The Fas-FADD death domain complex structure reveals the basis of DISC assembly and disease mutations

Abstract

The death-inducing signaling complex (DISC) formed by the death receptor Fas, the adaptor protein FADD and caspase-8 mediates the extrinsic apoptotic program. Mutations in Fas that disrupt the DISC cause autoimmune lymphoproliferative syndrome (ALPS). Here we show that the Fas-FADD death domain (DD) complex forms an asymmetric oligomeric structure composed of 5-7 Fas DD and 5 FADD DD, whose interfaces harbor ALPS-associated mutations. Structure-based mutations disrupt the Fas-FADD interaction in vitro and in living cells; the severity of a mutation correlates with the number of occurrences of a particular interaction in the structure. The highly oligomeric structure explains the requirement for hexameric or membrane-bound FasL in Fas signaling. It also predicts strong dominant negative effects from Fas mutations, which are confirmed by signaling assays. The structure optimally positions the FADD death effector domain (DED) to interact with the caspase-8 DED for caspase recruitment and higher-order aggregation.

Fig. 1

Biochemical and structural characterization of the Fas DD–FADD DD complexes. (a) Tandem mass spectrum of the hFas–hFADD complex showing the dissociated monomers and the ‘stripped’ complexes at high gas phase collisional activation. The peak centered at 5913 m/z was isolated and collision energy (50–100 V) was applied. Well-resolved peaks at high m/z correspond to the stripped complexes for the 5:5, 6:5 and 7:5 complexes. Inset, mass spectrum of the hFas–hFADD complex showing monomers and dimers of hFas and hFADD at lower m/z and the unresolved charge series at higher m/z. (b) A representative multi-angle light scattering (MALS) measurement of the hFas–hFADD complex. More MALS data are summarized in Supplementary Table 1. (c) Crystal structure of the core 5:5 mFas–hFADD complex. The Fas molecules are shown in warm colors and the FADD DD molecules are shown in cold colors. (d) Class averages of the mFas–hFadd complex (1^st and 4^th lines) with best matching projections from the current mFas–hFADD crystal structure (2^nd and 5^th lines) and from a previously published structure of the Fas–FADD complex (3^rd and 6^th lines) .

Fig. 2

Interactions in the Fas DD–FADD DD complex. (a) Schematic planar diagram showing the construction of the complex. The locations of the three types of contacts are shown. (b) Sequence alignment of human and mouse Fas DD and FADD DD. Major interfacial residues are colored in yellow for type Ia, cyan for type Ib, magenta for type IIa, green for type IIb, red for type IIIa and blue for type IIIb. Single letter codes below the sequences indicate mutations tested with red indicating defective and black indicating non-defective. Locations of ALPS mutations are also shown. (c) Arrangement of molecules around Fas2, viewing into the schematic diagram in (a) from behind the page. (d) Zoom-in of (c) showing residues at the three types of interactions in Fas2 (labeled without a text box) and the surrounding molecules (labeled with a text box). Residues in Fas2 are colored in yellow for type Ia, cyan for type Ib, magenta for type IIa, green for type IIb (residue name not labeled), red for type IIIa and blue for type IIIb. Side chains of residues in the surrounding molecules are shown as stick models colored by atom types. (e) Arrangement of molecules around FADD3, viewing into the schematic diagram in (a) from behind the page. (f) Zoom-in of (e) showing residues at the three types of interactions in FADD3 (labeled without a text box) and the surrounding molecules (labeled with a text box). Residues in FADD3 are colored in yellow for type Ia, cyan for type Ib, magenta for type IIa (residue name not labeled), green for type IIb, red for type IIIa and blue for type IIIb. Side chains of residues in the surrounding molecules are shown as stick models colored by atom types. (g) Side by side surface representations of Fas DD showing the locations of the three types of interactions (left) and the surface charge features (right). (h) Side by side surface representations of FADD DD showing the locations of the three types of interactions (left) and the surface charge features (right).

Fig. 3

Structure-based mutagenesis and analysis of ALPS mutations. (a) Analysis of structure-based mutations of Fas DD and FADD DD in the in vitro His-tag pulldown assay. (−) indicates a defective interaction. PD: pulldown; I: input. (b) Mapping of ALPS-associated mutation residues onto the surface of Fas DD. (c) Pulldown of ALPS-associated Fas DD mutants by His-tagged FADD DD. (−) indicates a defective interaction. PD: pulldown.

Fig. 4

Interactions in living cells and functional effects of Fas DD and FADD DD mutations. (a) 293T cells were transfected with CFP-tagged FADD DD and YFP-tagged wild-type or mutant full-length Fas and analyzed for FRET by flow cytometry. Bi-exponential plots of the FRET signals are shown for cells transfected with the indicated constructs. A plasmid encoding a truncated Fas protein without a death domain (Fas ΔDD) was used as a negative control. Change in geometric mean fluorescence intensity (ΔMFI) was calculated by subtracting the MFI of non-interacting proteins (Fas ΔDD with WT FADD DD) from the MFI in the FRET channel of the indicated interacting pair. At right the mean fluorescence intensity relative to FRET signal from the Fas ΔDD–FADD interaction (ΔMFI) is shown for the indicated Fas DD mutants interacting with wild-type FADD. The occurrences of the interactions in the complex are shown. (b) FRET analysis of 293T cells transfected with either wild-type or mutant FADD DD interacting with full-length Fas or Fas ΔDD. At right the ΔMFI is shown for the indicated FADD DD mutants interacting with wild-type Fas. The occurrences of the interactions in the complex are shown. Results shown in (a) and (b) are representative of three independent experiments. (c) Effect of Fas DD mutants on Fas-induced cell death in transfected Jurkat E6.1 cells. Cells were transfected with the indicated constructs and apoptosis was induced with anti-Fas or FasL-LZ. Cell death was assayed by flow cytometry. Results are the average +/− s.e.m. of specific cell death in three independent experiments. (d) The Fas DD–full-length FADD complex model constructed by superimposing FADD DD with the structure of full-length FADD . Fas DDs are shown in ribbon diagrams while FADD molecules are shown in surface representations. The location of the FADD DED for caspase-8 recruitment is shown for one of the FADD molecules. (e) A proposed model for post-receptor DISC formation that utilizes FasL hexamerization. All structures are shown in ribbon diagrams except for the intracellular death domains of Fas, which are shown in surface representations. The two FasL trimers are shown in gray. The six Fas extracellular domains are shown in the same color as their intracellular death domains. A 6^th Fas intracellular death domain (yellow) was added to the crystal structure. The extracellular and the intracellular domains are connected via straight lines of the same colors. Full-length FADD molecules are shown in ribbon diagrams. The light green bar represents the cellular membrane.