NMR structure and mutagenesis of the FADD (Mort1) death-effector domain

Eberstadt, Matthias; Huang, Baohua; Chen, Zehan; Meadows, Robert P.; Ng, Shi-Chung; Zheng, Lixin; Lenardo, Michael J.; Fesik, Stephen W.

doi:10.1038/31972

Cited by 225 publications

(243 citation statements)

References 26 publications

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“…We therefore mutated Phe-28 to glycine, since mutation of the corresponding residues in FADD (F25G) and MC159 (F30A) impairs their signaling function. 16,22 The F28G substitution, however, had no effect on caspase processing, DISC formation, subcellular localization or the antiapoptotic function of c-FLIP R (Figure 7). This suggests that the architecture of c-FLIP R was not disturbed and that Phe-28 The charge triads of c-FLIP R are dispensable for DISC recruitment.…”

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confidence: 96%

“…13,14 Similar to FADD, each DED of MC159 contains two prominent surface features important for protein interactions that distinguish them from other death motifs, namely a hydrophobic patch and a charge triad, also known as RxDL motif. [13][14][15][16] Although the structure of MC159 provides a template for understanding the mechanism of FLIP inhibition, it is unknown which structural motifs mediate DISC recruitment of cellular FLIP proteins. In the present study, we characterize for the first time the short isoform of murine c-FLIP and show that c-FLIP R but not c-FLIP short is the sole truncated isoform expressed in murine cells.…”

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confidence: 99%

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Mutational analyses of c-FLIPR, the only murine short FLIP isoform, reveal requirements for DISC recruitment

et al. 2008

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Cellular FLICE-inhibitory protein (c-FLIP) proteins are known as potent inhibitors of death receptor-mediated apoptosis by interfering with caspase-8 activation at the death-inducing signaling complex (DISC). Among the three human isoforms, c-FLIP long , c-FLIP short and c-FLIP R , the latter isoform is poorly characterized. We report here the characterization of murine c-FLIP R and show that it is the only short c-FLIP isoform expressed in mice. By generating several mutants, we demonstrate that both death effector domains (DEDs) are required for DISC binding and the antiapoptotic function of c-FLIP R . Surprisingly, the C-terminal tail is important for both protein stability and DISC recruitment. Three-dimensional modeling of c-FLIP R revealed a substantial similarity of the overall structures and potential interaction motifs with the viral FLIP MC159. We found, however, that c-FLIP R uses different structural motifs for its DISC recruitment. Whereas MC159 interferes with interaction and selfoligomerization of the DISC component FADD by its extensive hydrophilic surface, a narrow hydrophobic patch of c-FLIP R on the surface of DED2 is crucial for DISC association. Thus, despite the presence of similar tandem DEDs, viral and cellular FLIPs inhibit apoptosis by remarkably divergent mechanisms. Death receptors like CD95 (Fas/Apo-1) transduce death signals upon ligand binding and formation of a death-inducing signaling complex (DISC), which comprises the receptor, the adaptor protein Fas-associated death domain (FADD) and procaspase-8 (FLICE) or procaspase-10. 1 This assembly brings procaspase-8/10 in close proximity to the receptor and allows them to be activated by dimerization. 2-4 Activation of the initiator caspases can be counteracted by FLICEinhibitory proteins (FLIPs). 5,6 Next to viral FLIP (v-FLIP) proteins, 7 three cellular homologs (cellular FLICE-inhibitory proteins (c-FLIPs)) have been identified, namely c-FLIP long , c-FLIP short and c-FLIP R , which are generated by differential splicing. [8][9][10] The C-terminus of c-FLIP long contains a catalytically inactive caspase-like domain, whereas c-FLIP short and c-FLIP R have only a truncated C-terminus.As a characteristic feature, FLIP proteins contain tandem death effector domains (DEDs), which mediate their recruitment into the DISC. 10,11 The DED forms a bundle of six antiparallel a-helices similar to the death domain (DD) and the caspase recruitment domain (CARD), two other signaling motifs involved in homotypic protein interactions. 12 Curiously, despite their importance in apoptosis, there are currently no reported structures for cellular FLIP proteins. Only the structure of the tandem DEDs of the v-FLIP MC159 from Molluscum contagiosum has been reported showing a rigidly associated dumbbell-shaped molecule, which is tightly packed by an extensive hydrophobic interface between its two DEDs. 13,14 Similar to FADD, each DED of MC159 contains two prominent surface features important for protein interactions that distinguish them from other death mot...

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confidence: 96%

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confidence: 99%

Mutational analyses of c-FLIPR, the only murine short FLIP isoform, reveal requirements for DISC recruitment

et al. 2008

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“…[9][10][11][12] Both consist of six antiparallel alpha helices tethered together by a small linker peptide. DD interactions are mediated through charge-charge interactions whereas DED interactions appear to occur mostly through hydrophobic residues.…”

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confidence: 99%

Extensive regions of the FADD death domain are required for binding to the TRAIL receptor DR5

et al. 2005

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“…The aim of our present study is to express and purify the C-terminal region-containing FADD for further research. Due to the difficulty in the expression and purification of the whole FADD molecule and its instability, no full-length FADD could be obtained to qualify for structural determination (23). In the present paper, the DED domain, which was supposed to mediate FADD self-association, was deleted from mFADD (30,31).…”

Section: Discussionmentioning

confidence: 96%

“…The individual threedimensional structure of hFADD-DED (residues 1-83), mFADD-DD (residues 89-183), hFADD-DD (residues 93-192) and hFADD (residues 1-191) in solution were well resolved by NMR (21)(22)(23)(24), none of which contained the third functional domain involved in non-apoptotic processes.…”

Section: Introductionmentioning

confidence: 99%

Expression, Purification and Characterization of C-FADD

Chen

Huang

et al. 2009

Cell Mol Immunol

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FADD is an important proapoptotic adaptor in death receptor-induced apoptosis. Recently, FADD has been found to participate in a variety of non-apoptotic processes, such as development, cell cycle progression and survival. Its non-apoptotic activities were regulated by the phosphorylated status of the serine residue located at the C-terminal region, a domain distinct from the proapoptotic function related DED and DD domains. However, due to the difficulties in expression and crystallization of natural FADD, by far the molecular structures of all FADD variants did not contain the C-terminal region. To elucidate the structure-function relationship of C-terminal region, we need to obtain an FADD variant that containing C-terminal region. In this study, mouse FADD (80-205) containing DD domain and C-terminal region, designated as C-FADD, was expressed in E. coli with His-tag at the N-terminus and purified by Ni 2+ affinity chromatography. The purified protein existed as a homogenous monomer in glutaraldehyde cross-linking analysis and exhibited a typical α-helix spectrum in CD (circular dichroism) assay. In vitro His-tag pull-down assay demonstrated that the purified C-FADD possessed the CK I-binding activity which was important for its non-apoptotic function. Cellular & Molecular Immunology. 2009;6(4):295-301.

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NMR structure and mutagenesis of the FADD (Mort1) death-effector domain

Cited by 225 publications

References 26 publications

Mutational analyses of c-FLIPR, the only murine short FLIP isoform, reveal requirements for DISC recruitment

Mutational analyses of c-FLIPR, the only murine short FLIP isoform, reveal requirements for DISC recruitment

Extensive regions of the FADD death domain are required for binding to the TRAIL receptor DR5

Expression, Purification and Characterization of C-FADD

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