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. Author manuscript; available in PMC: 2010 Jul 22.
Published in final edited form as: Mol Cell. 2005 Dec 22;20(6):939–949. doi: 10.1016/j.molcel.2005.10.023

Crystal Structure of MC159 Reveals Molecular Mechanism of DISC Assembly and FLIP Inhibition

Jin Kuk Yang *,#, Liwei Wang *,#, Lixin Zheng †, Fengyi Wan †, Misonara Ahmed *, Michael J Lenardo †, Hao Wu *,✉
PMCID: PMC2908330  NIHMSID: NIHMS215864  PMID: 16364918

Summary

The death inducing signaling complex (DISC) comprising Fas, FADD and caspase-8/10 is assembled via homotypic associations between death domains (DDs) of Fas and FADD and between death effector domains (DEDs) of FADD and caspase-8/10. Caspase-8/10 and FLICE/caspase-8 inhibitory proteins (FLIPs) that inhibit caspase activation at the DISC level contain tandem DEDs. Here we report the crystal structure of a viral FLIP, MC159, at 1.2Å resolution. It reveals a non-canonical fold of DED1, a dumbbell-shaped structure with rigidly associated DEDs and a new mode of interaction in the DD superfamily. While the conserved hydrophobic patch of DED1 interacts with DED2, the corresponding region of DED2 mediates caspase-8 recruitment and contributes to DISC assembly. In contrast, MC159 cooperatively assembles with Fas and FADD via an extensive surface that encompasses the conserved charge triad. This interaction apparently competes with FADD self-association and disrupts higher order oligomerization required for caspase activation in the DISC.

Introduction

Death receptors (DRs) in the Tumor Necrosis Factor (TNF) Receptor (TNFR) superfamily mediate the extrinsic pathway of apoptosis and play critical roles in embryonic development, cellular homeostasis and immune regulation (French and Tschopp, 2003; Locksley et al., 2001; Peter and Krammer, 2003; Wajant, 2002). Current members of the DR subfamily include Fas (also known as CD95 and APO-1), TNF-R1, DR3, TRAIL-R1, TRAIL-R2, DR6, EDA-R and NGF-R (French and Tschopp, 2003). Signaling by these receptors is triggered by interaction with one or more specific ligands that are members of the TNF superfamily. Genetic mutations or abnormal expression of DRs and their ligands have been associated with many human diseases including the Autoimmune Lymphoproliferative Syndrome (ALPS), cancer, and tissue destructive diseases such as graft-versus-host disease, multiple sclerosis and stroke (French and Tschopp, 2003). Highly effective therapeutics for modulating DR signaling have emerged. These include antagonistic antibodies in tissue destructive diseases and agents that selectively trigger DR-mediated cell death in cancer cells (French and Tschopp, 2003). Therefore, a detailed understanding of the signaling and regulatory mechanisms of DRs has broad medical and biological importance.

DR signaling is mediated by highly specific protein-protein associations that generate oligomeric signaling assemblies such as the DISC (Kischkel et al., 1995; Wajant, 2002). DRs contain a death domain (DD) in their intracellular regions (Itoh and Nagata, 1993; Tartaglia et al., 1993; Wajant, 2002). For Fas, its DD recruits the Fas-associated death domain (FADD) adaptor protein via a homotypic interaction with the C-terminal DD of FADD (Chinnaiyan et al., 1995; Kischkel et al., 1995). FADD also contains an N-terminal DED that interacts homotypically with the tandem DEDs in the pro-domains of caspase-8 or -10 (Boldin et al., 1996; Muzio et al., 1996). These interactions form the ternary DISC containing Fas, FADD and caspase-8 or -10. Recruitment of pro-caspases into the DISC initiates proteolytic auto-processing (Medema et al., 1997; Salvesen and Dixit, 1999; Shi, 2004). This liberates active caspase-8 or -10 into the cytoplasm to cleave and activate effector caspases such as caspase-3 and caspase-7, leading to a cascade of events in apoptotic cell death (Salvesen, 2002).

In addition to caspase-8 and -10, most FLICE/caspase-8 inhibitory proteins (FLIPs) are also tandem DED-containing proteins, which regulate DISC assembly and caspase activation (Thome and Tschopp, 2001). Cellular FLIPs (cFLIPs), comprising the long and short isoforms, cFLIP(L) and cFLIP(S), are tightly regulated in expression in T cells and might be involved in the control of both T-cell activation and death (Goltsev et al., 1997; Han et al., 1997; Hu et al., 1997b; Inohara et al., 1997; Irmler et al., 1997; Rasper et al., 1998; Shu et al., 1997; Srinivasula et al., 1997; Thome and Tschopp, 2001). Viral FLIPs (vFLIPs) appear to have evolved to inhibit apoptosis of infected host cells and are present in the poxvirus molluscum contagiosum virus (MCV) (Bertin et al., 1997; Garvey et al., 2002b; Hu et al., 1997a; Shisler and Moss, 2001; Thome et al., 1997) and γ-herpesviruses such as 

  e q u i n e
  h e r p e s v i r u s - 2
  ( E H  V - 2 ) ,
h  e r p e s v i r u s
  s a i m i r i        ( H  V S ) ,
  t h e               K a p o s i -
  a s s o c i a t e d
  h u m a n
  h e r p e s v i r u s - 8
  ( H H  V - 8 )              and
  r h e s u s
  r h a d i n o v i r u s
  ( R R  V )

(Bertin et al., 1997; Hu et al., 1997a; Searles et al., 1999; Thome et al., 1997; Thome and Tschopp, 2001).

