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• W1985909474 abstract "Death receptor signaling is initiated by the assembly of the deathinducing signaling complex, which culminates in the activation of the initiator caspase, either caspase-8 or caspase-10. A family of viral and cellular proteins, known as FLIP, plays an essential role in the regulation of death receptor signaling. Viral FLIP (v-FLIP) and short cellular FLIP (c-FLIPS) inhibit apoptosis by interfering with death receptor signaling. The structure and mechanisms of v-FLIP and c-FLIPS remain largely unknown. Here we report a high resolution crystal structure of MC159, a v-FLIP derived from the molluscum contagiosum virus, which is a member of the human poxvirus family. Unexpectedly, the two tandem death effector domains (DEDs) of MC159 rigidly associate with each other through a hydrophobic interface. Structure-based sequence analysis suggests that this interface is conserved in the tandem DEDs from other v-FLIP, c-FLIPS, and caspase-8 and -10. Strikingly, the overall packing arrangement between the two DEDs of MC159 resembles that between the caspase recruitment domains of Apaf-1 and caspase-9. In addition, each DED of MC159 contains a highly conserved binding motif on the surface, to which loss-of-function mutations in MC159 map. These observations, in conjunction with published evidence, reveal significant insights into the function of v-FLIP and suggest a mechanism by which v-FLIP and c-FLIPS inhibit death receptor signaling. Death receptor signaling is initiated by the assembly of the deathinducing signaling complex, which culminates in the activation of the initiator caspase, either caspase-8 or caspase-10. A family of viral and cellular proteins, known as FLIP, plays an essential role in the regulation of death receptor signaling. Viral FLIP (v-FLIP) and short cellular FLIP (c-FLIPS) inhibit apoptosis by interfering with death receptor signaling. The structure and mechanisms of v-FLIP and c-FLIPS remain largely unknown. Here we report a high resolution crystal structure of MC159, a v-FLIP derived from the molluscum contagiosum virus, which is a member of the human poxvirus family. Unexpectedly, the two tandem death effector domains (DEDs) of MC159 rigidly associate with each other through a hydrophobic interface. Structure-based sequence analysis suggests that this interface is conserved in the tandem DEDs from other v-FLIP, c-FLIPS, and caspase-8 and -10. Strikingly, the overall packing arrangement between the two DEDs of MC159 resembles that between the caspase recruitment domains of Apaf-1 and caspase-9. In addition, each DED of MC159 contains a highly conserved binding motif on the surface, to which loss-of-function mutations in MC159 map. These observations, in conjunction with published evidence, reveal significant insights into the function of v-FLIP and suggest a mechanism by which v-FLIP and c-FLIPS inhibit death receptor signaling. Apoptosis plays a central role in the development and homeostasis of metazoans (1Horvitz H.R. Chembiochem. 2003; 4: 697-711Crossref PubMed Scopus (133) Google Scholar, 2Danial N.N. Korsmeyer S.J. Cell. 2004; 116: 205-219Abstract Full Text Full Text PDF PubMed Scopus (4056) Google Scholar, 3Wang X. Genes Dev. 2001; 15: 2922-2933Crossref PubMed Scopus (94) Google Scholar, 4Hay B.A. Huh J.R. Guo M. Nat. Rev. Genet. 2004; 5: 911-922Crossref PubMed Scopus (73) Google Scholar, 5Rathmell J.C. Thompson C.B. Cell. 2002; 109: S97-S107Abstract Full Text Full Text PDF PubMed Scopus (396) Google Scholar). Inappropriate regulation of apoptosis contributes to a number of human pathologies, including cancer, autoimmune diseases, and neurodegenerative disorders (6Thompson C.B. Science. 