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• W2017086893 abstract "TRAIL is a cytokine that induces apoptosis in a wide variety of tumor cells but rarely in normal cells. It contains an extraordinarily elongated loop because of an unique insertion of 12–16 amino acids compared with the other members of tumor necrosis factor family. Biological implication of the frame insertion has not been clarified. We have determined the crystal structure of TRAIL in a complex with the extracellular domain of death receptor DR5 at 2.2 Å resolution. The structure reveals extensive contacts between the elongated loop and DR5 in an interaction mode that would not be allowed without the frame insertion. These interactions are missing in the structures of the complex determined by others recently. This observation, along with structure-inspired deletion analysis, identifies the critical role of the frame insertion as a molecular strategy conferring specificity upon the recognition of cognate receptors. The structure also suggests that a built-in flexibility of the tumor necrosis factor receptor family members is likely to play a general and important role in the binding and recognition of tumor necrosis factor family members. TRAIL is a cytokine that induces apoptosis in a wide variety of tumor cells but rarely in normal cells. It contains an extraordinarily elongated loop because of an unique insertion of 12–16 amino acids compared with the other members of tumor necrosis factor family. Biological implication of the frame insertion has not been clarified. We have determined the crystal structure of TRAIL in a complex with the extracellular domain of death receptor DR5 at 2.2 Å resolution. The structure reveals extensive contacts between the elongated loop and DR5 in an interaction mode that would not be allowed without the frame insertion. These interactions are missing in the structures of the complex determined by others recently. This observation, along with structure-inspired deletion analysis, identifies the critical role of the frame insertion as a molecular strategy conferring specificity upon the recognition of cognate receptors. The structure also suggests that a built-in flexibility of the tumor necrosis factor receptor family members is likely to play a general and important role in the binding and recognition of tumor necrosis factor family members. tumor necrosis factor TNF receptor cysteine-rich domain The TNF1 and the TNF receptor (TNFR) superfamilies play important roles in regulating many biological functions, especially as prominent mediators of immune regulation, inflammatory responses, bone development, and homeostasis (1Cosman D. Stem Cells. 1994; 12: 440-455Crossref PubMed Scopus (119) Google Scholar, 2Lotz M. Setareh M. Kempis J. Schwarz H. J. Leukocyte Biol. 1996; 60: 1-7Crossref PubMed Scopus (57) Google Scholar, 3Simonet W.S. Lacey D.L. Dunstan C.R. Kelley M. Chang M.S. Luthy R. Nguyen H.Q. Wooden S. Bennett L. Boone T. Shimamoto G. DeRose M. Elliott R. Colombero A. Tan H.L. Trail G. Sullivan J. Davy E. Bucay N. Renshaw-Gegg L. Hughes T.M. Hill D. Pattison W. Campbell P. Sander S. Van G. Tarpley J. Derby P. Lee R. Boyle W.J. Cell. 1997; 89: 309-319Abstract Full Text Full Text PDF PubMed Scopus (4261) Google Scholar, 4Wong B.R. Josien R. Choi Y. J. Leukocyte Biol. 1999; 65: 715-724Crossref PubMed Scopus (198) Google Scholar). Ligands belonging to the TNF family are expected to function as a homotrimer as suggested by the crystal structures of a subset of the family (TNFα, TNFβ, CD40L, and TRAIL). The monomeric unit of these proteins contains two β-pleated sheets, which form a β-sandwich conforming to the jellyroll topology. The TNFR family members are transmembrane proteins with several exceptions that contain an extracellular domain only. The extracellular domain of the receptors is characterized by the concatenated cysteine-rich domains (CRDs) (5Bazan J.F. Curr. Biol. 1993; 3: 603-606Abstract Full Text PDF PubMed Scopus (94) Google Scholar) that are responsible for ligand binding. Although most of the intracellular domains of the receptor family members are not conserved, those of a subset of the receptors contain a common death domain. It is a protein-protein interaction motif that recruits cellular partners and ultimately activates a protease cascade leading to apoptosis. Despite the same structural scaffolds, namely the β-sandwich for the TNF family and the concatenated CRDs for the TNFR family, the recognition between cognate ligands and receptors is achieved in a highly specific manner with typical dissociation constants in nanomolar range. TRAIL is a recently identified member of the TNF family (6Wiley S.R. Schooley K. Smolak P.J. Din W.S. Huang C.P. Nicholl J.K. Sutherland G.R. Smith T.D. Rauch C. Smith C.A. Immunity. 