Demonstrating in vivo interaction of two important biomolecules and the relevance of the interaction to a biological process have been difficult issues in biomedical research. Here, we report the use of a homology modeling approach to establish the significance of protein interactions in governing the activation of programmed cell death in Caenorhabditis elegans. A protein interaction cascade has been postulated to mediate activation of cell death in nematodes, in which the BH3-domain-containing (Bcl-2 homology region 3) protein EGL-1 binds the cell-death inhibitor CED-9 and induces release of the death-activating protein CED-4 from inhibitory CED-4͞CED-9 complexes. We show here that an unusual gain-offunction mutation in ced-9 (substitution of glycine 169 to glutamate) that results in potent inhibition of most nematode cell deaths impairs the binding of EGL-1 to CED-9 and EGL-1-induced release of CED-4 from CED-4͞CED-9 complexes. Based on a modeled EGL-1͞CED-9 complex structure, we generated second-site compensatory mutations in EGL-1 that partially restore the binding of EGL-1 to CED-9(G169E) and EGL-1-induced release of CED-4 from CED-4͞CED-9(G169E) complexes. Importantly, these mutations also significantly suppress the death-protective activity of CED-9(G169E) in vivo. These results establish that direct physical interaction between EGL-1 and CED-9 is essential for the release of CED-4 and the activation of cell death. The structure-based design of second-site suppressors via homology modeling should be widely applicable for probing important molecular interactions that are implicated in fundamental biological processes. P rogrammed cell death is a tightly regulated cellular process crucial for metazoan development and homeostasis (1, 2). Improper regulation of programmed cell death can lead to a variety of diseases, including cancer and degenerative disorders (3). Genetic analysis of programmed cell death in Caenorhabditis elegans has identified four genes whose activities are essential for proper activation and execution of programmed cell death (4). Three of them promote cell death (ced-3, ced-4, and egl-1), and the fourth, ced-9, protects against cell death (5-7). Importantly, these genes encode proteins that share significant sequence and functional homology with mammalian cell death regulators. EGL-1 is similar to BH3-only pro-apoptotic proteins (7, 8), CED-3 is a member of the aspartate-specific cysteine protease family (caspases) (9, 10), CED-4 is similar to one of the apoptotic protease-activating factors (Apaf-1) (11, 12), and CED-9 is a member of a family of anti-apoptotic proteins first defined by the mammalian Bcl-2 protein (8,13,14). Some of the C. elegans proteins and their mammalian homologues have been shown to be functionally interchangeable (4), indicating that the cell death pathway is evolutionarily conserved.Bcl-2 was first identified as an inhibitor of apoptosis by virtue of its ability to protect against lymphocyte cell death (14-16). Subsequently, a family of Bcl-2-related proteins, ...