1995; 267: 1456-1462Crossref PubMed Scopus (6203) Google Scholar, 7Hanahan D. Weinberg R.A. Cell. 2000; 100: 57-70Abstract Full Text Full Text PDF PubMed Scopus (22579) Google Scholar, 8Yuan J. Yankner B.A. Nature. 2000; 407: 802-809Crossref PubMed Scopus (1607) Google Scholar, 9Green D.R. Evan G.I. Cancer Cell. 2002; 1: 19-30Abstract Full Text Full Text PDF PubMed Scopus (904) Google Scholar, 10Vaux D.L. Flavell R.A. Curr. Opin. Immunol. 2000; 12: 719-724Crossref PubMed Scopus (94) Google Scholar). The pathways of apoptosis are evolutionarily conserved, culminating in the activation of death proteases, known as caspases (named after cysteine protease with Asp substrate specificity) (11Riedl S.J. Shi Y. Nat. Rev. Mol. Cell. Biol. 2004; 5: 897-907Crossref PubMed Scopus (1582) Google Scholar, 12Thornberry N.A. Lazebnik Y. Science. 1998; 281: 1312-1316Crossref PubMed Scopus (6178) Google Scholar). In mammalian cells, apoptosis manifests in two forms, intrinsic and extrinsic, which are triggered by cell death stimuli from intra- and extracellular environments, respectively. The extracellular death stimuli, such as the Fas/CD95 ligand, directly activate the death receptors through ligand-induced assembly of a death-inducing signaling complex (DISC) 2The abbreviations used are: DISC, death-inducing signaling complex; TNFR1, tumor necrosis factor receptor 1; TRAIL, TNF-related apoptosis-inducing ligand; DD, death domain; DED, death effector domain; CARD, caspase recruitment domain; SeMet, seleno-methionine; MAD, multiwavelength anomalous dispersion; r.m.s.d., root-mean-square deviation. at the plasma membrane (13Nagata S. Annu. Rev. Genet. 1999; 33: 29-55Crossref PubMed Scopus (676) Google Scholar, 14Peter M.E. Krammer P.H. Cell Death Differ. 2003; 10: 26-35Crossref PubMed Scopus (895) Google Scholar). The assembly of DISC results in the activation of caspase-8, which subsequently activates the effector caspases, caspase-3 and -7 (11Riedl S.J. Shi Y. Nat. Rev. Mol. Cell. Biol. 2004; 5: 897-907Crossref PubMed Scopus (1582) Google Scholar). The death receptor family comprises at least eight members, in which the TNFR1, Fas, and TRAIL receptors have been shown to activate apoptosis for immune system functions such as surveillance and elimination of virally infected or cancerous cells (15Ozoren N. El-Deiry W.S. Semin. Cancer Biol. 2003; 13: 135-147Crossref PubMed Scopus (261) Google Scholar). The activated death ligands are homo-trimeric and thus induce oligomerization of the death receptors upon binding. The death receptors subsequently recruit the adaptor protein FADD (or TRADD for TNFR1) through direct interaction between a death domain (DD) present in both proteins (16Holler N. Tardivel A. Kovacsovics-Bankowski M. Hertig S. Gaide O. Martinon F. Tinel A. Deperthes D. Calderara S. Schulthess T. Engel J. Schneider P. Tschopp J. Mol. Cell. Biol. 2003; 23: 1428-1440Crossref PubMed Scopus (331) Google Scholar, 17Boldin M.P. Varfolomeev E.E. Pancer Z. Mett I.L. Camonis J.H. Wallach D. J. Biol. Chem. 1995; 270: 7795-7798Abstract Full Text Full Text PDF PubMed Scopus (941) Google Scholar, 18Chinnaiyan A.M. O'Rourke K. Tewari M. Dixit V.M. Cell. 1995; 81: 505-512Abstract Full Text PDF PubMed Scopus (2163) Google Scholar). FADD, in turn, uses its death effector domain (DED) to interact with the N-terminal tandem DEDs of procaspase-8 or procaspase-10, thereby linking these initiator caspases to the activated death receptors within the DISC (19Kischkel F.C. Lawrence D.A. Tinel A. LeBlanc H. Virmani A. Schow P. Gazdar A. Blenis J. Arnott D. Ashkenazi A. J. Biol. Chem. 2001; 276: 46639-46646Abstract Full Text Full Text PDF PubMed Scopus (430) Google Scholar, 20Muzio M. Chinnaiyan A.M. Kischkel F.C. O'Rourke K. Shevchenko A. Ni J. Scaffidi C. Bretz J.D. Zhang M. Gentz R. Mann M. Krammer P.H. Peter M.E. Dixit V.M. Cell. 1996; 85: 817-827Abstract Full Text Full Text PDF PubMed Scopus (2743) Google Scholar, 21Wang J. Chun H.J. Wong W. Spencer D.M. Lenardo M.