The DDs and DEDs involved in DR signaling are members of the DD superfamily, which also includes the caspase recruitment domains (CARDs) involved in the intrinsic cell death pathway and the Pyrin domains (PYDs) involved in inflammatory signaling (Kohl and Grutter, 2004; Reed et al., 2004). The DD superfamily contains homotypic protein-protein interaction modules. NMR structures of the first DD (Huang et al., 1996), DED (Eberstadt et al., 1998), CARD (Chou et al., 1998) and PYD (Hiller et al., 2003) have all revealed a common six-helical bundle structure. Despite their biological importance, there are currently no reported structures of tandem DEDs. Both the architectural features of the individual DEDs in tandem DEDs and the mode of their association are entirely unknown. Therefore, the molecular basis for the recruitment of caspase-8, caspase-10 and FLIPs remains to be revealed.

To elucidate the molecular basis for the functions of tandem DED-containing proteins, we determined the crystal structure of MC159, a vFLIP from MCV, at 1.2Å resolution. It reveals a dumbbell-shaped structure with rigidly associated DED1 and DED2 via hydrophobic interactions. DED1 is highly divergent from DED2 and other DED structures. The DED1/DED2 interaction is topologically different from the previously observed CARD/CARD and DD/DD interactions (Qin et al., 1999; Xiao et al., 1999). A hydrogen-bonded charge triad is present on the surface of DED1 and DED2, which may represent a general characteristic feature of DEDs, but not other members of the DD superfamily. We found that the conserved hydrophobic patch of DED2 mediates caspase-8 recruitment and DISC assembly. Similar mechanisms might be used by cFLIPs and herpesvirus vFLIPs to compete with caspase-8 recruitment. In contrast to hydrophobic interactions MC159 uses an extensive charge surface that encompasses the charge triad to cooperatively assemble with Fas and FADD. This interaction does not compete with caspase-8 recruitment, but apparently competes with FADD self-association and disrupts higher order oligomerization required for caspase activation in the DISC. Together, these structural, biochemical and mutational studies reveal important insights into the molecular mechanism of DISC assembly and vFLIP inhibition.

Results

Fas, FADD, and MC159 are cooperatively assembled into a ternary complex

We used recombinant proteins and a His-tag pull down assay to establish an in vitro system for assessing the recruitment of MC159. MC159 interacted effectively with Fas DD and FADD, as shown by the pull down of an apparent ternary complex using His-tagged MC159 (Figure 1A, lane 4). However, in the absence of Fas DD, His-tagged MC159 only weakly pulled down FADD (Figure 1A. lane 3) and in the absence of MC159, Fas DD only weakly interacted with FADD (Figure 1B, lanes 3 and 7). These data suggest that the binary Fas DD/FADD and FADD/MC159 interactions are weak and that the ternary complex is cooperatively assembled.

Figure 1.

Figure 1

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Cooperative assembly of Fas, FADD and MC159. A. Schematic domain organization of Fas, FADD and MC159. B. His-tag pull down of MC159, Fas DD and FADD. C. His-tag pull down of Fas DD and FADD. D. His-tag pull down of MC159 and FADD DED. E. His-tag pull down of Fas DD and FADD DD. Contaminates are marked by *.

Because isolated FADD DED interacted strongly with MC159 (Figure 1C, lane 4) and isolated FADD DD interacted strongly with Fas DD (Figure 1D, lane 2), it appears that the DED in full-length FADD auto-inhibits its DD from interacting effectively with Fas DD and that the DD in full-length FADD auto-inhibits its DED from interacting strongly with MC159. This auto-inhibition is relieved when the third component of the complex is present.

High-resolution structure of MC159: non-canonical fold of DED1 and the charge triad

To elucidate the molecular basis of DISC assembly and FLIP mediated inhibition of caspase activation, we determined the crystal structure of MC159, a vFLIP from MCV (Figure 2, Table 1). The structure of full-length MC159 was first solved at 3.8Å resolution by single-wavelength anomalous diffraction and fourfold non-crystallographic symmetry averaging (Figure 2A). It revealed that more than fifty residues at the C-terminal tail of MC159 were disordered in the crystal. By removing this region of MC159, a different crystal form that diffracted to 1.2Å resolution was obtained (Figure 2B). The original atomic model built from the low-resolution crystal form was used to determine the structure of the high-resolution crystal form. Structural analyses are based on information from the high-resolution structure.

Figure 2.

Figure 2

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Crystal structure of MC159. A. MC159 tetramer structure from the P2221 crystal form, shown in cyan, magenta, yellow and green, respectively for each monomer. B. Stereo ribbon drawing of the high-resolution structure of MC159 from the P212121 crystal form. DED1 is shown in cyan and DED2 in magenta. Helices H0–H7 of DED1 and H1–H6 of DED2 are labeled. H3 is absent in DED1. C. Electrostatic surface representation of MC159, showing the dumbbell shaped structure and the rich charges on one face of MC159. The top panel is related to B by an approximate 180° rotation along the vertical axis followed by a 90° rotation on the page. The lower panel is related to B by an approximate 90° rotation on the page. D. Stereo cylinder drawing, showing the superposition of MC159 DED1 (cyan) with FADD DED (red) and DED2 (magenta) with FADD DED (yellow). For the superposition between DED1 and FADD, helices H0 and H7 are only present in DED1, while H3 is only present in FADD. E. Stick model for the hydrogen bonding interactions in the charge triad of DED1.

Table 1.

Crystallographic statistics.

