Membrane-Targeted Soluble Form of BAK Unfolds Like an Umbrella Upon Pore-Formation

Abstract

Mitochondrial permeabilization by the pore-forming Bcl2 (B-cell lymphoma-2) proteins such as BAX or BAK constitutes a key regulatory step in the apoptotic processes. Based on the structural similarity of these to the pore-forming bacterial proteins such as colicin and the transmembrane domain of diphtheria toxin, it has been hypothesized that BAX or BAK undergoes conformational changes upon pore formation, in which the hydrophobic helical hairpin structure found in these proteins is unwrapped and inserts into the membrane. We have developed a liposomal system that recapitulates the membrane-permeabilization by BAK through pore-formation. Using a Ni(II)-nitriloacetic acid liposomal system which can conjugate hexa-histidine tagged proteins to the surface of the simulated outer mitochondrial membrane surface, we demonstrate that nanomolar concentrations of BAK when targeted to the membrane surface can efficiently permeabilize the membrane by forming large pores. Using pairs of spin labels introduced at various positions in BAK, we measured the distances between them in the BAK protein before and after pore-formation in the Ni-NTA-liposomal system. The distance between spin labeled residues 55R1 and 146R1, which are located at the αH1-αH2 loop and at the tip of the αH5-αH6 helical hairpin loop, respectively, changes from approximately 15 Å to 50 Å upon pore-formation. On the other hand, the distance between residues near the top of the hairpin, i.e., 75R1 and 122R1, which are located on αH2 and at the N-terminus of αH5, respectively, changes from approximately 20 Å to 25 Å. These results suggest that upon pore formation the layers covering the αH5-αH6 helical hairpin structure in BAK open up, exposing the helical hairpin structure for membrane insertion. These results are consistent with the aforementioned hypothesis regarding the conformational changes associated with the pore-forming Bcl2 proteins upon membrane permeabilization.