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13884-13888Crossref PubMed Scopus (311) Google Scholar). Thus the assembly of DISC relies on homotypic DD and DED interactions and effectively facilitates the activation of initiator caspases. To evade the host immune response, a number of viruses express distinct families of inhibitory proteins to suppress apoptosis and to promote their replication and survival in host cells (22Thome M. Tschopp J. Nat. Rev. Immunol. 2001; 1: 50-58Crossref PubMed Scopus (352) Google Scholar). One important family of such proteins is the viral FLICE-inhibitory proteins (v-FLIPs), which were initially identified in molluscum contagiosum virus and in several γ-herpesviruses (22Thome M. Tschopp J. Nat. Rev. Immunol. 2001; 1: 50-58Crossref PubMed Scopus (352) Google Scholar). v-FLIPs have been shown to block the activation of the death receptors at the level of DISC assembly (22Thome M. Tschopp J. Nat. Rev. Immunol. 2001; 1: 50-58Crossref PubMed Scopus (352) Google Scholar). Some of the representative v-FLIP proteins include molluscum contagiosum virus MC159, equine herpesvirus-2 E8, herpesvirus saimiri ORF16, bovine herpesvirus-4 E2, and Kaposi's sarcoma associated herpesvirus (KSHV/HHV8) K13 (23Bertin J. Armstrong R.C. Ottilie S. Martin D.A. Wang Y. Banks S. Wang G.H. Senkevich T.G. Alnemri E.S. Moss B. Lenardo M.J. Tomaselli K.J. Cohen J.I. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1172-1176Crossref PubMed Scopus (384) Google Scholar, 24Hu S. Vincenz C. Buller M. Dixit V.M. J. Biol. Chem. 1997; 272: 9621-9624Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar, 25Thome M. Schneider P. Hofmann K. Fickenscher H. Meinl E. Neipel F. Mattmann C. Burns K. Bodmer J.L. Schroter M. Scaffidi C. Krammer P.H. Peter M.E. Tschopp J. Nature. 1997; 386: 517-521Crossref PubMed Scopus (1145) Google Scholar, 26Wang G.H. Bertin J. Wang Y. Martin D.A. Wang J. Tomaselli K.J. Armstrong R.C. Cohen J.I. J. Virol. 1997; 71: 8928-8932Crossref PubMed Google Scholar, 27Djerbi M. Screpanti V. Catrina A.I. Bogen B. Biberfeld P. Grandien A. J. Exp. Med. 1999; 190: 1025-1032Crossref PubMed Scopus (386) Google Scholar). The hallmark of all v-FLIP proteins is two DED domains in tandem, which are also present in the prodomains of caspase-8 and caspase-10. The presence of DEDs in v-FLIP immediately suggests potential interaction with FADD and caspase-8. Indeed, the DEDs of MC159 and E8 have been shown to interact with the DED of FADD and the prodomain of caspase-8 (23Bertin J. Armstrong R.C. Ottilie S. Martin D.A. Wang Y. Banks S. Wang G.H. Senkevich T.G. Alnemri E.S. Moss B. Lenardo M.J. Tomaselli K.J. Cohen J.I. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1172-1176Crossref PubMed Scopus (384) Google Scholar, 24Hu S. Vincenz C. Buller M. Dixit V.M. J. Biol. Chem. 1997; 272: 9621-9624Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar, 25Thome M. Schneider P. Hofmann K. Fickenscher H. Meinl E. Neipel F. Mattmann C. Burns K. Bodmer J.L. Schroter M. Scaffidi C. Krammer P.H. Peter M.E. Tschopp J. Nature. 1997; 386: 517-521Crossref PubMed Scopus (1145) Google Scholar, 26Wang G.H. Bertin J. Wang Y. Martin D.A. Wang J. Tomaselli K.J. Armstrong R.C. Cohen J.I. J. Virol. 1997; 71: 8928-8932Crossref PubMed Google Scholar, 28Shisler J.L. Moss B. Virology. 