SeMet (residues 1–241) Native (residues 1–187)
Data collection
Space group P2221 P212121
Cell dimensions
    a, b, c 84.0Å, 108.2Å, 142.7Å 35.0Å, 62.9Å, 75.8Å
Resolution 30 - 3.8Å 15 - 1.2Å
Rsym 8.4% (35.3%) 5.9% (30.7%)
I/σI 20.0 (3.5) 48.5 (4.4)
Completeness 99.7 (98.6%) 99.6% (97.9%)
Redundancy 6.5 (4.5) 6.7 (4.2)
Refinement
Resolution 15 - 1.2Å
No. reflections 51,463
Rwork / Rfree 21.2% / 23.3%
No. atoms
    Protein / Water and other small molecule 1,492 / 322
Average B-factors
    Protein / Water and other small molecule 15.8Å2 / 33.5Å2
R.m.s deviations
    Bond lengths / angles 0.0037Å / 0.99°
Ramachandran Plot
    Most favored / additionally allowed 93.9% / 6.1%
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†

Highest resolution shell is shown in parenthesis.

The tandem DEDs of MC159 form a dumbbell-shaped structure with each DED as the weight at either end (Figure 2C). Although all members of the DD superfamily appear to exhibit the fold of a six-helical bundle, DED1 is highly divergent from DED2 and the known NMR structures of single DEDs from FADD (Eberstadt et al., 1998) and PEA-15 (Hill et al., 2002) (Figure 2D, Table S1 in the Supplemental Data). In particular, the topologically equivalent helix H3 is missing, which is replaced by a short loop connecting helices H2 and H4. This loop is mostly hydrophilic and does not contribute to the hydrophobic core of DED1. Two additional helices are present, helix H0 at the N-terminus and helix H7 that covers helix H5, helps to close the hydrophobic core of the domain and brings the chain to the beginning of DED2. In addition, DED1 has a shorter helix H2 and a different orientation in helix H6. Only about half of the residues in DED1 were aligned within 3Å in Cα distance to DED2, FADD DED and PEA-15 DED (Table S1 in Supplemental Data). In fact, a DALI structural homology search (Holm and Sander, 1995) showed that DED1 structure is almost as different to these DED structures as to other structures in the DD superfamily such as those of DDs, PYDs and CARDs (Table S1 in the Supplemental Data).

On the other hand, DED2 is a bona fide DED. It showed extensive structural homology with the known DED structures from FADD and PEA-15, which were also the top two matches from a structural homology search with DALI (Figure 2D, Table S1 in the Supplemental Data). Among the 97 residues in DED2, 74 residues were aligned with FADD and PEA-15 to within 3.0Å in Cα distance, giving rise to RMSDs of 1.4Å in both cases (Table S1 in the Supplemental Data). All six canonical helices are present in DED2. In comparison with FADD DED, the helices H1, H4, H5 and H6 of DED2 are more conserved structurally while helices H2 and H3 show more variations in position. Helix H6 is exceptionally long in DED2; it comprises 21 residues and runs to the end of the construct at residue 187. Approximately half of helix H6 does not pack against the core of DED2; instead it protrudes prominently. In the low-resolution crystal form, helix H6 is involved in the organization of four MC159 molecules in an almost perfect 222 non-crystallographic symmetry (Figure 2A). However, MC159 is a monomer in solution and the tetrameric association does not appear to have any biological significance. Previous truncation studies have shown that a large part of helix H6 could be truncated (residues 1–179) without affecting its apoptosis protection function (Garvey et al., 2002a) and mutations of exposed residues on the remaining part of H6 did not affect ternary complex formation with Fas and FADD (see below).

The high-resolution structure of MC159 revealed a hydrogen-bonded charge triad on the surface of DED1 and DED2 (Figure 2E, Table S2 in Supplemental Data), which contributes to the highly charged features on one face of the structure (Figure 2C). The charge triad, which we refer to as the E/D-RxDL motif, involves the Arg and Asp residues in the RxDL motif previously noted to be functionally crucial (residues 69–72 in DED1 and residues 166–169 in DED2) (Garvey et al., 2002b), situated in helix H6 and the preceding loop, and a negatively charged residue (E24 in DED1 and E111 in DED2) in helix H2. Extensive hydrogen bonding interactions are observed among these charged side chains. In DED1, for example, Arg69 situates in between Asp71 and Glu24. All three polar atoms in the Arg69 side chain make hydrogen bonds: Nε and Nη2 with the Oδ1 and Oδ2 atoms of Asp71 and Nη1 with Oε1 and Oε2 of Glu24 with perfect hydrogen bonding distances. These hydrogen bonds likely help to maintain a precise organization of the side chains, which may be functionally important (see below). It is also possible that they play a local structural role in maintaining the conformation of this region of the DEDs.

The charge triad is highly conserved in most single and tandem DEDs (Figure 3A), but not present in other members of the DD superfamily, suggesting that it is a characteristic feature of DEDs alone. Interestingly, the charge triad is conspicuously altered or missing in three of the four DEDs in caspase-8 and caspase-10. In caspase-8 DED2, the E/D-RxDL motif has a Glu, Lys and Ser at the three charged residue positions and x is missing. It is possible that the change from Arg to Lys lowers the hydrogen bonding potential so it now only interacts with one negatively charged residue in the triad. For caspase-10, the E/D-RxDL motif is missing in both DED1 and DED2. It is possible that this motif does not play an important role in caspase recruitment but is important in other functions of DED-containing proteins (see below).

Figure 3.

