Demise of the Umbrella Model in BAK-Mediated Mitochondrial Membrane Permeabilization During Apoptosis

Abstract

BCL-2 proteins, including BAX and BAK, play critical roles in apoptosis. Upon activation by cell death signals, BAX or BAK forms large oligomeric pores in the mitochondrial outer membrane, permeabilizing mitochondria. This allows the escape of the cell death factors such as cytochrome c from the intermembrane space into the cytoplasm, where they execute downstream cell death events. Using a proteo-liposomal system that recapitulates the pore-forming processes by BAX or BAK (Oh et al. JBC 2010; 285, 28924-28937), we previously demonstrated that spin labels attached in the BH3 (Bcl-2 homology domain 3) domain were juxtaposed within 5-10 A distance in the oligomeric form in the membrane, providing direct evidence for the existence of the BH3:BH3 contact interface between nearby monomers. We have further determined the conformational changes in BAK upon membrane insertion by applying the site-directed spin labeling method of EPR. Pairs of spin labels introduced at various positions in BAK allowed us to measure the inter-domain distances before and after the pore-formation. The results show that BAK unfolds and inserts into the membrane in an unexpected way, contrary to the predictions by the “umbrella model” in which the sandwiching outer layers of the BAK protein open up to expose the core α5-α6 helical hairpin for membrane insertion. Our results also indicate that residue 83 in the BH3 domain is in close proximity to the central region of α5 helix (i.e., residue 135) but is removed from its amino-terminus (i.e., residue 122) within a monomer in the membrane-inserted oligomeric pore. These results identify α5 helix as the previously unknown domain in contact with the BH3 domain in the membrane bound state of BAK (ibid), providing further insights into the mechanism of BAK (or BAX) dimerization and oligomerization.