2001; 282: 14-25Crossref PubMed Scopus (68) Google Scholar). Through homotypic DED interactions, the v-FLIPs are thought to hinder efficient recruitment and subsequent activation of caspase-8 at the death receptors. Long and short cellular homologues of the v-FLIP have also been identified as c-FLIPL and c-FLIPS, respectively (22Thome M. Tschopp J. Nat. Rev. Immunol. 2001; 1: 50-58Crossref PubMed Scopus (352) Google Scholar). In addition to the tandem DEDs, c-FLIPL contains a caspase-like domain. For both c-FLIPS and c-FLIPL, their tandem DEDs appear to antagonize death receptor signaling in the same manner when expressed at high levels (24Hu S. Vincenz C. Buller M. Dixit V.M. J. Biol. Chem. 1997; 272: 9621-9624Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar, 29Chang D.W. Xing Z. Pan Y. Algeciras-Schimnich A. Barnhart B.C. Yaish-Ohad S. Peter M.E. Yang X. EMBO J. 2002; 21: 3704-3714Crossref PubMed Scopus (482) Google Scholar 31Shu H.B. Halpin D.R. Goeddel D.V. Immunity. 1997; 6: 751-763Abstract Full Text Full Text PDF PubMed Scopus (367) Google Scholar, 33Srinivasula S.M. Ahmad M. Ottilie S. Bullrich F. Banks S. Wang Y. Fernandes-Alnemri T. Croce C.M. Litwack G. Tomaselli K.J. Armstrong R.C. Alnemri E.S. J. Biol. Chem. 1997; 272: 18542-18545Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar, 34Inohara N. Koseki T. Hu Y. Chen S. Nunez G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10717-10722Crossref PubMed Scopus (278) Google Scholar, 35Irmler M. Thome M. Hahne M. Schneider P. Hofmann K. Steiner V. Bodmer J.L. Schroter M. Burns K. Mattmann C. Rimoldi D. French L.E. Tschopp J. Nature. 1997; 388: 190-195Crossref PubMed Scopus (2228) Google Scholar, 36Han D.K. Chaudhary P.M. Wright M.E. Friedman C. Trask B.J. Riedel R.T. Baskin D.G. Schwartz S.M. Hood L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11333-11338Crossref PubMed Scopus (222) Google Scholar). Although the DED-containing proteins are known to control apoptosis from death receptors, the underlying mechanisms remain largely unknown. This is in part due to the technical difficulty in working with membrane-associated protein complexes. As a consequence, there is a lack of comprehensive structural information on the relevant proteins and protein complexes. At present, we only have limited structural information on the isolated DED of FADD (37Eberstadt M. Huang B. Chen Z. Meadows R.P. Ng S.-C. Zheng L. Lenardo M.J. Fesik S.W. Nature. 1998; 392: 941-945Crossref PubMed Scopus (204) Google Scholar) and Pea-15 (38Hill J.M. Vaidyanathan H. Ramos J.W. Ginsberg M.H. Werner M.H. EMBO J. 2002; 21: 6494-6504Crossref PubMed Scopus (86) Google Scholar), and the DD of Fas (39Huang B. Eberstadt M. Olejniczak E.T. Meadows R.P. Fesik S.W. Nature. 1996; 384: 638-641Crossref PubMed Scopus (322) Google Scholar) and FADD (40Berglund H. Olerenshaw D. Sankar A. Federwisch M. McDonald N.Q. Driscoll P.C. J. Mol. Biol. 2000; 302: 171-188Crossref PubMed Scopus (83) Google Scholar, 41Jeong E.J. Bang S. Lee T.H. Park Y.I. Sim W.S. Kim K.S. J. Biol. Chem. 1999; 274: 16337-16342Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). All structures conform to a common fold comprising six antiparallel α-helices, which is also shared by caspase recruitment domain (CARD) (42Fesik S.W. Cell. 2000; 103: 273-282Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar). To gain insights into the mechanism by which v-FLIP inhibits death receptor signaling, we determined the crystal structure of MC159 at 1.4-Å resolution. This structure reveals a number of unexpected findings that significantly assist the deciphering of mechanisms by which v-FLIP and c-FLIPS function. Protein Purification—A cDNA fragment encoding MC159-(1-183) was subcloned into pGEX-2T (Amersham Biosciences) and transformed into