Figure 3

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Mode of interaction between DED1 and DED2. A. Sequence alignment of MC159 with other tandem DED containing proteins and structure-based sequence alignment with FADD. Residues with more than 40Å2 surface area buried at the DED1/DED2 interface are shaded in red (completely buried) and magenta (partially buried) and residues with 30–40Å2 surface area buried at the DED1/DED2 interface are shaded in brown. Residues at the charge triad E/D/-RxDL motif positions are shaded in green and residues at the conserved hydrophobic patch of DED2 are shaded in blue. Residues of MC159 involved in FADD interaction are marked by *. Residues of caspase-8 involved in FADD interaction are marked by #. Sequences identical to MC159 are shaded in yellow. Secondary structures of MC159 and FADD are shown above and below the sequences, respectively. Different shades of blue bars indicate whether a residue is buried (dark blue) or exposed (light blue). CASP-8: caspase-8; CASP-10: caspase-10; cFLIP: cFLIP(S); EHV-2 E8: vFLIP from equine herpesvirus-2. B. Regions of MC159 involved in the interaction are shown in blue for DED1 (residues 22–35 and 57–66), red for DED2 (residues 94–105 and 135–145) and green for the DED1–DED2 linker (residues 86–94). C. Detailed interaction between DED1 and DED2. Potential hydrogen bonds are shown by dotted lines. D, E. Comparison of DED1/DED2 interaction with the Apaf-1 CARD/pro-caspase-9 CARD interaction. Apaf-1 and pro-caspase-9 in the Apaf-1/pro-caspase-9 complex are each superimposed with DED1, respectively, in D and E. F, G. Comparison of DED1/DED2 interaction with the Pelle DD/Tube DD interaction. Pelle and Tube in the Pelle/Tube complex are each superimposed with DED1, respectively, in F and G.

The interaction between DED1 and DED2 reveals a conserved rigid tandem DED association and a new mode of interaction in the DD superfamily

Unlike beads on a string, the structure of MC159 revealed that DED1 and DED2 are rigidly associated with each other into a compact structure (Figure 2B, 2C). The DED1/DED2 interface is extensive, burying approximately 1380Å2 surface area. The two DEDs are related, very approximately, by a translation across the contact interface so that one side of DED1 is contacting the equivalently opposite side of DED2. The translational relationship between DED1 and DED2 is made possible by helix H7 of DED1.

The interaction at the DED1/DED2 interface is mostly hydrophobic, mediated by helices H2 and H5 of DED1 and helices H1 and H4 of DED2 (Figure 3B, 3C). There are a total of 195 interfacial atomic contacts, among which 117 are between non-polar atoms, 17 are between polar atoms and 61 are mixed contacts between polar and non-polar atoms. The surface patch formed by H2 and H5 of DED1 is the equivalent conserved hydrophobic patch identified on the surface of FADD DED important for caspase-8 interaction (Eberstadt et al., 1998) (Figure 3A). The H1 and H4 surface of DED2 is equivalent to the second hydrophobic patch identified in FADD DED structure on the opposite side, which does not appear to be important in FADD signaling (Eberstadt etal.,1998). In addition to hydrophobic interactions, three interfacial hydrogen-bonding networks are observed (Figure 3C). The first one is the salt bridge between Asp34, localized between H2 and H4 of DED1, and Arg97 in H1 of DED2. The second one involves the side chains of H23 and S26 in H2 of DED1, Glu105 in H1 of DED2, and Arg135 in the H3–H4 loop of DED2. The last one is the salt bridge between Glu62 in H5 of DED1 and Lys98 in H1 of DED2. The interfacial residues, especially those that are completely buried at the DED1/DED2 interface and contribute large surface areas, such as F30, L31, F92, L93 and R97, are mostly conserved in tandem DEDs (Figure 3A). This suggests that all known tandem DEDs form a similar rigid compact structure as MC159.

The observed interaction between DED1 and DED2 represents the first example of a DED/DED interaction. However, because DED1 is highly divergent from a typical DED fold, the interaction may not be representative of other DED/DED interactions. The interaction also differs from the two known modes of interaction within the DD superfamily, the interaction seen in the Pelle DD/Tube DD complex (Xiao et al., 1999) and that in the Apaf-1 CARD/pro-caspase-9 CARD complex (Qin et al., 1999) (Figure 3D, E, F, G). The interaction between Pelle and Tube is completely different, involving the H4 region of Pelle and the H6 region of Tube as well as an interaction at the C-terminal tail of Tube. The Apaf-1 CARD/pro-caspase-9 CARD interaction is somewhat similar; instead of the H2 and H5 helices as in DED1, the H2 and H3 helices of Apaf-1 interacts with the H1 and H4 helices of pro-caspase-9. Interestingly, these three cases of homotypic interactions within the DD superfamily are all asymmetric, in contrast to what might have been expected for homotypic interactions.

MC159 mutants that fail to protect cells from apoptosis do not form a ternary complex with Fas and FADD

Previous mutational studies on MC159 have suggested that FADD recruitment does not seem sufficient for its anti-apoptotic function because almost all MC159 mutants that were deficient in protecting cells from Fas and TNFR1-induced apoptosis appear to have retained their abilities to interact with FADD (Garvey et al., 2002a). Having established that FADD does not interact strongly with MC159 in the absence of Fas, we considered the possibility that the binary FADD/MC159 interaction may not serve as a good indicator for MC159 recruitment to the DISC. Instead, we used our in vitro assay of ternary complex formation with Fas and FADD to characterize these previously generated MC159 mutants (Table 2). Strikingly, all tested mutants that failed to protect cells from apoptosis, including M2, M3, M5, M6, M8, M16, M17, M21 and M24, were also unable to form ternary complexes with Fas and FADD (Figure S1, lanes 2, 4, 6, 11, 15, 13, 17, 19, 24 in the Supplemental Data). In contrast, MC159 mutants that retained full or partial ability to protect cells, such as M7, M9, M10, M18 and M27, were able to form the ternary complex (Figure S1, lanes 28, 30, 32, 37, 39 in the Supplemental Data). Therefore, the ability of MC159 to protect cells from apoptosis is closely correlated with its recruitment to Fas and FADD. Hence, in contrast to previous suggestion, it appears that DISC recruitment of MC159 is both necessary and sufficient for its apoptotic protection function.

