Demonstration of the in vivo interaction of key cell death regulators by structure-based design of second-site suppressors.
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.
Parrish
J
J
Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, CO 80309, USA.
Metters
H
H
Chen
L
L
Xue
D
D
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D051017:Apoptosis Regulatory Proteins; D017173:Caenorhabditis elegans / Q000166:cytology / Q000378:metabolism; D029742:Caenorhabditis elegans Proteins; D002135:Calcium-Binding Proteins / Q000378:metabolism / Q000502:physiology*; D015801:Helminth Proteins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D008969:Molecular Sequence Data; D011485:Protein Binding; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D019253:Proto-Oncogene Proteins c-bcl-2; D012097:Repressor Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D051017:Apoptosis Regulatory Proteins; D029742:Caenorhabditis elegans Proteins; D002135:Calcium-Binding Proteins; C089224:Ced-4 protein, C elegans; C087233:Ced-9 protein, C elegans; C112486:EGL-1 protein, C elegans; D015801:Helminth Proteins; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D012097:Repressor Proteins