A high-resolution dimeric structure reveals a critical role of α-helix 9 in apoptotic Bax pore assembly in the mitochondrial membrane

Bax functions as a proapoptotic protein perforating the mitochondrial membrane to release mitochondrial proteins and DNA that kill the cell. Structures of full-length Bax monomer in solution and core domain dimers in solution and bound to lipid/detergent bicelles reveal how Bax proteins must be activated, unfold, refold and dimerize prior to forming oligomeric pores in the membrane. In particular, amphipathic core dimer forms part of the pore wall between the nonpolar lipid bilayer and the aqueous conduit. However, atomic resolution structures of other parts of the pore were scarce. Here, we elucidate a structure of Bax C-terminal α-helix 9 (α9) in lysolipid micelles using NMR. According to this high-resolution structure the α9 regions form an amphipathic helical dimer with an extended nonpolar interface and several uncharged polar residues on the surface. Structure-guided mutagenesis and functional assessment demonstrate that the nonpolar interactions are important for Bax dimerization in and perforation of the mitochondrial membrane. Surprisingly the polar residues are also important because they form bifurcated hydrogen bonds between helical turns to stabilize each helix and thereby the dimer. Molecular dynamics simulations of an oligomer constructed with wall-forming core and transmembrane α9 dimers linked by flexible α6-α7-α8 bridges in a mitochondrial lipid bilayer generate an atomic resolution model for a stable Bax pore capable to release cytochrome C. Thus, we made an important step toward elucidating molecular mechanisms of apoptotic Bax perforation.