Table 2.

Characterization of previously generated (denoted as M followed by a number) and additional (denoted as MC followed by a number) MC159 mutants for ternary complex formation with Fas and FADD.

Name Mutation Sites† Ternary Complex Formation Apoptosis Protection†
M2 H23A, E24A, D25A − −
M3 H33A, D34A − −
M5 E62A − −
M6 R69A, D71A − −
M7 K81A, E82A + +
M8 R95A, R97A − −
M9 E105A, E106A +/− +/−
M10 R135A, E138A + +
M16 E18A, E19A, D21A − −
M17 F30A − −
M18 E111A, R113A +/− +
M21 L72A, L73A − −
M24 L31A − −
M27 M160A, L161A + +/−
M29 L157A − +/−
MC1 S9A, L10A, P11A, F12A +
MC2 E24D +
MC3 E24Q −
MC4 P37A +
MC5 T40A, T41A, T43A, Q44A −
MC6 C47A, S48A +
MC7 S50A, Q51A, Q52A +
MC8 Q51A, Q52A +
MC9 T56A, L57A −
MC10 Q68A +
MC11 R69K −
MC12 D71N −
MC13 K74A, S75A +
MC14 L79A, S80A −
MC15 Q86A, L87A, L88A +
MC16 T94A, R95A −
MC17 D108A, S109A, S110A +
MC18 E111Q +/−
MC19 L117G +
MC20 F118G +
MC21 L117G, F118G +
MC22 C120A, N121A, L122A,
N123A, P124A, S125A		 −
MC23 S127A, T128A +
MC24 S131A, E132A, S133A +
MC25 E144A, N145A −
MC26 V146, L148 +
MC27 S150G, P151G +
MC28 S155, V156 +
MC29 R166K +
MC30 D168N +
MC31 Q171A, Q172A +
MC32 V174A, E175A +
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†

Apoptosis protection data for previously generated mutants were as reported in (Garvey et al., 2002a). The mutation sites for the previously generated mutants were determined by re-sequencing of the mutant plasmids and are different in M8 (previously reported as R95A, Y96A and R97A), M9 (previously reported as E105A, E106A and D108A) and M18 (previously reported as E111A, L112A and R113A).

The DED2 hydrophobic patch of caspase-8 is required for FADD interaction

The MC159 structure provides a template for examining the structure and function relationships of tandem DED-containing proteins. Because earlier mutagenesis experiment on FADD showed that its hydrophobic patch comprising the conserved F25 and L26 residues are important for caspase-8 recruitment (Eberstadt et al., 1998), we speculated that the same hydrophobic patch on caspase-8 may be involved in FADD association. Since the corresponding hydrophobic patch on DED1 is buried at the DED1/DED2 interface, only one conserved hydrophobic patch is present on tandem DED structures (Figure 2C, 3A). To elucidate whether this conserved hydrophobic patch of DED2 is involved in interaction with FADD, we performed immunoprecipitation between caspase-8 and FADD (Figure 4A). An active site mutant of caspase-8 (C360S) was used to avoid caspase-8 auto-processing. As predicated, the single site hydrophobic patch mutant of caspase-8, F122G, exhibited weakened interaction with FADD. The double mutant, F122G and L123G, completely abolished its interaction with FADD, demonstrating that these surface exposed hydrophobic residues are absolutely required for the interaction of caspase-8 with FADD.

Figure 4.

Figure 4

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The hydrophobic patch of caspase-8, but not MC159, is involved in FADD interaction and MC159 does not compete with caspase-8 recruitment. A. Immunoprecipitation of wildtype and hydrophobic patch mutants of caspase-8 (C360S) and FADD. B. Ternary complex formation of hydrophobic patch mutants of MC159 with Fas and FADD. Contaminating bands are indicated by *. Please note that the last two lanes were cut and pasted from another gel. C. MC159 does not compete with caspase-8 recruitment. Human lymphoma H9 cells were transfected with GFP-fusion constructs for expressing GFP (Vct), MC159:GFP and cFLIP(S):GFP as indicated. The DISC was induced by anti-Fas stimulation as indicated and its components were detected from the immunoprecipitates and cell lysates by probing against caspase-8 (upper panels) and GFP (lower panels). Please note that the last lane of the lysates was cut and pasted due to misloading in sample lanes. Data are representative of two independent experiments. D. Caspase-8 recruitment in the presence of increasing amount of MC150 transfection.

The DED2 hydrophobic patch of MC159 is not required for ternary complex formation and MC159 does not compete with caspase-8 for DISC recruitment

To determine whether the same hydrophobic patch on DED2 of MC159 is also important for its recruitment, we examined the ability of the hydrophobic patch mutants of MC159 in ternary complex formation with Fas and FADD. In contrast to the requirement for the DED2 hydrophobic patch in caspase-8 recruitment, both single (L117G, F118G) and double (L117G and F118G) hydrophobic patch mutants of MC159 had essentially no effect on its ability to form a ternary complex with Fas and FADD (Figure 4B). This suggests that the hydrophobic patch is not required for MC159 recruitment.

To elucidate whether MC159 competes with caspase-8 for recruitment to FADD, we performed DISC assay in H9 cells with either GFP-MC159 or GFP-cFLIP(S) transfection. Upon stimulation by anti-Fas antibody, the cell lysates were immunoprecipitated, resolved by SDS-PAGE and probed with either anti-caspase-8 or anti-GFP antibodies. While cFLIP(S) transfection strongly inhibited caspase-8 recruitment, MC159 transfection did not attenuate caspase-8 recruitment to the DISC (Figure 4C). In fact, a detailed titration on the transfection of GFP-MC159 fusion plasmid indicated that MC159 might have subtly enhanced caspase-8 recruitment (Figure 4D). These and the hydrophobic patch mutation data suggest that caspase-8 and MC159 apparently utilize different structural features for FADD interaction.

