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

Abstract

Graphical Abstract Highlights d BH3 binding to BAX groove essential for late but not early steps in BAX activation d Mutations identified in BAX helices 1 and 6 near a proposed second BH3 binding site d They reduced BH3 binding to this site and stabilized BAX in inactive conformations d They revealed allosteric changes controlling BAX mitochondrial membrane association SUMMARYTo 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 a1 and a6. Our mutagenesis studies reveal that late steps like oligomerization require activation through the groove but probably not earlier steps like MOM association. Conversely, a1 or a6 obstruction and alanine mutagenesis scanning implicate these helices early in BAX activation. The a1 and a6 mutations lowered BH3 binding, altered the BAX conformation, and reduced its MOM translocation and integration; their exposure of the BAX a1-a2 loop allosterically sequestered its a9 membrane anchor in the groove. The crystal structure of an a6 mutant revealed additional allosteric effects. The results suggest that the a1 and a6 region drives MOM association and integration, whereas groove binding favors subsequent steps toward oligomerization.(E) Upon a death stimulus, unlike the active conformation assumed by WT BAX and K21E, the groove mutants translocate to the MOM but retain an inactive conformation. Bax/Bak DKO MEFs expressing the BAX variants were pre-incubated with caspase inhibitor Q-VD.oph (25 mM) for 1 h, then treated with 5 mM etoposide. After 16 h, the cells were permeabilized with 0.025% digitonin and treated with PK (20 min on ice) before fractionation, carbonate extraction, and immunoblotting for BAX. (F) Unlike WT and K21E BAX, the groove mutants do not expose the N-terminal 6A7 epitope upon a death stimulus. DKO MEFs expressing the human BAX variants, treated as in (E), were solubilized in 1% (3-((3-cholamidopropyl) dimethylammonio)-1 (CHAPS), and activated BAX was immunoprecipitated with antibody 6A7. All immunoblots are representative of at least two independent experiments.

Robust autoactivation for apoptosis by BAK but not BAX highlights BAK as an important therapeutic target

“…6b). As previously observed 38 , in response to cBID, low levels of BAX but not BAX R109D released cytochrome c from Bak −/− MLM (Fig. 6b, lane 3 vs. 10).…”

“…Recombinant BAX R109D and BAX R34A were generated by site-directed mutagenesis of human wild-type or cysnull BAX, respectively 38,44 , and the recombinant wild-type and mutant BAX proteins expressed and purified as described 9 .…”

“…Membrane insertion of BAX (expressed in cells or recombinant protein added to MLM) was assessed by sodium carbonate extraction, as described 38 . BAK and BAX conformation change was assessed by limited proteolysis, as described 45 .…”

“…To enhance the likelihood of BAX BH3 exposure, we also tested the groove mutant BAX R109D (Fig. 6a), which has a low affinity for cBID 12,38 . We reasoned the low affinity for BH3 domains may also reduce BH3:groove homodimer formation.…”

“…S1a) 12,14,35,36 . Activation sites on the receptor BAK and BAX molecules might include the canonical α2-α5 surface groove 12,14,37 or a modeled α1/α6 trigger site 32,[38][39][40] (Fig. S1b).…”