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

Kyoung Joon Oh, Pawan Singh, Kyungro Lee, Kelly Foss, Shinyoub Lee, Minji Park, Steffi Lee, Sreevidya Aluvila, Matthew Park, Puja Singh, Ryung S. Kim, Jindrich Symersky, D. Eric Walters

Research output: Contribution to journalArticle

65 Citations (Scopus)

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 Ni2+-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 α5-α6 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 Å 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.

Original languageEnglish (US)
Pages (from-to)28924-28937
Number of pages14
JournalJournal of Biological Chemistry
Volume285
Issue number37
DOIs
StatePublished - Sep 10 2010

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Oligomers
His-His-His-His-His-His
Membranes
Proteins
Nitrilotriacetic Acid
Apoptosis
Spin Labels
Monomers
Mitochondrial Membranes
Cytochromes c
Cell death
Cytoplasm
Cell Death
Labeling
Paramagnetic resonance
Lipids
Amino Acids

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology
  • Medicine(all)

Cite this

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. / Oh, Kyoung Joon; Singh, Pawan; Lee, Kyungro; Foss, Kelly; Lee, Shinyoub; Park, Minji; Lee, Steffi; Aluvila, Sreevidya; Park, Matthew; Singh, Puja; Kim, Ryung S.; Symersky, Jindrich; Walters, D. Eric.

In: Journal of Biological Chemistry, Vol. 285, No. 37, 10.09.2010, p. 28924-28937.

Research output: Contribution to journalArticle

Oh, Kyoung Joon ; Singh, Pawan ; Lee, Kyungro ; Foss, Kelly ; Lee, Shinyoub ; Park, Minji ; Lee, Steffi ; Aluvila, Sreevidya ; Park, Matthew ; Singh, Puja ; Kim, Ryung S. ; Symersky, Jindrich ; Walters, D. Eric. / 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. In: Journal of Biological Chemistry. 2010 ; Vol. 285, No. 37. pp. 28924-28937.
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T1 - 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

AU - Oh, Kyoung Joon

AU - Singh, Pawan

AU - Lee, Kyungro

AU - Foss, Kelly

AU - Lee, Shinyoub

AU - Park, Minji

AU - Lee, Steffi

AU - Aluvila, Sreevidya

AU - Park, Matthew

AU - Singh, Puja

AU - Kim, Ryung S.

AU - Symersky, Jindrich

AU - Walters, D. Eric

PY - 2010/9/10

Y1 - 2010/9/10

N2 - 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 Ni2+-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 α5-α6 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 Å 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.

AB - 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 Ni2+-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 α5-α6 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 Å 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.

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