An extensive surface of MC159 and the charge triad region of FADD are both required for ternary complex formation

Among the previously generated MC159 mutants, most are on buried residues and therefore may cause structural perturbations. Only M2 (H23A, E24A, D25A), M3 (H33A, D34A), M6 (R69A, D71A) and M16 (E18A, E19A, D21A) contain surface exposed residues and are defective in protection against apoptosis (Table 2). These residues may directly participate in FADD interaction. To thoroughly map the molecular determinants of MC159 recruitment, we generated 32 additional MC159 mutants on surface exposed residues, most of which contain charged or large (either hydrophobic or hydrophilic) side chains. We characterized them using the in vitro assay on ternary complex formation. 9 out of the 32 mutants were defective in ternary complex formation (Table 2).

Mapping of MC159 mutations that were defective in ternary complex formation (Figure 3A) showed the involvement of an extensive region of the structure in its recruitment (Figure 5A). This suggests that each MC159 may contact at least two adjacent FADD molecules. On one side of the structure, a region near the charge triad in DED1, comprising mutants M2 (H23A, E24A, D25A), M6 (R69A, D71A), M16 (E18A, E19A, D21A), and MC5 (T40A, T41A, T43A and Q44A), is important (Figure 5B). For residues within the charge triad in this region, even very conserved substitutions, E24Q, R69K and D71N, abolished ternary complex formation. On the same side of the structure, the charge triad in DED2 does not seem to be as important, as E111Q, R166K and D168N mutations at worst weakened ternary complex formation. Therefore, on this side of the MC159 structure, it appears only DED1 is important for its recruitment. Most of the remaining mutations map to the opposite face of MC159 (Figure 5C). They comprise mutants M3 (H33A, D34A), MC9 (T56A, L57A), MC14 (L79A, S80A), MC16 (T94A, R95A), MC22 (C120A, N121A, L122A, N123A, P124A, S125A) and MC25 (E144A, N145A). Residues from both DED1 and DED2 participate interactions at this side.

Figure 5.

Figure 5

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Interaction between MC159 and FADD and a proposed model for the inhibition of caspase activation by MC159. A. Mapping of surface mutations of MC159 that are defective in FADD interaction onto the stereo ribbon diagram of its structure. Residues E18, E19, D21: magenta; H23, E24, D25: red; H33, D34: cyan; T40, T41, T43, Q44: dark blue; T56, L57: green; R69, D71: yellow; L79, S80: blue; T94, R95: gold; C120, N121, L122, N123, P124, S125: yellow; E145, N145: red. B. C. Mapping of the same mutations onto the molecular surface of MC159. Residues numbers are shown. B is shown in the same orientation as in A and C are related to B by a 180° rotation about the vertical axis. D. Mapping of FADD mutations, R72E and D74K within the charge triad, onto its surface. The location of the conserved hydrophobic patch (F25, L26) is also shown. E. A proposed model for the inhibition of caspase activation at the DISC by MC159 via disruption of FADD self-association. The pentagons represent monomeric Fas/FADD/caspase-8 complexes and filled yellow circles represent MC159 molecules. The Fas/FADD/caspase-8 complexes likely interact with each other via both trimeric associations as dictated by trimeric FasL and dimeric associations from FADD self-association. Schematically, the blue and red faces of the pentagons mediate trimeric associations and the face adjacent to the blue mediates dimeric associations. These two types of associations could mediate the infinite oligomerization of DISC during signaling. MC159 likely interacts with at least two neighboring FADD molecules and disrupts DISC oligomerization by competing with the same face used for the dimeric FADD self-association.

The importance of the charge triad of MC159 in FADD interaction suggests that the same charge triad region of FADD may be reciprocally important for interaction with MC159. To test this hypothesis, we performed site-directed mutagenesis on the charge triad region of FADD, R72E and D74K, and tested the ability of these FADD mutants to form a ternary complex with MC159. As predicted, mutants R72E and D74K completely abolished and weakened ternary complex formation, respectively (data not shown), suggesting that the charge triad region of FADD is involved in MC159 recruitment (Figure 5D).

DISCUSSION

Cooperative ternary complex assembly in caspase-8 and MC159 recruitment

Using recombinant proteins, we showed that Fas DD, FADD and MC159 are cooperatively assembled. Similarly, the ternary complex of Fas DD, FADD and caspase-8 are also cooperatively assembled (data not shown). These molecular assembly processes likely recapitulate the early event in Fas signaling in vivo, the formation of the “signaling protein oligomeric transduction structures” (SPOTS) (Siegel et al., 2004). Like the ternary complex formation, assembly of SPOTS is absolutely dependent on Fas DD and FADD, and is enhanced by caspase-8, regardless of its catalytic activity (Siegel et al., 2004). Because it is detrimental to activate caspases in the absence of distinct signaling events, this cooperative assembly appears to act as a safety measure in preventing accidental DISC formation. Other oligomeric death inducing complexes such as complex II formed by TRADD, FADD and caspase-8 (Micheau and Tschopp, 2003) and PIDDosome formed by PIDD, RAIDD and caspase-2 (Tinel and Tschopp, 2004) likely obey the same assembly mechanism.

