title: Assembly of the Bak apoptotic pore: a critical role for the Bak protein α6 helix in the multimerization of homodimers during apoptosis.
abstract: Bak and Bax are the essential effectors of the intrinsic pathway of apoptosis. Following an apoptotic stimulus, both undergo significant changes in conformation that facilitates their self-association to form pores in the mitochondrial outer membrane. However, the molecular structures of Bak and Bax oligomeric pores remain elusive. To characterize how Bak forms pores during apoptosis, we investigated its oligomerization under native conditions using blue native PAGE. We report that, in a healthy cell, inactive Bak is either monomeric or in a large complex involving VDAC2. Following an apoptotic stimulus, activated Bak forms BH3:groove homodimers that represent the basic stable oligomeric unit. These dimers multimerize to higher-order oligomers via a labile interface independent of both the BH3 domain and groove. Linkage of the α6:α6 interface is sufficient to stabilize higher-order Bak oligomers on native PAGE, suggesting an important role in the Bak oligomeric pore. Mutagenesis of the α6 helix disrupted apoptotic function because a chimera of Bak with the α6 derived from Bcl-2 could be activated by truncated Bid (tBid) and could form BH3:groove homodimers but could not form high molecular weight oligomers or mediate cell death. An α6 peptide could block Bak function but did so upstream of dimerization, potentially implicating α6 as a site for activation by BH3-only proteins. Our examination of native Bak oligomers indicates that the Bak apoptotic pore forms by the multimerization of BH3:groove homodimers and reveals that Bak α6 is not only important for Bak oligomerization and function but may also be involved in how Bak is activated by BH3-only proteins.
authors.affiliation: From the Walter and Eliza Hall Institute of Medical Research, Melbourne 3052, Australia,; the Department of Medical Biology, University of Melbourne, Melbourne 3010, Australia.
authors.forename: Stephen
authors.initials: S
authors.lastname: Ma
authors.affiliation: From the Walter and Eliza Hall Institute of Medical Research, Melbourne 3052, Australia,; the Department of Medical Biology, University of Melbourne, Melbourne 3010, Australia.
authors.forename: Colin
authors.initials: C
authors.lastname: Hockings
authors.affiliation: From the Walter and Eliza Hall Institute of Medical Research, Melbourne 3052, Australia.
authors.forename: Khatira
authors.initials: K
authors.lastname: Anwari
authors.affiliation: From the Walter and Eliza Hall Institute of Medical Research, Melbourne 3052, Australia,; the Department of Medical Biology, University of Melbourne, Melbourne 3010, Australia.
authors.forename: Tobias
authors.initials: T
authors.lastname: Kratina
authors.affiliation: From the Walter and Eliza Hall Institute of Medical Research, Melbourne 3052, Australia,; the Department of Medical Biology, University of Melbourne, Melbourne 3010, Australia.
authors.forename: Stephanie
authors.initials: S
authors.lastname: Fennell
authors.affiliation: the Biochemistry Section, Surgical Neurology Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892, and.
authors.forename: Michael
authors.initials: M
authors.lastname: Lazarou
authors.affiliation: the Department of Biochemistry, La Trobe Institute for Molecular Science, La Trobe University, Melbourne 3086, Australia.
authors.forename: Michael T
authors.initials: MT
authors.lastname: Ryan
authors.affiliation: From the Walter and Eliza Hall Institute of Medical Research, Melbourne 3052, Australia,; the Department of Medical Biology, University of Melbourne, Melbourne 3010, Australia.
authors.forename: Ruth M
authors.initials: RM
authors.lastname: Kluck
authors.affiliation: From the Walter and Eliza Hall Institute of Medical Research, Melbourne 3052, Australia,; the Department of Medical Biology, University of Melbourne, Melbourne 3010, Australia,. Electronic address: dewson@wehi.edu.au.
authors.forename: Grant
authors.initials: G
authors.lastname: Dewson
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D051023:BH3 Interacting Domain Death Agonist Protein / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D051379:Mice; D018345:Mice, Knockout; D055503:Protein Multimerization / Q000502:physiology*; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D050996:Voltage-Dependent Anion Channel 2 / Q000235:genetics / Q000378:metabolism; D050998:bcl-2 Homologous Antagonist-Killer Protein / Q000235:genetics / Q000378:metabolism*
chemical_list: D051023:BH3 Interacting Domain Death Agonist Protein; C494773:Bak1 protein, mouse; C494815:Bid protein, mouse; C494766:Vdac2 protein, mouse; D050996:Voltage-Dependent Anion Channel 2; D050998:bcl-2 Homologous Antagonist-Killer Protein
keywords: Apoptosis; Bak; Bax; Bcl-2 Family Proteins; Blue Native PAGE; Mitochondria; Oligomerization; Protein Conformation
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