Mutagenesis of the BH3 domain of BAX identifies residues critical for dimerization and killing.
The BCL-2 family of proteins is comprised of proapoptotic as well as antiapoptotic members (S. N. Farrow and R. Brown, Curr. Opin. Genet. Dev. 6:45-49, 1996). A prominent death agonist, BAX, forms homodimers and heterodimerizes with multiple antiapoptotic members. Death agonists have an amphipathic alpha helix, called BH3; however, the initial assessment of BH3 in BAX has yielded conflicting results. Our BAX deletion constructs and minimal domain constructs indicated that the BH3 domain was required for BAX homodimerization and heterodimerization with BCL-2, BCL-XL, and MCL-1. An extensive site-directed mutagenesis of BH3 revealed that substitutions along the hydrophobic face of BH3, especially charged substitutions, had the greatest affects on dimerization patterns and death agonist activity. Particularly instructive was the BAX mutant mIII-1 (L63A, G67A, L70A, and M74A), which replaced the hydrophobic face of BH3 with alanines, preserving its amphipathic nature. BAXmIII-1 failed to form heterodimers or homodimers by yeast two-hybrid or immunoprecipitation analysis yet retained proapoptotic activity. This suggests that BAX's killing function reflects mechanisms beyond its binding to BCL-2 or BCL-XL to inhibit them or simply displace other protein partners. Notably, BAXmIII-1 was found predominantly in mitochondrial membranes, where it was homodimerized as assessed by homobifunctional cross-linkers. This characteristic of BAXmIII-1 correlates with its capacity to induce mitochondrial dysfunction, caspase activation, and apoptosis. These data are consistent with a model in which BAX death agonist activity may require an intramembranous conformation of this molecule that is not assessed accurately by classic binding assays.
Wang
K
K
Departments of Medicine and Pathology, Division of Molecular Oncology, Howard Hughes Medical Institute, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Gross
A
A
Waksman
G
G
Korsmeyer
S J
SJ
D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis; D001665:Binding Sites; D002460:Cell Line; D002461:Cell Line, Transformed; D003432:Cross-Linking Reagents; D019281:Dimerization; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011233:Precipitin Tests; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D012441:Saccharomyces cerevisiae; D051028:bcl-2-Associated X Protein
C494826:BAX protein, human; C494828:Bax protein, rat; D003432:Cross-Linking Reagents; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D051028:bcl-2-Associated X Protein