MC159 as a template for tandem DED structures

Unlike beads on a string, DED1 and DED2 are rigidly associated to form a single compact structure, in which each domain performs its unique structural role in the context of the tandem DEDs and is not interchangeable in position with each other. This integral structure of tandem DEDs explains the previous observation that both DEDs of MC159 are required for its anti-apoptotic function (Garvey et al., 2002a). On an isolated domain level, DED1 and DED2 differ significantly. While DED2 appears to be a bona fide DED and closely resembles the DED structures in FADD (Eberstadt et al., 1998) and PEA-15 (Hill et al., 2002), DED1 appears to be equally distant to these known DED structures as to other structures in the DD superfamily such as those of DDs, PYDs and CARDs. The association between DED1 and DED2 in tandem DEDs reveals a first mode of interaction between DEDs, which differs significantly from observed CARD/CARD and DD/DD interactions.

Structural analyses reveal two distinctive surface features in DED structures. The first feature is the conserved hydrophobic patches. In DED1 this conserved hydrophobic patch is used to support the DED1/DED2 interface and is buried at this interface. In contrast, the conserved DED2 hydrophobic patch is surface exposed and available for interactions. The second distinctive surface feature is the conserved hydrogen-bonded charge triad, which we here refer to as the E/D-RxDL motif. It is a signature of DEDs and is not present in DDs, CARDs or PYDs. This region of the DED surface also appears to be a hot spot for protein-protein interactions.

Molecular mechanism for the recruitment of caspase-8, and possibly caspase-10, cFLIPs and herpesvirus vFLIPs: the conserved hydrophobic patch

The first structure of a tandem DED-containing protein provides a template for understanding the molecular basis of procaspase recruitment and FLIP inhibition during DR signaling. The conserved hydrophobic patch on DEDs was first identified from the NMR structure of FADD, which comprises two central large hydrophobic residues, F25 and L26 (Eberstadt et al., 1998). It was shown to be important for the interaction of FADD with caspase-8. Here we show that the conserved DED2 hydrophobic patch of caspase-8 is important for its recruitment to FADD.

We speculate that the use of the hydrophobic patch for DISC recruitment is likely to be true for caspase-10, cFLIP and herpesvirus vFLIPs as well. In these tandem DED-containing proteins, the Phe residue in the hydrophobic patch is strictly conserved as Phe or Tyr, and the Leu residue is strictly conserved as Leu or Ile (Figure 3A) (Tibbetts et al., 2003). Therefore, cFLIP would compete directly with caspase-8 for DISC recruitment, as shown here (Figure 4C). In addition, herpesvirus vFLIPs would also likely inhibit caspase activation by direct competition with caspase-8 for DISC recruitment.

However, this scenario is not true for MC159. As we showed here, mutations of the DED2 hydrophobic patch residues did not affect MC159 recruitment. The lack of involvement of the hydrophobic patch for MC159 recruitment may be rationalized by its less conserved substitutions at this site; the conserved Phe is replaced by a Leu and the conserved Leu is replaced by a Phe. In FADD, it appears that the Phe position has to be an aromatic residue as changes to Tyr and Trp retained its ability to interact with caspase-8 while a change to Val abolished the interaction (Eberstadt et al., 1998). It is likely then that the Phe to Leu substitution in MC159 also abolishes its interaction with the hydrophobic patch of FADD. The same Phe to Leu change is also observed in MC160, another vFLIP-like protein from MCV (Shisler and Moss, 2001). The only other DED-containing protein that does not have the conserved hydrophobic patch is PEA-15, a protein not involved in death receptor signaling and regulation.

Molecular mechanism for MC159 recruitment and inhibition of caspase activation: the charge triad and the disruption of FADD self association

In contrast to the involvement of the hydrophobic patch, we showed here that MC159 uses an extensive surface encompassing the charge triad for interaction with FADD. This is consistent with the previous observation that the RxDL motifs of MC159 play important roles in MC159 function (Garvey et al., 2002a). The extensive nature of the interaction surface suggests that each MC159 contacts at least two adjacent FADD molecules. Reciprocally, we showed that the charge triad of FADD is important for interaction with MC159. However, since MC159 does not compete with caspase-8 for recruitment, how does it inhibit caspase activation and apoptosis?

To answer this question, we have to analyze the basic requirement for DISC assembly. Because FasL is intrinsically trimeric, the assembled DISC should at least be trimeric. In addition, because FADD self associates, higher order of aggregation is generated. If we assume that FADD self associates via a dimeric interaction, this dimerization of trimers would provide a mode of infinite aggregation (Figure 5E) into variable sized SPOTS that are observable under light microscope. This existence of the dimeric symmetry would be consistent with the requirement for dimerization in caspase-8 activation (Boatright et al., 2003; Donepudi et al., 2003). In addition, the formation of higher order oligomers is essential for caspase activation as trimeric soluble FasL is rather inefficient in inducing caspase activation while hexameric FasL is much more efficient (Holler et al., 2003).

Interestingly, we have shown recently that FADD also self-associates via the RxDL motif in the charge triad region (Muppidi et al., 2005). Therefore, MC159 would compete with and disrupt FADD self-association. In the absence of FADD self-association, higher order oligomerization of DISC would be abolished and therefore caspase activation would be inhibited (Figure 5E). This model of MC159 function elegantly explains the molecular basis for its anti-apoptotic activity. It is supported by the observation that MC159 reduces the size of SPOTS during Fas signaling (Siegel et al., 2004). Similarly, it blocks the formation of FADD “death effector filaments”, which are self-oligomerized FADD molecules upon overexpression, and inhibits caspase activation and apoptosis (Garvey et al., 2002b; Siegel et al., 1998). Therefore, unlike cFLIPs and herpesvirus vFLIPs, MC159 uses a non-competitive mechanism for inhibiting caspase activation at the DISC level.

