STRUCTURAL BIOLOGY OF CED-9-MEDIATED SUPPRESSION OF CED-4

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Apoptosis plays a central role in the development and homeostasis of all multi-cellular organisms. Alterations in apoptotic pathways have been implicated in many debilitating human diseases including cancer. Genetic studies pioneered by Robert Horvitz have led to the identification of four genes that control the onset of apoptosis in the model organism Caenorhabditis elegans. The protein products of these four genes, EGL-1, CED-9, CED-4, and CED-3 act in a linear pathway to execute cell death and constitute a classic paradigm for the understanding of apoptosis. Despite genetic studies, the molecular mechanisms by which Egl1, CED9, CED4, and CED3 control the initiation of apoptosis remain largely unknown. Towards these goals, we have initiated systematic biochemical and X-ray crystallographic analyses of protein complexes involved in this paradigm. CED4 remains constitutively associated with CED9 as an inactive complex prior to apoptosis. We have characterized a binary complex between the full-length CED4 and a large functional domain of CED9. We recently obtained crystals of this complex in two different spacegoups. One crystal form diffracts X-ray weakly. The best crystals of the other crystal form diffract X-ray to about 3.5 ¿ at CHESS A-1 station. We would like to collect a native data set at CHESS A-1 station and attempt to solve the structure by molecular replacement, as the atomic coordinates for CED-9 are available. This structure will reveal important regulatory mechanisms of CED-4 by CED-9 and has significant impact on our understanding of the apoptotic pathway in both C.elegans and mammals.