BL21(DE3) Escherichia coli-competent cells. Cells were grown to an A600 of 1.0 at 37 °C before induction with 0.5 mm isopropyl 1-thio-β-d-galactopyranoside at 23 °C overnight. The N-terminal glutathione S-transferase-tagged MC159 protein was purified as previously described (43Chai J. Shiozaki E. Srinivasula S.M. Wu Q. Datta P. Alnemri E.S. Shi Y. Cell. 2001; 104: 769-780Abstract Full Text Full Text PDF PubMed Scopus (491) Google Scholar). The preparation of the seleno-methionine (SeMet)-substituted MC159 (residues 1-183) was as described (44Li W. Srinivasula S.M. Chai J. Li P. Wu J.-W. Zhang Z. Alnemri E.S. Shi Y. Nat. Struct. Biol. 2002; 9: 436-441Crossref PubMed Scopus (249) Google Scholar). Crystallization and Data Collection—Similar rod-shaped crystals of native and SeMet MC159 protein (residues 1-183) were grown overnight at room temperature using the hanging drop vapor diffusion method. The best SeMet crystals were generated by streak seeding under the condition of 0.2 m sodium acetate, 0.1 m Tris hydrochloride, pH 8.5, and 14% polyethylene glycol 3350, with 0.5 μl of 1 m ammonium sulfate additive. Data were collected at the X25 beam line of Brookhaven National Synchrotron Light Source. MAD data were collected at the Selenium K edge to a maximum resolution of 1.5 Å. A native data set was collected at 1.4 Å using a crystal grown at the same conditions as the SeMet crystals except 16% (w/v) polyethylene glycol 3350 was used. Both crystals were cryoprotected in a buffer containing 0.2 m sodium acetate, 0.1 m Tris hydrochloride, pH 8.5, 18% w/v polyethylene glycol 3350, 0.15 m ammonium acetate, and 10% glycerol. The crystals had a primitive orthorhombic space group with unit cell dimensions of a = 35 Å, b = 63 Å, c = 76 Å, and a = b = c = 90°. Unit cell dimensions were consistent with one molecule per asymmetric unit. Structure Determination—For the MAD experiments, data were processed using the HKL suite of programs (45Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38592) Google Scholar). Systematic absences for reflections of classes h00, 0k0, and 00l in both the SeMet and native data strongly indicated that the space group was P212121. The SHELX suite of programs (46Sheldrick G.M. Acta Crystallogr. Sect. A. 1990; 46: 467-473Crossref Scopus (19249) Google Scholar) was used to extract experimental phase information. The MAD data were prepared for structure determination with SHELXC, and four out of five possible selenium atom sites were found by SHELXD. The contrast in the solvent-flattened electron density maps in SHELXE unambiguously distinguished between the correct and inverted heavy atom locations (contrast of 0.445 and 0.268, respectively). The electron density map, generated from SHELXE, was clearly interpretable, and given the high resolution of the MAD data, the structure was automatically build from solvent-flattened MAD phases calculated to 1.5-Å resolution using the program ARP/wARP (47Perrakis A. Morris R. Lamzin V.S. Nat. Struct. Biol. 1999; 6: 458-463Crossref PubMed Scopus (2563) Google Scholar). Further manual building using the program O (48Jones T.A. Zou J.-Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. A. 1991; 47: 110-119Crossref PubMed Scopus (13014) Google Scholar) and refinement using the program CNS (49Brunger A.T. Adams P.D. Clore G.M. Delano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16978) Google Scholar) completed the structure. The final atomic model of MC159, refined to 1.4-Å resolution, contains residues 7-183. There is no electron density for residues 1-6, and we presume that these residues are flexible and disordered in the crystals. Overall Structure of the Tandem DEDs of MC159—The full-length MC159 