PMID:11027303

Citation	Parrish, J, Metters, H, Chen, L and Xue, D (2000) Demonstration of the in vivo interaction of key cell death regulators by structure-based design of second-site suppressors. Proc. Natl. Acad. Sci. U.S.A. 97:11916-21
Abstract	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-4/CED-9 complexes. We show here that an unusual gain-of-function 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-4/CED-9 complexes. Based on a modeled EGL-1/CED-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-4/CED-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.
Links	PubMed PMC17269 Online version:10.1073/pnas.210391597
Keywords	Amino Acid Sequence; Amino Acid Substitution; Animals; Apoptosis/drug effects; Apoptosis/physiology; Apoptosis Regulatory Proteins; Caenorhabditis elegans/cytology; Caenorhabditis elegans/metabolism; Caenorhabditis elegans Proteins; Calcium-Binding Proteins/metabolism; Calcium-Binding Proteins/physiology; Helminth Proteins/chemistry; Helminth Proteins/metabolism; Helminth Proteins/physiology; Molecular Sequence Data; Protein Binding; Proto-Oncogene Proteins/chemistry; Proto-Oncogene Proteins/metabolism; Proto-Oncogene Proteins/physiology; Proto-Oncogene Proteins c-bcl-2; Repressor Proteins/metabolism; Sequence Homology, Amino Acid
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