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
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Rent or buy this article
Get just this article for as long as you need it
Prices may be subject to local taxes which are calculated during checkout
PDB entry 7LK4 is presently on hold at https://www.ebi.ac.uk/pdbe/entry/pdb/7lk4.
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.
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.
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.
Dewson G, Kluck RM. Mechanisms by which Bak and Bax permeabilise mitochondria during apoptosis. J Cell Sci. 2009;122:2801–8.
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.
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.
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.
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.
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.
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.
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.
Suzuki M, Youle RJ, Tjandra N. Structure of Bax: coregulation of dimer formation and intracellular localization. Cell. 2000;103:645–54.
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.
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.
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.
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.
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.
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.
Lawrence MC, Colman PM. Shape Complementarity at Protein-Protein Interfaces. J Mol Biol. 1993;234:946–50.
Epa VC, Colman PM. Shape and electrostatic complementarity at viral antigen-antibody complexes. Curr Top Microbiol Immunol. 2001;260:45–53.
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.
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.
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.
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.
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.
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.
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.
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.
Dengler MA, Gibson L, Adams JM. BAX mitochondrial integration is regulated allosterically by its alpha1-alpha2 loop. Cell Death Differ. 2021;28:3270–81.
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.
Cooper A, Dryden DTF. Allostery without Conformational Change - a Plausible Model. Eur Biophys J Biophy. 1984;11:103–9.
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.
McCoy AJ, Grosse-Kunstleve RW, Adams PD, Winn MD, Storoni LC, Read RJ. Phaser crystallographic software. J Appl Crystallogr. 2007;40:658–74.
Emsley P, Lohkamp B, Scott WG, Cowtan K. Features and development of Coot. Acta Crystallogr D Biol Crystallogr. 2010;66:486–501.
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.
Webb B, Sali A. Comparative Protein Structure Modeling Using Modeller. Curr Protoc Bioinformatics. 2016;54:5.6.1-5.6.37.
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.
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.
Bussi G, Donadio D, Parrinello M. Canonical sampling through velocity rescaling. J Chem Phys. 2007;126:014101.
Parrinello M, Rahman A. Canonical sampling through velocity rescaling. J Appl Phys. 1981;52:9.
Essmann U, Perera L, Berkowitz M, Darden T, Lee H, Pedersen LG. A smooth particle mesh Ewald method. J Chem Phys. 1995;103:19.
Hess B. P-LINCS: A Parallel Linear Constraint Solver for Molecular Simulation. J Chem Theory Comput. 2008;4:116–22.
Humphrey W, Dalke A, Schulten K. VMD: visual molecular dynamics. J Mol Graph. 1996;14:33–38. 27-38
Bakan A, Meireles LM, Bahar I. ProDy: protein dynamics inferred from theory and experiments. Bioinformatics. 2011;27:1575–7.
Amadei A, Linssen ABM, Berendsen HJC. Essential Dynamics of Proteins. Proteins. 1993;17:412–25.
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.
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.
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.
The authors declare no competing interests.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Edited by G. Melino
Rights and permissions
About this article
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