protein (residues 1-241) consists of two tandem DEDs and a C-terminal extension. Both sequence analysis and limited proteolysis indicated that the C-terminal extension of MC159 is flexible and disordered in solution. Because flexible sequences may impede crystallization, we generated and purified a truncated version (residues 1-183) of MC159, which contains the two tandem DEDs but lacks the C-terminal extension. We crystallized the truncated MC159 and determined its structure using multiwavelength anomalous dispersion (MAD). The atomic model was refined to 1.4-Å resolution (Table 1).TABLE 1Statistics of crystallographic analysisSeMetData collectionNativePeakInflectionHigh remoteLow remoteWavelength (Å)0.97950.97900.97940.96410.9800Resolution (Å)50-1.450-1.7550-1.7550-1.7550-1.5Total observations153,552102,394102,687102,27796,746Unique reflections33,49517,76417,71717,70828,066R-sym (%)3.67.36.86.84.6Completeness (%)98.099.398.998.899.4RefinementResolution (Å)50-1.450-1.5Reflections (|F| > 0)31,04426267Completeness (%)91.293.4R-factor (%)20.619.5R-free (%)23.621.7No. atoms13761376No. waters174175r.m.s.d. bond lengths0.0140.014r.m.s.d. bond angles1.621.64r.m.s.d. B-factors2.802.61Ramachandran plotMost favored (%)95.198.3 Open table in a new tab The truncated MC159 adopts a dumbbell-shaped structure, with the two DEDs at two opposing ends (Fig. 1, A and B). The two DED domains can be superimposed onto each other with a root-mean-squared deviation (r.m.s.d.) of 1.78 Å over 60 aligned Cα atoms (Fig. 1C). The first DED domain (DED1, residues 7-78) of MC159 only contains five α-helices and represents a departure from the canonical death motif fold (Fig. 1A). Consequently, DED1 of MC159 is superimposed with the DED of FADD (37Eberstadt M. Huang B. Chen Z. Meadows R.P. Ng S.-C. Zheng L. Lenardo M.J. Fesik S.W. Nature. 1998; 392: 941-945Crossref PubMed Scopus (204) Google Scholar) with an r.m.s.d. of 1.84 Å over only 45 aligned Cα atoms. These 45 aligned amino acids come from helices α1a, α2a, α5a, and α6a in DED1. Compared with the DED of FADD (37Eberstadt M. Huang B. Chen Z. Meadows R.P. Ng S.-C. Zheng L. Lenardo M.J. Fesik S.W. Nature. 1998; 392: 941-945Crossref PubMed Scopus (204) Google Scholar) and the second DED (DED2, residues 93-183) of MC159, the third α-helix is reduced to a rigid loop in DED1 of MC159. Sequence analysis suggests that this structural feature might be unique to MC159, because, compared with FADD and other proteins, MC159 lacks a few conserved residues in the region that corresponds to the α3a helix (Fig. 2). In contrast, the second DED domain (DED2, residues 93-183) of MC159 can be superimposed with the DED of FADD with an r.m.s.d. of 1.57 Å over 80 aligned Cα atoms, which cover all six α-helices and most of the intervening loop sequences (Fig. 1D).FIGURE 2Structure-based sequence alignment of the DED regions from v-FLIPs, c-FLIP, FADD, and caspase-8 and caspase-10. Secondary structural elements are indicated above the MC159 sequences. Conserved residues are highlighted in yellow. The conserved binding elements on the surface of DED1 and DED2 are indicated, with the residues under red background. Residues that contribute to the interactions between DED1 and DED2 are identified with blue triangles. The highly conserved residues that play important stabilizing roles at the DED interface are identified by black dots underneath the sequences. The effect of alanine-scanning mutagenesis is indicated by colored squares above the sequences: red, loss-of-function; yellow, partial loss-of-function; green, no effect (52Garvey T.L. Bertin J. Siegel R.M. Wang G.H. Lenardo M.J. Cohen J.I. J. Virol. 