Homogeneous Oligomers of Pro-apoptotic BAX Reveal Structural Determinants of Mitochondrial Membrane Permeabilization

Abstract

Highlights d Fos-12 induces homogeneous BAX oligomers that recapitulate physiologic activation d SAXS, HXMS, and crosslinking analyses reveal conformational features of BAX O d BAX O distinguishes between the structural determinants of activation and poration d BAX O uncovered roles for a6 and a9 in the execution phase of mitochondrial apoptosis

The conformational stability of pro-apoptotic BAX is dictated by discrete residues of the protein core

“…In each case, we observed striking, regiospecific consequences of replacing the bulky hydrophobic residue with alanine. Specifically, the L113A mutant showed progressive deprotection over time of α6, which is directly engaged by L113 and implicated in both the mechanism of BH3 triggering 27 and membrane interaction by its conserved arginine residues 28 (Fig. 4a ).…”

“…Distinct and step-specific roles of discrete residues along the BAX activation continuum has emerged as a remarkable feature of this pro-apoptotic mechanism. For example, we recently demonstrated that mutagenesis of the α6 surface residue I133 disrupted BH3 binding to the trigger site and thus the initiation of BAX activation but had no adverse effect on the execution phase steps of mitochondrial translocation, self-association, and membrane poration 28 . In contrast, mutagenesis of R134, a surface residue immediately adjacent to I133 and also a component of the N-terminal trigger site had no effect on BH3-mediated initiation of BAX activation and instead impaired membrane tropism and oligomeric poration 28 .…”

“…For example, we recently demonstrated that mutagenesis of the α6 surface residue I133 disrupted BH3 binding to the trigger site and thus the initiation of BAX activation but had no adverse effect on the execution phase steps of mitochondrial translocation, self-association, and membrane poration 28 . In contrast, mutagenesis of R134, a surface residue immediately adjacent to I133 and also a component of the N-terminal trigger site had no effect on BH3-mediated initiation of BAX activation and instead impaired membrane tropism and oligomeric poration 28 . Here we find that, depending on the stage-specific structural context of monomer vs. dimer, BAX α5 core residues 113–116 and their network of hydrophobic interactions either have individual or cumulative functional importance.…”

Toxicity of Melastoma dodecandrum Lour. and its effects on lipopolysaccharide‑induced inflammation and oxidative stress

“…When ROS induces irreversible DNA damage, p53 is involved in the regulation of apoptosis through upregulation of Bax and downregulation of Bcl-2 (30). Both Bax and Bcl-2 belong to the Bcl-2 family and regulate apoptosis by modulating mitochondrial membrane permeability (31). Bax activation and Bcl-2 inhibition promote mitochondrial permeability transition pore opening, resulting in the release of Cyt-C from the mitochondria into the cytoplasm, which in turn activates caspase-9 and its downstream effector caspase 3 to enhance apoptosis (30).…”

Amphipathic Bax core dimer forms part of apoptotic pore wall in the mitochondrial membrane

“…(i) Site-specific crosslinking, double electron-electron resonance (DEER) spectroscopy and Förster resonance energy transfer have verified the dimeric interaction between the 2 to 5 regions of two full-length Bax proteins integrated into the native or model mitochondrial membrane (Bleicken et al, 2014;Dewson et al, 2012;Gahl et al, 2014;Zhang et al, 2016). (ii) Accessibility of cysteine residues placed in the nonpolar surface of the 2 to 5 dimer to a charged sulfhydryl reactive labeling agent (IASD), electron paramagnetic resonance (EPR) detected accessibility of spin probes attached to the nonpolar surface to molecular oxygen enriched in the lipid bilayer or paramagnetic quenchers soluble in water, fluorescence emission and quenching of environment sensitive dyes attached to the nonpolar surface, and H-D exchange of the 4 to 5 region in a membrane-bound Bax oligomer are consistent with their locations within the membrane (Annis et al, 2005;Bleicken et al, 2018;Bleicken et al, 2010;Flores-Romero et al, 2017;Hauseman et al, 2020;Westphal et al, 2014;Zhang et al, 2016). (iii) In contrast to the stable conformation of the 2 to 5 dimer, the DEER spectral data from the 6 to 9 region indicate a dynamic conformation that would allow the 6 from one protomer to engage the cytosolic surface of the MOM, and the 6 from the other protomer to engage the intermembrane surface, with the two 9 membrane anchors in opposite orientations (Bleicken et al, 2014).…”