Bax plays a central role in the mitochondrial pathway of apoptosis. Upon activation, cytosolic Bax monomers oligomerize on the surface of mitochondria and change conformation concertedly to punch holes into the outer membrane. The subsequent release of cytochrome c initiates cell death. However, the structure of membrane-inserted Bax and its mechanism of action remain largely unknown. Here, we propose a 3D model of active Bax at the membrane based on double electron-electron resonance (DEER) spectroscopy in liposomes and isolated mitochondria. We show that active Bax is organized at the membrane as assemblies of dimers. In addition to a stable dimerization domain, each monomer contains a more flexible piercing domain involved in interdimer interactions and pore formation. The most important structural change during Bax activation is the opening of the hairpin formed by helices 5 and 6, which adopts a clamp-like conformation central to the mechanism of mitochondrial permeabilization.

Bax is a key regulator of apoptosis that, under cell stress, accumulates at mitochondria, where it oligomerizes to mediate the permeabilization of the mitochondrial outer membrane leading to cytochrome c release and cell death. However, the underlying mechanism behind Bax function remains poorly understood. Here, we studied the spatial organization of Bax in apoptotic cells using dual-color single-molecule localization-based super-resolution microscopy. We show that active Bax clustered into a broad distribution of distinct architectures, including full rings, as well as linear and arc-shaped oligomeric assemblies that localized in discrete foci along mitochondria. Remarkably, both rings and arcs assemblies of Bax perforated the membrane, as revealed by atomic force microscopy in lipid bilayers. Our data identify the supramolecular organization of Bax during apoptosis and support a molecular mechanism in which Bax fully or partially delineates pores of different sizes to permeabilize the mitochondrial outer membrane.