title: Direct activation of full-length proapoptotic BAK.
abstract: Proapoptotic B-cell lymphoma 2 (BCL-2) antagonist/killer (BAK) and BCL-2-associated X (BAX) form toxic mitochondrial pores in response to cellular stress. Whereas BAX resides predominantly in the cytosol, BAK is constitutively localized to the outer mitochondrial membrane. Select BCL-2 homology domain 3 (BH3) helices activate BAX directly by engaging an α1/α6 trigger site. The inability to express full-length BAK has hampered full dissection of its activation mechanism. Here, we report the production of full-length, monomeric BAK by mutagenesis-based solubilization of its C-terminal α-helical surface. Recombinant BAK autotranslocates to mitochondria but only releases cytochrome c upon BH3 triggering. A direct activation mechanism was explicitly demonstrated using a liposomal system that recapitulates BAK-mediated release upon addition of BH3 ligands. Photoreactive BH3 helices mapped both triggering and autointeractions to the canonical BH3-binding pocket of BAK, whereas the same ligands crosslinked to the α1/α6 site of BAX. Thus, activation of both BAK and BAX is initiated by direct BH3-interaction but at distinct trigger sites. These structural and biochemical insights provide opportunities for developing proapoptotic agents that activate the death pathway through direct but differential engagement of BAK and BAX.
authors: Department of Pediatric Oncology and the Program in Cancer Chemical Biology, Dana-Farber Cancer Institute, Boston, MA 02215, USA.
authors: Elizaveta S
authors: ES
authors: Leshchiner
authors: Craig R
authors: CR
authors: Braun
authors: Gregory H
authors: GH
authors: Bird
authors: Loren D
authors: LD
authors: Walensky
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000502:physiology; D006801:Humans; D051379:Mice; D018345:Mice, Knockout; D008930:Mitochondria, Liver / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D050998:bcl-2 Homologous Antagonist-Killer Protein / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D051028:bcl-2-Associated X Protein / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: C494772:BAK1 protein, human; C494826:BAX protein, human; C494773:Bak1 protein, mouse; C494827:Bax protein, mouse; D011994:Recombinant Proteins; D050998:bcl-2 Homologous Antagonist-Killer Protein; D051028:bcl-2-Associated X Protein
references: Nature. 1995 Apr 20;374(6524):733-6
references: 7715730
references: Science. 1997 Feb 14;275(5302):983-6
references: 9020082
references: Mol Cell. 2011 Nov 18;44(4):517-31
references: 22036586
references: Cell. 2000 Jul 7;102(1):33-42
references: 10929711
references: Nature. 2008 Oct 23;455(7216):1076-81
references: 18948948
references: J Biol Chem. 2010 Sep 10;285(37):28924-37
references: 20605789
references: Nat Cell Biol. 2006 Dec;8(12):1348-58
references: 17115033
references: Curr Protoc Chem Biol. 2011 Sep 1;3(3):99-117
references: 23801563
references: J Biol Chem. 2011 Mar 11;286(10):8213-8230
references: 21196599
references: Methods Enzymol. 2008;446:77-106
references: 18603117
references: Mol Cell. 2005 Feb 4;17(3):393-403
references: 15694340
references: Nat Chem Biol. 2012 Jul;8(7):639-45
references: 22634637
references: Nature. 2005 Jun 2;435(7042):677-81
references: 15902208
references: J Cell Biol. 2003 Sep 1;162(5):877-87
references: 12952938
references: Science. 2001 Apr 27;292(5517):727-30
references: 11326099
references: Cancer Cell. 2002 Sep;2(3):183-92
references: 12242151
references: Mol Cell. 2009 Nov 25;36(4):696-703
references: 19941828
references: Cell. 2008 Dec 12;135(6):1074-84
references: 19062087
references: Nature. 1995 Apr 20;374(6524):731-3
references: 7715729
references: J Am Soc Mass Spectrom. 1994 Nov;5(11):976-89
references: 24226387
references: Lancet Oncol. 2010 Dec;11(12):1149-59
references: 21094089
references: Proc Natl Acad Sci U S A. 2007 Dec 4;104(49):19512-7
references: 18040043
references: J Struct Biol. 2009 Apr;166(1):32-7
references: 19135534
references: Nature. 1995 Apr 20;374(6524):736-9
references: 7715731
references: PLoS One. 2012;7(3):e31510
references: 22442658
references: Science. 2007 Feb 9;315(5813):856-9
references: 17289999
references: J Cell Biol. 2011 Jul 11;194(1):39-48
references: 21727192
references: J Biol Chem. 2006 May 26;281(21):14764-75
references: 16571718
references: Nat Protoc. 2007;2(8):1896-906
references: 17703201
references: Mol Cell. 2000 Dec;6(6):1389-99
references: 11163212
references: Genes Dev. 2000 Aug 15;14(16):2060-71
references: 10950869
references: Mol Cell. 2008 May 9;30(3):369-80
references: 18471982
references: Methods Enzymol. 2003;374:461-91
references: 14696385
references: Cell. 2002 Nov 1;111(3):331-42
references: 12419244
references: Genes Dev. 1996 Nov 15;10(22):2859-69
references: 8918887
references: Methods Enzymol. 2008;446:387-408
references: 18603135
references: Science. 2003 Jul 25;301(5632):513-7
references: 12881569
references: EMBO J. 1995 Nov 15;14(22):5589-96
references: 8521816
references: Science. 2004 Sep 3;305(5689):1466-70
references: 15353804
references: Science. 1997 Feb 21;275(5303):1129-32
references: 9027314
references: EMBO J. 2003 Apr 1;22(7):1497-507
references: 12660157
references: Cell. 2000 Nov 10;103(4):645-54
references: 11106734
references: J Biomol NMR. 1996 Dec;8(4):477-86
references: 9008363
references: J Biol Chem. 2005 Feb 11;280(6):4738-44
references: 15550399
references: Mol Cell. 2009 Nov 13;36(3):487-99
references: 19917256
references: Mol Cell. 2010 Nov 12;40(3):481-92
references: 21070973
references: EMBO J. 1999 May 4;18(9):2330-41
references: 10228148
references: Mol Cell. 2006 Oct 20;24(2):199-210
references: 17052454
references: Nature. 1996 May 23;381(6580):335-41
references: 8692274
references: J Biol Chem. 2011 Jan 7;286(1):491-501
references: 21041309
references: Mol Cell. 2005 Feb 18;17(4):525-35
references: 15721256
references: Proc Natl Acad Sci U S A. 1998 Apr 28;95(9):4997-5002
references: 9560217
references: Methods Enzymol. 2008;446:369-86
references: 18603134
references: Mol Cell. 2001 Sep;8(3):705-11
references: 11583631
references: Chem Biol. 2010 Dec 22;17(12):1325-33
references: 21168768
references: Mol Cell. 2006 Dec 8;24(5):677-688
references: 17157251
references: J Med Chem. 2008 Nov 13;51(21):6902-15
references: 18841882