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 and autoimmune diseases. The extrinsic cell death pathway, which involves the formation of the death-inducing signaling complex (DISC) and culminates in the activation of caspase-8 or-10, has been extensively documented at the cellular level. However, there is a serious lack of molecular andstructural characterization; consequently, the underlying mechanisms remain poorly understood. In this grant application, systematic characterization of the extrinsic cell death pathway is proposed, including X-ray crystallographic and biochemical analyses of FLIP, FADD, Fas, Caspase-8, and their cognate complexes. Significant progress has been achieved; the work proposed here has the following specific aims: (1) Determination of the high-resolution structure of v-FLIP and c-FLIPS. The structural information on v-FLIP and c-FLIPS will serve as a basis for understanding their biological function. The structure will reveal whether and how the two tandem DED domains interact with each other. (2) Determination of the structure of v-FLIP or c-FLIPS in complex with caspase-8. In addition to revealing the molecular basis of homotypic interactions between DED domains, this structure may provide important clues on how FLIP antagonizes the activation of caspase-8. (3) Determination of the structure of the Fas-FADD complex. This structure will reveal the structural basis for the recognition of Fas by the adaptor protein FADD and provide an explanation to mounting biochemical evidence. (4) Determination of the structure of the Fas-FADD-caspase-8 complex. This structure will reveal the architecture of assembly for the three essential components of DISC and provide important clues on how caspase-8 is activated within DISC. (5) Biochemical characterization of mechanisms of apoptotic activation and inhibition. This specific aim, together with the closely related structural studies, will reveal comprehensive mechanisms that govern the activation of caspase-8 by DISC and the suppression of this process by FLIPs.