title: Conformational changes in BAK, a pore-forming proapoptotic Bcl-2 family member, upon membrane insertion and direct evidence for the existence of BH3-BH3 contact interface in BAK homo-oligomers.
abstract: During apoptosis, the pro-apoptotic Bcl-2 family proteins BAK and BAX form large oligomeric pores in the mitochondrial outer membrane. Apoptotic factors, including cytochrome c, are released through these pores from the mitochondrial intermembrane space into the cytoplasm where they initiate the cascade of events leading to cell death. To better understand this pivotal step toward apoptosis, a method was developed to induce membrane permeabilization by BAK in the membrane without using the full-length protein. Using a soluble form of BAK with a hexahistidine tag at the C terminus and a liposomal system containing the Ni(2+)-nitrilotriacetic acid lipid analog that can bind hexahistidine-tagged proteins, BAK oligomers were formed in the presence of the activator protein p7/p15Bid. In this system, we determined the conformational changes in BAK upon membrane insertion by applying the site-directed spin labeling method of EPR to 13 different amino acid locations. Upon membrane insertion, the BH3 domains were reorganized, and the alpha5-alpha6 helical hairpin structure was partially exposed to the membrane environment. The monomer-monomer interface in the oligomeric structure was also mapped by measuring the distance-dependent spin-spin interactions for each residue location. Spin labels attached in the BH3 domain were juxtaposed within 5-10 A distance in the oligomeric form in the membrane. These results are consistent with the current hypothesis that BAK or BAX forms homodimers, and these homodimers assemble into a higher order oligomeric pore. Detailed analyses of the data provide new insights into the structure of the BAX or BAK homodimer.
authors.affiliation: Department of Biochemistry, Chicago Medical School, Rosalind Franklin University of Medicine and Science, North Chicago, Illinois 60064, USA. kyoung.oh@rosalindfranklin.edu
authors.forename: Kyoung Joon
authors.initials: KJ
authors.lastname: Oh
authors.forename: Pawan
authors.initials: P
authors.lastname: Singh
authors.forename: Kyungro
authors.initials: K
authors.lastname: Lee
authors.forename: Kelly
authors.initials: K
authors.lastname: Foss
authors.forename: Shinyoub
authors.initials: S
authors.lastname: Lee
authors.forename: Minji
authors.initials: M
authors.lastname: Park
authors.forename: Steffi
authors.initials: S
authors.lastname: Lee
authors.forename: Sreevidya
authors.initials: S
authors.lastname: Aluvila
authors.forename: Matthew
authors.initials: M
authors.lastname: Park
authors.forename: Puja
authors.initials: P
authors.lastname: Singh
authors.forename: Ryung-Suk
authors.initials: RS
authors.lastname: Kim
authors.forename: Jindrich
authors.initials: J
authors.lastname: Symersky
authors.forename: D Eric
authors.initials: DE
authors.lastname: Walters
mesh_terms: D000818:Animals; D051023:BH3 Interacting Domain Death Agonist Protein; D006801:Humans; D051379:Mice; D051336:Mitochondrial Membranes / Q000737:chemistry / Q000378:metabolism; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship; D050998:bcl-2 Homologous Antagonist-Killer Protein / Q000737:chemistry* / Q000378:metabolism; D051028:bcl-2-Associated X Protein / Q000737:chemistry / Q000378:metabolism
chemical_list: C494772:BAK1 protein, human; D051023:BH3 Interacting Domain Death Agonist Protein; C494773:Bak1 protein, mouse; C494827:Bax protein, mouse; C494815:Bid protein, mouse; D050998:bcl-2 Homologous Antagonist-Killer Protein; D051028:bcl-2-Associated X Protein