Structural study of the RIPoptosome core reveals a helical assembly for kinase recruitment.

Cell death has been actively studied in the medical research field for several decades due to its importance and involvement in many human diseases such as cancer and neurodegenerative diseases.1,2 As a result, it is now known that there are two major types of programmed cell death, apoptosis and necroptosis.3−5 Apoptosis is a major molecular program to eliminate potentially dangerous and unnecessary cells by intrinsic (mitochondrial) or extrinsic (death ligand-mediated) pathways. Necroptosis (also called programmed necrosis) contributes to the regulation of the immune system, cancer development, and stress-mediated cellular responses.6−8 Necrotic cell death was initially viewed as an accidental and unregulated event. However, many studies have shown that necrotic cell death can be programmed and regulated via unique signaling pathways.4,6,8,9 Apoptosis and necroptosis may be intertwined. For example, signaling events initiated by the binding of secreted and cell surface death ligands such as TNFα, FasL, and TRAIL to death receptors may lead to either apoptosis or necroptosis under different cellular contexts. Receptor interaction protein kinase 1 (RIP1) and 3 (RIP3), Fas-associated protein with death domain (FADD), and caspase-8 are the main downstream signaling components.6,10 RIP1, FADD, and caspase-8 assemble into a complex termed the RIPoptosome, which is responsible for apoptosis induction.11,12 However, when caspase activity is inhibited by pharmacological agents or during viral infections, RIP1 associates with RIP3 to form a complex termed the necrosome, which activates necroptosis.8,13 RIP1 contains a C-terminal death domain (DD) that allows the recruitment of FADD through homotypic DD–DD interactions (Figure ​(Figure1A).1A). FADD is an adaptor protein that contains an N-terminal death effector domain (DED) and a C-terminal DD. The N-terminal DED of FADD interacts with the caspase-8 DED. DED and DD both belong to the death domain superfamily, which also includes the caspase recruitment domain (CARD) and pyrin domain (PYD).14−16 While previous structural studies of the necrosome formed by RIP1 and RIP3 revealed a functional amyloid-like organization,17 the structural basis for the assembly of RIP1 and FADD within the RIPoptosome remains completely unknown. Figure 1 In vitro reconstitution and EM analysis of the RIP1 DD/FADD DD complex. (A) Domain organization of RIP3 and the RIPoptosome components, RIP1, FADD, and caspase-8. Interacting domains are indicated by blue circles. DD, death domain; DED, death effector ... In this study, we reconstituted the RIP1 DD/FADD DD complex, the core of the RIPoptosome, and elucidated the overall structure of the complex by negative-stain electron microscopy (EM) and modeling, providing new insight into the mechanism underlying RIP1-mediated apoptosis and necroptosis. Our study shows that RIP1 DD and FADD DD form a stable complex in vitro with a structure similar to that of the Fas DD/FADD DD complex. The structure of the RIP1 DD/FADD DD complex is another example of the conserved interactions between domains in the DD superfamily. The results presented here suggest that a helical assembly using three distinct types of interactions is the general mechanism that underlies the assembly of domains of the DD superfamily.