2002; 76: 697-706Crossref PubMed Scopus (69) Google Scholar). Amino acid residues that are > 40% solvent-exposed, 10-40% solvent-exposed, and <10% solvent-exposed are indicated by open, half-filled, and filled circles above the sequences, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT) DED1 and DED2 are connected by a stretch of 14 amino acids (residues 79-92), which form a short helix α7 and two surface loops. Unexpectedly, the two DED domains of MC159 rigidly associate with each other through a hydrophobic interface, involving ∼1450 Å 2The abbreviations used are: DISC, death-inducing signaling complex; TNFR1, tumor necrosis factor receptor 1; TRAIL, TNF-related apoptosis-inducing ligand; DD, death domain; DED, death effector domain; CARD, caspase recruitment domain; SeMet, seleno-methionine; MAD, multiwavelength anomalous dispersion; r.m.s.d., root-mean-square deviation. of buried surface area. The close stacking between the two DED domains of MC159 creates a deep surface groove that encircles the molecule (Fig. 1B). This surface groove is lined with a number of acidic amino acids that give rise to a highly negatively charged surface. In contrast, the two opposing ends of the MC159 structure are less charged and contain a number of hydrophobic residues. Conserved Interactions at the DED1-DED2 Interface—Of the three classes of signaling motifs in apoptosis, only DED is known to occur in tandem, and the tandem DEDs are present in all known v-FLIPs, c-FLIPL, c-FLIPS, caspase-8, and caspase-10 (Fig. 2). One important finding from the structure of MC159 is the unexpected revelation that the two tandem DED domains pack closely against each other through a large hydrophobic interface. At this interface, α2a in DED1 plays a dominant role by binding to α1b and α4b of DED2 (Figs. 2 and 3A). Another helix from DED1, α5a, also contributes to this interface by making additional interactions at the periphery (Fig. 3A). At the center of the interface, Phe-30 and Leu-31, both from α2a in DED1, make multiple van der Waals contacts to a complementary hydrophobic surface in DED2, formed by Val-101, Val-137, and Leu-141 and the aliphatic portion of side chains of Arg-97 and Lys-98 (Fig. 3A). At the periphery, Leu-27 and Val-66 in DED1 make additional interactions with hydrophobic residues in DED2. These hydrophobic interactions are further buttressed by five inter-domain hydrogen bonds (Fig. 3A). In particular, Asp-34 in DED1 accepts a pair of charge-stabilized hydrogen bonds from Arg-97 in DED2. Structural and sequence analysis indicates that the interactions at the DED1-DED2 interface are highly conserved among all proteins that contain tandem DED domains (Figs. 2 and 3A). Among the residues that mediate interactions at the center of the interface, Phe-30, Leu-31, Arg-97, Val-137, and Leu-141, are either preserved or replaced by similar residues in all proteins that contain tandem DED domains (Fig. 2). Moreover, the hydrogen bonds between Asp-34 and Arg-97 are also predicted to be conserved in a majority of these proteins (Fig. 2). This analysis strongly suggests that the rigid structure of MC159 and the interface determinants between the tandem DEDs are representative of v-FLIPs, c-FLIP, caspase-8, and caspase-10. Similarity with CARD-CARD Interactions—Because there is no prior structural information on any DED·DED complex or on any tandem DED-containing protein, we initially assumed that the packing arrangement between the two DED domains of MC159 might represent a novel arrangement. To confirm this assumption, we used the atomic coordinates of the MC159 protein to search the protein data base using the program DALI (50Holm L. Sander C. J. Mol. Biol. 1993; 233: 123-138Crossref PubMed Scopus (3566) Google Scholar) as well as our own automated homology search program (P. D. J.). To our surprise, the packing interactions between the two DEDs of MC159 were found to be significantly homologous to those between the CARD domains of Apaf-1 and caspase-9 (51Qin H. Srinivasula S.M. Wu G. Fernandes-Alnemri T. Alnemri E.S. Shi Y. Nature. 