Project Summary (Lin Project) Apoptosis is a cell death program that normally eliminates dysfunctional cells, and hence is essential to human health. Insufficient apoptosis leads to cancer, while excessive apoptosis worsens brain damage after stroke. To develop effective treatments for these diseases we need a comprehensive understanding of how apoptosis is regulated. Mitochondrial membrane perforation is the commitment step of apoptosis that is regulated by interactions among the Bcl-2 family proteins, such as the pro-apoptotic Bax and the anti-apoptotic Bcl-2. While anti-cancer drugs that selectively target and inhibit the anti-apoptotic proteins have shown promise in clinical trials, limited structural information on the pro-apoptotic proteins has become a bottleneck for development of neural protective agents that can reduce stroke damage. In particular, structures of active Bax oligomers that can form giant pores in the mitochondrial membrane to release deadly mitochondrial factors are unknown. Since native Bax proteins have multiple ways to interact and can generate different types of heterogeneous oligomers that are not amenable to structure determination, an alternative and innovative experimental approach is required to determine the structures of these different oligomers and the giant pores they can form. We hypothesize that: (A) Homogeneous small oligomers suitable for structure determination can be created by using truncated or mini-Bax proteins that retain key interacting regions seen in intact Bax oligomers; (B) These small oligomers will be ideal tools to dissect the mechanism by which intact Bax proteins perforate the mitochondrial membrane, and by which anti-apoptotic compounds target Bax to prevent this mitochondrial leakage and neuronal cell death. Our project for the Oklahoma COBRE in Structural Biology Phase II application will test our novel hypotheses in the following Specific Aims: (1) What are the structures of the mini- Bax oligomers and how do they contribute to the giant pore assembly by intact Bax? (2) How do anti-apoptotic compounds block Bax pore formation in the mitochondrial membrane? In Aim 1, we will use molecular biology techniques to construct mini-Bax proteins. We will perform disulfide crosslinking experiments and size exclusion chromatography to select the mini-oligomer forming proteins and a mitochondrial protein or liposomal dye release assay will be used to identify the mini-pore forming oligomers. We will conduct crystallization trials with the pore-forming, size-selected homogeneous mini-oligomers and solve their structures using X-ray diffraction. If suitable crystals are not obtained, we will measure small-angle X-ray scattering of the mini-Bax proteins in solution, detergent micelle, or lipid nanodisc to reveal the mini-oligomeric structures. Moreover, we will use electron cryo-microscopy to reveal the mini-pore structures in membranes, and mutagenesis to assess how these mini-structures contribute to mega pore assembly by intact Bax in vitro and in vivo. Our collaborator Dr. David Andrews has discovered several compounds that block Bax oligomerization at an undefined dimer stage and protect primary neurons from glutamate excitotoxicity in an in vitro model for stroke damage. In Aim 2, we will explore if these compounds inhibit the oligomerization but not dimerization of our mini-Bax proteins and if so, whether this inhibition is sufficient to abolish mini-pore formation. We will also explore if these compounds force intact Bax into a dead-end dimer that cannot induce mitochondrial membrane perforation and if so, we will solve the dead-end dimer structure. The anticipated project outcomes are the structural details of the molecular interactions that are critical for pro-apoptotic Bax pore assembly in the mitochondrial membrane, and the regulation of this process by anti-apoptotic compounds. The impact of these significant outcomes to the apoptosis field will be vital, because they will reveal the relevant targets and mechanisms for developing better neural protective drugs to effectively combat brain damage from stroke.