Experimental Procedures

Protein Expression and Purification

MC159 from molluscum contagiosum virus (residues 1–241 and residues 1–187) was expressed as His-tagged proteins in E. coli using 20°C induction and purified by Ni-affinity and gel filtration chromatography. Human Fas DD, human FADD, human FADD DD and human FADD DED proteins, either His-tagged or with no tags, were expressed similarly using low temperature inductions. FADD and FADD DED constructs both contain the F25Y mutation that retains FADD function but improves its solution behavior (Eberstadt et al., 1998).

Crystallization and Structure Determination of MC159

Selenomethionine-substituted full-length MC159 were crystallized using the hanging drop vapor diffusion method under 50–150 mM sodium/potassium phosphate at pH 5.0. The crystals were highly variable; upon screening many crystals, a single-wavelength anomalous diffraction data set was collected at 3.8Å resolution using the SGX ID beam line at the Advanced Photon Source (Table 1). Diffraction data were processed and scaled using HKL2000 (Otwinowski and Minor, 1997). The crystal belongs to space group P2221 with cell dimensions of a=84.0Å, b=108.2Å and c=142.7Å. Selenium sites were determined using direct method calculations in SnB (Weeks and Miller, 1999) and fed into SOLVE (Terwilliger, 2004) for phase determination. In combination with 4-fold non-crystallographic symmetry averaging in RESOLVE (Terwilliger, 2004), an interpretable map was obtained. Facilitated by the known selenium sites as markers for methionine residues, all four molecules in the crystallographic asymmetric unit were traced and built (residues 2–187) using O (Jones et al., 1991).

The structure revealed that a large segment of the C-terminal tail (residues 188–241) is disordered in the crystal. This C-terminal tail is not important for the function of MC159 (Garvey et al., 2002a). A new construct (residues 1–187), made based on the ordered part of the molecule, gave soluble proteins and was crystallized using the hanging drop vapor diffusion method under 1.0–1.2 M sodium acetate in 0.1 M imidazole at pH 6.5. The crystals belong to space group P212121 and diffracted beyond 1.2Å resolution at the X4A beamline of the National Synchrotron Light Source. The structure was determined by molecular replacement calculations in COMO (Jogl et al., 2001) using the partially refined model from the initial crystal form. All crystallographic refinement calculations were performed in CNS (Brunger et al., 1998) and the structures were analyzed and displayed using Setor (Evans, 1993), Molscript/Raster3D (Kraulis, 1991; Merritt and Murphy, 1994) and Grasp (Nicholls et al., 1991).

Mutagenesis and His-tag Pull down

Mutagenesis of MC159 was performed with the QuikChange Site-Directed Mutagenesis Method (Stratagene). Wildtype and mutant His-tagged MC159 proteins were first purified using Ni-affinity to assess their expression levels. Then cell lysates containing estimated equivalent quantities of MC159 proteins were mixed with those of non-tagged Fas DD and FADD. The mixtures were incubated at room temperature for 2 hours and His-tag pull down experiments were performed to determine whether they form ternary complexes with Fas and FADD.

Immunoprecipitation and Western blot (IP/WB)

Human embryonic kidney tumor line 293T cells were seeded at 106/well in complete DMEM medium in 6-well plates and cultured overnight. The cells were transfected with 3 µg of plasmid DNA using Fugene 6 (Roche) reagent according to the manufacturer’s instructions. The cells were cultured for 24 ~48 hours, harvested and lysed on ice by 0.5 ml of the TNTG lysis buffer (30 mM Tris.Cl, pH 8.0, 150 mM NaCl, 1% Triton X-100, and 10% glycerol) with 1 × protease inhibitor cocktail (Roche) for 30 minutes. The lysates were centrifuged at 10,000×g, 8 °C for 10 minutes. The protein-normalized lysates were subjected to immunoprecipitation by adding 10 µg/ml appropriate antibody, 30 µl of protein G-agarose (Roche), and rotating > 2 hours in cold room. The precipitates were washed 5 times with cold TNTG lysis buffer and then subjected to 4~20% Tris/Glycine-SDS PAGE under reduced and denatured conditions. The resolved protein bands were transferred onto nitrocellulose membranes and probed with 1 µg/ml of appropriate primary antibodies in PBS-T (phosphate buffered saline with 0.1% Tween-20) at room temperature for 2 hours, and then with appropriate HRP-conjugated secondary antibodies for 1 hour. The blots were then developed by the SuperSignaling system (Pierce) following the manufacturer’s instructions.

For DISC assay, 8×106 human lymphoma H9 cells were transfected with up to 20ug plasmids by electroporation using the BTX Electro Cell Manipulatro-600. The transfected cells were cultured in 10 ml of complete RPMI medium for 36 hours and viable cells were purified by gradient centrifugation. Each of 5×106 transfected cells was subjected to mock or anti-Fas (Apo-1.3, 1ug/ml) stimulation for 10 minutes at 37°C. The stimulated cells were washed twice with cold PBS and followed with the IP/WB protocols described above.

Supplementary Material

Suppl

Acknowledgements

We thank Dr. Stephen R. Wasserman for the use of SGX-CAT at Sector 31 of the Advanced Photon Source, which was constructed and operated by Structural GenomiX, Inc., Randy Abramowitz and John Schwanof for use of the X4A beamline at NSLS, Dr. Jeffrey Cohen for providing MC159 mutants and Samantha Jayawickrama for some of the mutagenesis work. J.K.Y is a postdoctoral fellow of the Serono Foundation. L.X.Z., F.Y.W., and M.J.L. are supported by Intramural Research Program of the National Institute of Allergy and Infectious Diseases, NIH.

Footnotes

Accession Numbers

The coordinates have been deposited in the RCSB Protein Data Bank with the PDB codes of 2BBZ for the 3.8Å resolution MC159 structure and 2BBR for the 1.2Å resolution MC159 structure.

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