1999; 399: 547-555Crossref Scopus (362) Google Scholar) (Fig. 3B), with an r.m.s.d. of 1.8 Å over 85 aligned Cα atoms in both domains. The relative domain orientation and the interface are both very similar. In both cases, the α2 helix in DED1 of MC159 or in Apaf-1 CARD plays a dominant role in binding to helices α1 and α4 in DED2 of MC159 or in caspase-9 CARD (51Qin H. Srinivasula S.M. Wu G. Fernandes-Alnemri T. Alnemri E.S. Shi Y. Nature. 1999; 399: 547-555Crossref Scopus (362) Google Scholar). The inter-domain hydrogen bonds from Asp-34 of DED1 to Arg-97 of DED2 (Fig. 3A) are also reminiscent of the CARD-CARD interactions, where acidic residues from Apaf-1 hydrogen bond to basic residues in caspase-9 (51Qin H. Srinivasula S.M. Wu G. Fernandes-Alnemri T. Alnemri E.S. Shi Y. Nature. 1999; 399: 547-555Crossref Scopus (362) Google Scholar). This analysis indicates that, despite a lack of apparent sequence homology, the DED1-DED2 interface observed in MC159 is very similar to the CARD-CARD interface between Apaf-1 and caspase-9. This observation further suggests that the tandem DED domains in FLIP and caspases might have arisen from gene transfer (from another interacting protein) rather than gene duplication (within the same protein). Classification of Mutations in MC159—As one of the first v-FLIP proteins to be identified, MC159 had been subjected to intense investigation. In particular, an extensive alanine-scanning mutagenesis was performed on MC159 to identify important amino acids that are critical for its anti-apoptotic function in vivo (52Garvey T.L. Bertin J. Siegel R.M. Wang G.H. Lenardo M.J. Cohen J.I. J. Virol. 2002; 76: 697-706Crossref PubMed Scopus (69) Google Scholar). This effort led to the identification of a comprehensive set of amino acids in both DEDs that are required to protect HeLa and Jurkat cells from apoptosis induced by TNFR1, Fas, and TRAIL receptors (52Garvey T.L. Bertin J. Siegel R.M. Wang G.H. Lenardo M.J. Cohen J.I. J. Virol. 2002; 76: 697-706Crossref PubMed Scopus (69) Google Scholar). In conjunction with the structure, the mutagenesis information gives us an unprecedented opportunity to map the critical residues onto the surface of MC159 and to further identify critical surface motifs necessary for the anti-apoptotic function of MC159. The mutations may disrupt the function of MC159 by two distinct possibilities: to destabilize the structure or to affect a functional surface that is required for binding to other protein(s). To differentiate between these two possibilities, we first examined the solvent accessibility of all residues that were targeted for alanine scanning mutagenesis (Fig. 2) and classified the mutations into two categories: structural or non-structural (Table 2). Then we correlated the classification with the in vivo phenotypes (Table 2) and mapped all mutations onto the surface of the MC159 structure (Fig. 4, A and B). As expected, some mutations affect residues that are deeply buried, such as mutant 5 (E62A), mutant 17 (F30A), mutant 21 (L72A/L73A), mutant 24 (L31A), and mutant 40 (L28A/L29A/L31) (Table 2). These mutations are likely to destabilize the structure of MC159, thus crippling its function. Interestingly, amo" @default.
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• W1985909474 title "Crystal Structure of a Viral FLIP" @default.
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