BAX Activation: Mutations Near Its Proposed Non-canonical BH3 Binding Site Reveal Allosteric Changes Controlling Mitochondrial Association

Abstract

Summary: To elicit apoptosis, BAX metamorphoses from an inert cytosolic monomer into homo-oligomers that permeabilize the mitochondrial outer membrane (MOM). A long-standing puzzle is that BH3 domains apparently activate BAX by not only its canonical groove but also a proposed site involving helices α1 and α6. Our mutagenesis studies reveal that late steps like oligomerization require activation through the groove but probably not earlier steps like MOM association. Conversely, α1 or α6 obstruction and alanine mutagenesis scanning implicate these helices early in BAX activation. The α1 and α6 mutations lowered BH3 binding, altered the BAX conformation, and reduced its MOM translocation and integration; their exposure of the BAX α1-α2 loop allosterically sequestered its α9 membrane anchor in the groove. The crystal structure of an α6 mutant revealed additional allosteric effects. The results suggest that the α1 and α6 region drives MOM association and integration, whereas groove binding favors subsequent steps toward oligomerization. : BAX activation for apoptosis may require BH3 binding to its canonical groove and/or a less-characterized α1 and α6 site. Dengler et al. identify α1 and α6 mutations that impair early steps in BAX activation and reveal allosteric changes that control exposure of its membrane anchor and association with the mitochondrial outer membrane. Keywords: BCL-2 family, BAX activation, apoptosis, membrane association, oligomerization, allosteric changes, crystal structure, protein-protein association, protein conformation