title: Identification of an activation site in Bak and mitochondrial Bax triggered by antibodies.
abstract: During apoptosis, Bak and Bax are activated by BH3-only proteins binding to the α2-α5 hydrophobic groove; Bax is also activated via a rear pocket. Here we report that antibodies can directly activate Bak and mitochondrial Bax by binding to the α1-α2 loop. A monoclonal antibody (clone 7D10) binds close to α1 in non-activated Bak to induce conformational change, oligomerization, and cytochrome c release. Anti-FLAG antibodies also activate Bak containing a FLAG epitope close to α1. An antibody (clone 3C10) to the Bax α1-α2 loop activates mitochondrial Bax, but blocks translocation of cytosolic Bax. Tethers within Bak show that 7D10 binding directly extricates α1; a structural model of the 7D10 Fab bound to Bak reveals the formation of a cavity under α1. Our identification of the α1-α2 loop as an activation site in Bak paves the way to develop intrabodies or small molecules that directly and selectively regulate these proteins.
authors.affiliation: Molecular Genetics of Cancer Division, The Walter and Eliza Hall Institute of Medical Research, Victoria 3052, Australia.
authors.forename: Sweta
authors.initials: S
authors.lastname: Iyer
authors.affiliation: Molecular Genetics of Cancer Division, The Walter and Eliza Hall Institute of Medical Research, Victoria 3052, Australia.
authors.forename: Khatira
authors.initials: K
authors.lastname: Anwari
authors.affiliation: Molecular Genetics of Cancer Division, The Walter and Eliza Hall Institute of Medical Research, Victoria 3052, Australia.
authors.forename: Amber E
authors.initials: AE
authors.lastname: Alsop
authors.affiliation: Development and Stem Cells Program, Monash Biomedicine Discovery Institute and Department of Anatomy and Developmental Biology, Monash University, Melbourne, Victoria 3800, Australia.
authors.forename: Wai Shan
authors.initials: WS
authors.lastname: Yuen
authors.affiliation: Cancer and Haematology Division, The Walter and Eliza Hall Institute of Medical Research, Victoria 3052, Australia.
authors.forename: David C S
authors.initials: DC
authors.lastname: Huang
authors.affiliation: Development and Stem Cells Program, Monash Biomedicine Discovery Institute and Department of Anatomy and Developmental Biology, Monash University, Melbourne, Victoria 3800, Australia.
authors.forename: John
authors.initials: J
authors.lastname: Carroll
authors.affiliation: Department of Chemistry and Physics, La Trobe Institute for Molecular Sciences, La Trobe University, Victoria 3086, Australia.
authors.forename: Nicholas A
authors.initials: NA
authors.lastname: Smith
authors.affiliation: Department of Chemistry and Physics, La Trobe Institute for Molecular Sciences, La Trobe University, Victoria 3086, Australia.
authors.forename: Brian J
authors.initials: BJ
authors.lastname: Smith
authors.affiliation: Cell Signalling and Cell Death Division, The Walter and Eliza Hall Institute of Medical Research, Victoria 3052, Australia.
authors.forename: Grant
authors.initials: G
authors.lastname: Dewson
authors.affiliation: Molecular Genetics of Cancer Division, The Walter and Eliza Hall Institute of Medical Research, Victoria 3052, Australia.
authors.forename: Ruth M
authors.identifier: 0000-0002-7101-1925
authors.initials: RM
authors.lastname: Kluck
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D017209:Apoptosis / Q000502:physiology*; D002478:Cells, Cultured; D045304:Cytochromes c / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D018604:Epitope Mapping / Q000379:methods; D000939:Epitopes / Q000378:metabolism*; D005260:Female; D005347:Fibroblasts; D055786:Gene Knockout Techniques; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D008928:Mitochondria / Q000378:metabolism; D062105:Molecular Docking Simulation; D056004:Molecular Dynamics Simulation; D016297:Mutagenesis, Site-Directed; D009865:Oocytes; D011485:Protein Binding / Q000502:physiology; D000072756:Protein Conformation, alpha-Helical; D055503:Protein Multimerization / Q000502:physiology; 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: D000911:Antibodies, Monoclonal; C494772:BAK1 protein, human; C494826:BAX protein, human; C494773:Bak1 protein, mouse; C494827:Bax protein, mouse; D000939:Epitopes; D050998:bcl-2 Homologous Antagonist-Killer Protein; D051028:bcl-2-Associated X Protein; D045304:Cytochromes c