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, 457 (7232), 1019-22

The Fas-FADD Death Domain Complex Structure Unravels Signalling by Receptor Clustering

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The Fas-FADD Death Domain Complex Structure Unravels Signalling by Receptor Clustering

Fiona L Scott et al. Nature.

Abstract

The death inducing signalling complex (DISC) formed by Fas receptor, FADD (Fas-associated death domain protein) and caspase 8 is a pivotal trigger of apoptosis. The Fas-FADD DISC represents a receptor platform, which once assembled initiates the induction of programmed cell death. A highly oligomeric network of homotypic protein interactions comprised of the death domains of Fas and FADD is at the centre of DISC formation. Thus, characterizing the mechanistic basis for the Fas-FADD interaction is crucial for understanding DISC signalling but has remained unclear largely because of a lack of structural data. We have successfully formed and isolated the human Fas-FADD death domain complex and report the 2.7 A crystal structure. The complex shows a tetrameric arrangement of four FADD death domains bound to four Fas death domains. We show that an opening of the Fas death domain exposes the FADD binding site and simultaneously generates a Fas-Fas bridge. The result is a regulatory Fas-FADD complex bridge governed by weak protein-protein interactions revealing a model where the complex itself functions as a mechanistic switch. This switch prevents accidental DISC assembly, yet allows for highly processive DISC formation and clustering upon a sufficient stimulus. In addition to depicting a previously unknown mode of death domain interactions, these results further uncover a mechanism for receptor signalling solely by oligomerization and clustering events.

Figures

Figure 1
Figure 1. Overall structure of the Fas/FADD DD complex
(left) The structure shows a tetrameric arrangement of Fas/FADD DD complexes. Contacts between the complexes are solely mediated by Fas molecules. (right) Cartoon representation of the Fas/FADD complex structure. Color coding: one Fas/FADD DD complex is displayed in green (Fas) and blue (FADD) ribbons, while the remaining three complexes are colored red, magenta and blue (surface representation).
Figure 2
Figure 2. Fas/FADD DD complex: Fas/FADD and Fas/Fas interactions are dependent on Fas opening
a, Primary Fas/FADD DD complex. FADD (blue) adopts the characteristic death domain fold with helices one and six participating in the main interaction site. In Fas (green) only helices one to four approximately adopt a death domain-like fold, while a long helix (stem helix) is found in place of helices five and six, which together with helix one provides the main interaction residues for FADD binding. Additionally a helix at the C-terminus of Fas (C-helix) is observed. b, Conformational change in Fas. Comparison of the structure of unbound Fas DD (closed form in orange; pdb entry: 1DDF) and Fas in the Fas/FADD complex. Due to formation of the stem helix residues of helix five and six shift significantly. Additionally, the rearrangement of helix six exposes part of the hydrophobic core of Fas. c, Cartoon illustration of Fas opening. d, Primary Fas/FADD interface. View onto interfaces governing primary complex formation. Surface representation shows complementary hydrophobic patches (yellow) on FADD and Fas surrounded by polar residues (magenta). The hydrophobic interface on Fas becomes exposed upon Fas opening. e, Fas-Dimer unit. Another consequence of Fas opening is the formation of Fas-dimer units, which interact via the stem and C-helix. (right) Cartoon representation of the dimer. f, The Fas/FADD DD complex is weak. Dilution experiment of the isolated Fas/FADD complex shows cooperative dissociation of the complex below concentrations as high as 50 μM (plot derived from quantitative SDS-PAGE analysis of Fas-DD retained on Ni-NTA resin from various Fas-DD/FADD-DD-His6 complex dilutions).
Figure 3
Figure 3. The Fas/FADD bridge in the DISC: binding of full length FADD and the key role of Fas opening in vivo
a, Overlay of the structure of full length FADD* (pdb entry: 2gf5; orange) onto the Fas/FADD complex structure. The last helix of the DD of unbound FADD (red dot) shifts to avoid clashing with the newly formed Fas C-helix in the Fas/FADD complex. b, Conformational change in full length FADD. Proteins were expressed separately, and His-tagged versions of the DD of FADD, or full length FADD, were mixed with untagged Fas. Ni-NTA chromatography demonstrates that full length FADD shows reduced initial binding to the Fas DD, when compared to the FADD DD protein. c, While initial binding of full length FADD to Fas DD is reduced, prolonged incubation leads to the formation of DISC-like structures. Incubation of both proteins overnight led to the formation of ring-like structures with a strong tendency to form clusters as determined using electron microscopy. Displayed are single ring-like structures and clusters from several images. Due to their strong tendency to self-adhere no consistent monolayer for thorough evaluation could be generated to date. d/e, Propagating Fas opening results in hyperactive Fas. (d) Location of Ile313 in closed (unbound, orange) and open (complex, green) form of Fas. (e) Huh7 cells transfected with Fas I313D show elevated cell death, assessed by Annexin V reactivity, compared to Fas wt upon stimulation with Fas Antibody (left, standard deviations, n=3), FasL (middle), and also in the absence of a stimulus (right). Equal cell surface Fas expression was confirmed by FACS and immunoblot (data not shown). *Full length FADD refers to the well characterized FADD F25Y mutant (see Supplementary Methods)
Figure 4
Figure 4. Model of DISC formation: mechanism of receptor signaling through clustering
Schematic illustrating DISC formation. a, Model for Fas opening. Like in any two state model, it can be assumed that the closed and open form of Fas exist in equilibrium. In the absence of an apoptotic signal the equilibrium between closed and open forms of Fas strongly favors the closed form while open forms are unstable. Upon an apoptotic stimulus the equilibrium shifts to favor the open form of Fas. Fas molecules are brought together by Fas ligand in a permissive environment dependent on several factors including lipid rafts and membrane constitution. The close proximity of Fas DDs now allows for stem helices to interact leading to stabilization of the open form. b, Opening and formation of the Fas/Fas bridge links trimeric DD units defined in earlier studies (Supplementary Table 2), which are formed by globular regions of the open Fas. FADD can now bind to the open Fas molecules further stabilizing the bridge. The consequence is processive interlinked DISC formation and clustering in which open Fas molecules interacting via their globular domains are linked by a multitude of weak Fas/FADD bridges leading to overall stable DISC clusters. This permits activation of caspase-8, presumable by proximity enforced dimerization, and induction of Apoptosis.

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References

    1. Ashkenazi A, Dixit VM. Apoptosis control by death and decoy receptors. Curr Opin Cell Biol. 1999;11:255–60. - PubMed
    1. Peter ME, Krammer PH. The CD95(APO-1/Fas) DISC and beyond. Cell Death Differ. 2003;10:26–35. - PubMed
    1. Taylor RC, Cullen SP, Martin SJ. Apoptosis: controlled demolition at the cellular level. Nat Rev Mol Cell Biol. 2008;9:231–41. - PubMed
    1. Fesik SW. Insights into programmed cell death through structural biology. Cell. 2000;103:273–82. - PubMed
    1. Park HH, et al. The death domain superfamily in intracellular signaling of apoptosis and inflammation. Annu Rev Immunol. 2007;25:561–86. - PMC - PubMed

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