Homogenous Oligomers of Pro-Apoptotic BAX Reveal Structural Determinants of Mitochondrial Membrane Permeabilization

Abstract

BAX is a dynamic protein that transforms from a cytosolic monomer into a toxic oligomer that permeabilizes the mitochondrial outer membrane to achieve apoptotic cell death. How BAX monomers assemble into a higher-order conformation, and the structural determinants essential to membrane permeabilization, remain a mechanistic mystery. A key hurdle has been the inability to generate a pure, homogeneous, and stable BAX oligomer for analysis. Here, we report the production and characterization of a full-length BAX oligomer that recapitulates physiologic BAX activation. Hydrogen-deuterium exchange mass spectrometry and next-generation chemical crosslinking analyses revealed striking conformational consequences of oligomerization. Small angle X-ray scattering provided further insight into the macromolecular structure of this BAX oligomer. Importantly, these homogenous BAX oligomers enabled the assignment of specific roles to particular residues and α-helices that mediate individual steps of BAX activation, including the critical “execution-phase” known as mitochondrial outer membrane permeabilization (MOMP). Unexpectedly, we find that the BAX C terminus is not required for self association during early BAX oligomerization, and has two distinguishable roles in inducing MOMP. Additionally we find that the distinctive amphipathicity of BAX α6, which shares the cation-rich and hydrophobic features of lytic peptide helices, is a key mechanistic driver of membrane disruption. Our results provide the first glimpse of a full-length and functional BAX oligomer, revealing structural requirements for the elusive execution-phase of mitochondrial apoptosis and enabling new methods for further dissection of the structure and function of BAX.