DED Interaction of FADD and Caspase-8 in the Induction of Apoptotic Cell Death

Fas-associated death domain (FADD) is an adapter molecule that bridges the interaction between receptor-interacting protein 1 (RIP1) and aspartate-specific cysteine protease-8 (caspase-8). As the primary mediator of apoptotic cell death, caspase-8 has two N-terminal death-effector domains (DEDs) and it interacts with other proteins in the DED subfamily through several conserved residues. In the tumor necrosis receptor-1 (TNFR-1)-dependent signaling pathway, apoptosis is triggered by the caspase-8/FADD complex by stimulating receptor internalization. However, the molecular mechanism of complex formation by the DED proteins remains poorly understood. Here, we found that direct DED-DED interaction between FADD and caspase-8 and the structure-based mutations (Y8D/I128A, E12A/I128A, E12R/I128A, K39A/I128A, K39D/I128A, F122A/I128A, and L123A/I128A) of caspase-8 disrupted formation of the stable DED complex with FADD. Moreover, the monomeric crystal structure of the caspase-8 DEDs (F122A/I128A) was solved at 1.7 Å. This study will provide new insight into the interaction mechanism and structural characteristics between FADD and caspase-8 DED subfamily proteins.


Bio-Transmission Electron Microscope
A small drop of each protein in solution was placed on a formvar carbon-coated copper grid and negatively stained with 2% uranyl acetate for 1 min. The BIO-TEM image was recorded in a Tecnai G2 Spirit electron microscope (USA) equipped with a CCD camera at a magnification of 67,000X.

GST Pull-Down Assay
For the GST pull-down assay, 50 μg of purified His-tagged proteins and their mutants were mixed with 50 μg of purified GST or GST-tagged proteins. Then, this was followed by incubation for 12 h at 4°C with gentle rotation. Pre-washed glutathione sepharose 4B bead was added with buffer A or PBS for 2 h at 4°C. The bead was centrifuged at 600 ×g for 3 min and washed with buffer A or PBS. The protein bound to the bead was eluted [50 mM Tris-HCl (pH 7.5), 30 mM glutathione] and then resolved on a 15% SDS polyacrylamide gel. The protein was subsequently analyzed by western blot using anti-His or anti-GST.

BIAcore Biosensor Analysis
Measurement of the apparent dissociation constant (K D ) between FADD and caspase-8 DEDs was carried out using a Biacore T100 Biosensor (GE Healthcare Biosciences, Sweden). The purified FADD DED was covalently bound to the Series S sensor chip CM5 (carboxylated dextran matrix) using an amine-coupling method. The FADD DED (50 μg/ml) in 10 mM sodium acetate (pH 5.0) was coupled via injection for 15 min at 10 μl/min, followed by the injection of 1 M ethanolamine to deactivate residual amine. For kinetic measurement at 25°C, caspase-8 DED samples with concentrations ranging from 250 to 5,000 nM were prepared by dilution in HBS-EP+ buffer (10 mM of HEPES (pH 7.4), 150 mM of NaCl, 3 mM of EDTA and 0.05% v/v surfactant P20). The immobilized ligand was regenerated by injecting 50 mM NaOH at a rate 10 μl/min during the cycles.

Crystallization
A crystal of the caspase-8 DEDs (residues 1-188, F122A and I128A) was obtained by the hanging-drop vapor diffusion method. Each drop of the caspase-8 DEDs in buffer A was mixed with a reservoir solution consisting of 20% w/v polyethylene glycol 3,350 and 0.2 M ammonium acetate (pH 7.1). The crystal appeared after about 2-3 days at 293 K and continued to grow up to a maximum size within 2 weeks.

