mesh Ewald method. J Chem Phys. 1995;103:19.

    Google Scholar 

  42. Hess B. P-LINCS: A Parallel Linear Constraint Solver for Molecular Simulation. J Chem Theory Comput. 2008;4:116–22.

    Article  CAS  PubMed  Google Scholar 

  43. Humphrey W, Dalke A, Schulten K. VMD: visual molecular dynamics. J Mol Graph. 1996;14:33–38. 27-38

    Article  CAS  PubMed  Google Scholar 

  44. Bakan A, Meireles LM, Bahar I. ProDy: protein dynamics inferred from theory and experiments. Bioinformatics. 2011;27:1575–7.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Amadei A, Linssen ABM, Berendsen HJC. Essential Dynamics of Proteins. Proteins. 1993;17:412–25.

    Article  CAS  PubMed  Google Scholar 

  46. Uren RT, Dewson G, Chen L, Coyne SC, Huang DC, Adams JM, et al. Mitochondrial permeabilization relies on BH3 ligands engaging multiple prosurvival Bcl-2 relatives, not Bak. J Cell Biol. 2007;177:277–87.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank Mike Lawrence and Mai Margetts for advice, reagents and protocols for the Brevibacillus expression system II. We acknowledge support of the staff at the Collaborative Crystallisation Centre and at the Australian Synchrotron beamline MX1.

Funding

Our work is supported by the NHMRC through fellowships (1116934 to PMC, 1079700 to PEC) and grants (1113133, 2001406, 1141874) the Australian Cancer Research Foundation, the Leukemia and Lymphoma Society (US) (SCOR grant 7001–03), Lady Tata Memorial Trust Fellowship (SI), Jack Brockhoff Foundation and Marian and E.H. Flack Trust Early Career Research Grant (SI), the Victorian State Government Operational Infrastructure Support and the Australian Government NHMRC IRISS (9000587). Part of this work used resources from the National Computational Infrastructure, which is supported by the Australian Government and provided through Intersect Australia under LIEF grants LE170100032 and through the HPC-GPGPU Facility which was established with the assistance of LIEF grant LE170100200.

Author information

Author notes

  1. These authors contributed equally: Adeline Y. Robin, Michelle S. Miller.

Authors and Affiliations

  1. Structural Biology Division, The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, Melbourne, VIC, 3052, Australia

    Adeline Y. Robin, Michelle S. Miller, Ahmad Z. Wardak, Daisy Lio, Richard W. Birkinshaw, Peter E. Czabotar & Peter M. Colman

  2. Blood Cells and Blood Cancer Division, The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, Melbourne, VIC, 3052, Australia

    Sweta Iyer, Melissa X. Shi & Ruth M. Kluck

  3. Department of Chemistry and Physics, La Trobe Institute for Molecular Sciences, La Trobe University, Melbourne, VIC, 3086, Australia

    Nicholas A. Smith & Brian J. Smith

Authors
  1. Adeline Y. Robin
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Michelle S. Miller
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Sweta Iyer
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Melissa X. Shi
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Ahmad Z. Wardak
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Daisy Lio
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Nicholas A. Smith
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Brian J. Smith
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Richard W. Birkinshaw
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Peter E. Czabotar
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Ruth M. Kluck
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Peter M. Colman
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

AYR, MSM, SI, MXS, AZW, DL, NAS and RWB designed and performed experiments, BJS, PEC, RMK and PMC designed and supervised experiments, wrote the paper with input from all the authors. All authors read and approved the final paper.

Corresponding authors

Correspondence to Ruth M. Kluck or Peter M. Colman.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Edited by G. Melino

Supplementary information

Rights and permissions

Reprints and Permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Robin, A.Y., Miller, M.S., Iyer, S. et al. Structure of the BAK-activating antibody 7D10 bound to BAK reveals an unexpected role for the α1-α2 loop in BAK activation. Cell Death Differ 29, 1757–1768 (2022). https://doi.org/10.1038/s41418-022-00961-w

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41418-022-00961-w

Search

Advanced search

Quick links