Mutation to Bax beyond the BH3 domain disrupts interactions with pro-survival proteins and promotes apoptosis

J Biol Chem. 2011 Mar 4;286(9):7123-31. doi: 10.1074/jbc.M110.161281. Epub 2011 Jan 3.

Abstract

Pro-survival members of the Bcl-2 family of proteins restrain the pro-apoptotic activity of Bax, either directly through interactions with Bax or indirectly by sequestration of activator BH3-only proteins, or both. Mutations in Bax that promote apoptosis can provide insight into how Bax is regulated. Here, we describe crystal structures of the pro-survival proteins Mcl-1 and Bcl-x(L) in complex with a 34-mer peptide from Bax that encompasses its BH3 domain. These structures reveal canonical interactions between four signature hydrophobic amino acids from the BaxBH3 domain and the BH3-binding groove of the pro-survival proteins. In both structures, Met-74 from the Bax peptide engages with the BH3-binding groove in a fifth hydrophobic interaction. Various Bax Met-74 mutants disrupt interactions between Bax and all pro-survival proteins, but these Bax mutants retain pro-apoptotic activity. Bax/Bak-deficient mouse embryonic fibroblast cells reconstituted with several Bax Met-74 mutants are more sensitive to the BH3 mimetic compound ABT-737 as compared with cells expressing wild-type Bax. Furthermore, the cells expressing Bax Met-74 mutants are less viable in colony assays even in the absence of an external apoptotic stimulus. These results support a model in which direct restraint of Bax by pro-survival Bcl-2 proteins is a barrier to apoptosis.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Apoptosis / drug effects
  • Apoptosis / physiology*
  • Biphenyl Compounds / pharmacology
  • Cell Survival / physiology
  • Cells, Cultured
  • Crystallography
  • Fibroblasts / cytology
  • Fibroblasts / physiology
  • Humans
  • Mice
  • Mitochondria / physiology
  • Mutagenesis / physiology
  • Myeloid Cell Leukemia Sequence 1 Protein
  • Nitrophenols / pharmacology
  • Piperazines / pharmacology
  • Protein Structure, Secondary
  • Protein Structure, Tertiary
  • Proto-Oncogene Proteins c-bcl-2 / chemistry
  • Proto-Oncogene Proteins c-bcl-2 / genetics
  • Proto-Oncogene Proteins c-bcl-2 / metabolism
  • Saccharomyces cerevisiae / physiology
  • Sulfonamides / pharmacology
  • bcl-2-Associated X Protein* / chemistry
  • bcl-2-Associated X Protein* / genetics
  • bcl-2-Associated X Protein* / metabolism

Substances

  • ABT-737
  • BAX protein, human
  • Biphenyl Compounds
  • Mcl1 protein, mouse
  • Myeloid Cell Leukemia Sequence 1 Protein
  • Nitrophenols
  • Piperazines
  • Proto-Oncogene Proteins c-bcl-2
  • Sulfonamides
  • bcl-2-Associated X Protein

Associated data

  • PDB/3PK1
  • PDB/3PL7