Data Collection and Structure Determination
Data for the caspase-8 DEDs were collected by the Pohang Light Source (PLS), Republic of Korea. Before data collection, the crystal was transferred to a cryoprotectant solution containing precipitant solution with 20% glycerol. After a brief soaking in the cryoprotectant solution, the crystal was flash-cooled to 100 K under liquid nitrogen stream. The diffraction data were processed using the HKL-2000 package [19]. The tertiary structure of the caspase-8 DEDs was determined by the molecular replacement method using the program AMoRe [20]. The model was improved by iterative model building using the program O [21]. The search was carried out using data obtained between 50.0 and 1.7 Å resolution (Table 1).
To obtain a soluble FADD DED and caspase-8 DED complex, recombinant FADD DED and caspase-8 DEDs were isolated by co-expression. The soluble FADD and caspase-8 proteins were purified to homogeneity and their binding was detected using SDS-PAGE. We found a co-expression complex peak by FPLC, which indicated a molecular weight of over 200 kDa. (Fig. 1D). The FADD DED and caspase-8 DED complex band from the gelfiltration showed the formation of a high-order oligomeric DED complex.
These results revealed that wild-type FADD DED interacts with caspase-8 DEDs in vitro. In addition, the four  (Fig. 2C).
To define the molecular basis of DED interactions in the complex, we performed electron microscopy (EM) on the wild-type FADD DED and caspase-8 DED co-expression complex and its morphology is shown (Fig. 2D). A microscopic image of negatively stained hetero-oligomeric protein complex was recorded, and protein oligomer was shown to be homogeneous in overall shape.
The binding affinity of caspase-8 DEDs for FADD DED was estimated using surface plasmon resonance (SPR) spectroscopy (Fig. 3). We found that wild-type caspase-8 DEDs physically bind strongly to wild-type FADD DED with an apparent K D of 38 nM. The interaction is stronger than those between FADD DED and mutated caspase-8 DEDs (I128A or R5E/I128A) (K D of 61 or 236 nM). Seven mutations including Y8, E12, K39, F122, and L123 residues disrupted FADD DED homotypic interaction with large K D and negative RU values.
We attempted to make a crystal of the FADD DED and caspase-8 DED complex but were not successful. However, the crystal of mutant caspase-8 DEDs (1-188, F122A/I128A) was obtained using the hanging-drop vapor diffusion method. It was grown using a reservoir of 20% w/v polyethylene glycol 3,350 and ammonium acetate (pH 7.1) and belongs to the C2 space group. The diffraction data were collected at a high resolution of 1.7 Å. The statistics for the X-ray diffraction data collection are summarized ( Table 1). The monomeric His-tagged caspase-8 DEDs (F122A/I128A) consist of only α-helices and loop regions and they have a dumbbell-shaped structure with rigidly associated DED1 and DED2 through hydrophobic interaction (Fig. 4). The interface amino acids (R5, Y8, E12, and K39) between FADD DED and caspase-8 DEDs are located on the binding pocket of the caspase-8 DEDs. The side chains for R5, Y8, E12, and K39 are exposed to DED1 of the caspase-8 surface. Other DED2 binding sites (F122A, L123 and I128A) of caspase-8 DEDs are located on the FL motif.
To investigate whether FADD interacts with caspase-8, truncated FADD DED and caspase-8 DEDs were characterized and purified as fusion E. coli. In the present study, co-expression, SEC, and Biacore biosensor analysis of the interaction between FADD DED and caspase-8 DEDs were performed. They support direct homotypic interactions between their DEDs. Moreover, these results indicate that several residues on the interface play a pivotal role in the assembly and regulation of homo-and hetero-complex formation of the death complex, which is crucial for understanding programmed apoptotic cell death mediated by FADD and caspase-8.
This study showed that mutations of some key binding residues (E12 and K39) in caspase-8 DED1 disrupted the interaction with FADD DED (Figs. 2 and 3). The interaction sites (Y8, E12, K39, F122, and L123) in caspase-8 are required for formation of DED homotypic interactions. Alteration of some DED residues in FADD, caspase-8, and Fas blocks receptor-mediated apoptotic cell death through several mechanisms [23]. Many extrinsic signaling pathways stimulate programmed cell death in tumor cells. These events are independent of tumor-suppressor protein p53 [24]. Thus, FADD and caspase-8 might be useful targets in cancer research studies. The present study provides crucial data on the structures of FADD and caspase-8 as well as the binding affinity of caspase-8 toward FADD, and finally, the homotypic interactions of DEDs. Our findings provide additional insight into the mechanisms underlying the interactions between DED superfamily proteins.