title: Mutation to Bax beyond the BH3 domain disrupts interactions with pro-survival proteins and promotes apoptosis.
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.
authors.affiliation: Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Australia. czabotar@wehi.edu.au
authors.forename: Peter E
authors.initials: PE
authors.lastname: Czabotar
authors.forename: Erinna F
authors.initials: EF
authors.lastname: Lee
authors.forename: Geoff V
authors.initials: GV
authors.lastname: Thompson
authors.forename: Ahmad Z
authors.initials: AZ
authors.lastname: Wardak
authors.forename: W Douglas
authors.initials: WD
authors.lastname: Fairlie
authors.forename: Peter M
authors.initials: PM
authors.lastname: Colman
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D001713:Biphenyl Compounds / Q000494:pharmacology; D002470:Cell Survival / Q000502:physiology; D002478:Cells, Cultured; D003461:Crystallography; D005347:Fibroblasts / Q000166:cytology / Q000502:physiology; D006801:Humans; D051379:Mice; D008928:Mitochondria / Q000502:physiology; D016296:Mutagenesis / Q000502:physiology; D064549:Myeloid Cell Leukemia Sequence 1 Protein; D009596:Nitrophenols / Q000494:pharmacology; D010879:Piperazines / Q000494:pharmacology; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000502:physiology; D013449:Sulfonamides / Q000494:pharmacology; D051028:bcl-2-Associated X Protein / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: C501332:ABT-737; C494826:BAX protein, human; D001713:Biphenyl Compounds; C579094:Mcl1 protein, mouse; D064549:Myeloid Cell Leukemia Sequence 1 Protein; D009596:Nitrophenols; D010879:Piperazines; D019253:Proto-Oncogene Proteins c-bcl-2; D013449:Sulfonamides; D051028:bcl-2-Associated X Protein