Alteration of tBiD-Induced Apoptotic Bax Poration in Mitochondrial Membranes by Mutations and Small Molecules

Abstract

Mitochondria-dependent apoptosis is regulated by the Bcl-2 protein family, of which Bax is a critical effector and its deficiency leads to cancer development and drug resistance. While the pro-survival Bcl-2 proteins are well-established targets for cancer treatment, the pro-death Bax activation in cancer cells or inhibition in normal cells has been less explored. To therapeutically target Bax, we must know how Bax is activated and how active Bax causes the apoptotic mitochondrial outer membrane (MOM) permeabilization. Current models propose that BH3 proteins transiently interact with Bax at the canonical groove or the trigger pocket to activate Bax, and the active Bax proteins embed in the MOM, and form oligomeric pores via the BH3-in-groove and other dimeric interactions. To test these models and determine how these molecular interactions contribute to Bax activation and pore formation in the MOM, we reconstituted the poration with purified proteins and mitochondria-mimic liposomes. Our results show that the BH3 protein tBid activates Bax via two binding sites that may be allosterically coupled. Further, the nonpolar and polar interactions of the lipid bilayer with two helical dimeric domains of the full-length oligomeric Bax proteins contribute to Bax pore formation. Furthermore, small molecule compounds that mimic the BH3 domain of tBid and bind the canonical groove in Bax inhibit the tBid-Bax interaction and reduce the Bax pore formation. Therefore, our study has not only revealed how Bax protein is activated and forms pores but suggested a potential for the small molecule BH3 mimetics to modulate the activity of this pro-death protein and to become novel cancer therapeutics.