1995; 3: 673-682Abstract Full Text PDF PubMed Scopus (2627) Google Scholar, 7Pitti R.M. Marsters S.A. Ruppert S. Donahue C.J. Moore A. Ashkenazi A. J. Biol. Chem. 1996; 271: 12687-12690Abstract Full Text Full Text PDF PubMed Scopus (1629) Google Scholar). It is a type II membrane protein that is processed proteolytically at the cell surface to form a soluble ligand (residues 114–281) (8Mariani S.M. Krammer P.H. Eur. J. Immunol. 1998; 28: 1492-1498Crossref PubMed Scopus (154) Google Scholar). An unique feature of TRAIL distinguished from those of other TNF family members is an insertion of 12–16 amino acids (depending on the proteins compared with) near the N terminus (9Cha S.-S. Kim M.-S. Choi Y.H. Sung B.-J. Shin N.K. Shin H.-C. Sung Y.C. Oh B.-H. Immunity. 1999; 11: 253-261Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar), called AA loop, which is previously known to be important for the receptor binding of TNFs (10Banner D.W. D'Arcy A. Janes W. Gentz R. Schoenfeld H.J. Broger C. Loetscher H. Lesslauer W. Cell. 1993; 73: 431-445Abstract Full Text PDF PubMed Scopus (971) Google Scholar, 11Van Ostade X. Tavernier J. Fiers W. Protein Eng. 1994; 7: 5-22Crossref PubMed Scopus (88) Google Scholar). TRAIL interacts with at least four different receptors, which share a high sequence homology in the extracellular domains. Two of these are apoptosis-inducing receptors, DR4 (12Pan G. O'Rourke K. Chinnaiyan A.M. Gentz R. Ebner R. Ni J. Dixit V.M. Science. 1997; 276: 111-113Crossref PubMed Scopus (1538) Google Scholar) and DR5 (13Pan G. Ni J. Wei Y.F., Yu, G. Gentz R. Dixit V.M. Science. 1997; 277: 815-818Crossref PubMed Scopus (1369) Google Scholar, 14Sheridan J.P. Marsters S.A. Pitti R.M. Gurney A. Skubatch M. Baldwin D. Ramakrishnan L. Gray C.L. Baker K. Wood W.I. Goddard A.D. Godowski P. Ashkenazi A. Science. 1997; 277: 818-821Crossref PubMed Scopus (1512) Google Scholar, 15Walczak H. Degli-Esposti M.A. Johnson R.S. Smolak P.J. Waugh J.Y. Boiani N. Timour M.S. Gerhart M.J. Schooley K.A. Smith C.A. Goodwin R.G. Rauch C.T. EMBO J. 1997; 16: 5386-5397Crossref PubMed Scopus (1009) Google Scholar), both of which contain an intracellular death domain. The other two are nonsignaling decoy receptors, DcR1 (13Pan G. Ni J. Wei Y.F., Yu, G. Gentz R. Dixit V.M. Science. 1997; 277: 815-818Crossref PubMed Scopus (1369) Google Scholar, 14Sheridan J.P. Marsters S.A. Pitti R.M. Gurney A. Skubatch M. Baldwin D. Ramakrishnan L. Gray C.L. Baker K. Wood W.I. Goddard A.D. Godowski P. Ashkenazi A. Science. 1997; 277: 818-821Crossref PubMed Scopus (1512) Google Scholar, 16Degli-Esposti M.A. Smolak P.J. Walczak H. Waugh J. Huang C.P. DuBose R.F. Goodwin R.G. Smith C.A. J. Exp. Med. 1997; 186: 1165-1170Crossref PubMed Scopus (554) Google Scholar) and DcR2 (17Marsters S.A. Sheridan J.P. Pitti R.M. Huang A. Skubatch M. Baldwin D. Yuan J. Gurney A. Goddard A.D. Godowski P. Ashkenazi A. Curr. Biol. 1997; 7: 1003-1006Abstract Full Text Full Text PDF PubMed Scopus (570) Google Scholar, 18Degli-Esposti M.A. Dougall W.C. Smolak P.J. Waugh J.Y. Smith C.A. Goodwin R.G. Immunity. 1997; 7: 813-820Abstract Full Text Full Text PDF PubMed Scopus (738) Google Scholar), which can bind to TRAIL but cannot trigger apoptosis. DcR1 lacks a cytoplasmic domain entirely (19Schneider P. Bodmer J.L. Thome M. Hofmann K. Holler N. Tschopp J. FEBS Lett. 1997; 416: 329-334Crossref PubMed Scopus (245) Google Scholar), whereas DcR2 has a nonfunctional truncated death domain (14Sheridan J.P. Marsters S.A. Pitti R.M. Gurney A. Skubatch M. Baldwin D. Ramakrishnan L. Gray C.L. Baker K. Wood W.I. Goddard A.D. Godowski P. Ashkenazi A. Science. 1997; 277: 818-821Crossref PubMed Scopus (1512) Google Scholar, 20Pan G. Ni J., Yu, G. Wei Y.F. Dixit V.M. FEBS Lett. 1998; 424: 41-45Crossref PubMed Scopus (277) Google Scholar). Another receptor, osteoprotegerin, binds to TRAIL with a dissociation constant of 400 nm, which is the lowest affinity of the known TRAIL receptors (21Truneh A. Sharma S. Silverman C. Khandekar S. Reddy M.P. Deen K.C. McLaughlin M.M. Srinivasula S.M. Livi G.P. Marshall L.A. Alnemri E.S. Williams W.V. Doyle M.L. J. Biol. Chem. 2000; 275: 23319-23325Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar). In contrast to TNFs and FasL, TRAIL has been known to induce apoptosis in a variety of tumor cells and some virally infected cells but not in normal cells (6Wiley S.R. Schooley K. Smolak P.J. Din W.S. Huang C.P. Nicholl J.K. Sutherland G.R. Smith T.D. Rauch C. Smith C.A. Immunity. 1995; 3: 673-682Abstract Full Text PDF PubMed Scopus (2627) Google Scholar, 7Pitti R.M. Marsters S.A. Ruppert S. Donahue C.J. Moore A. Ashkenazi A. J. Biol. Chem. 1996; 271: 12687-12690Abstract Full Text Full Text PDF PubMed Scopus (1629) Google Scholar). Consistently, administration of TRAIL to mice bearing human tumors actively suppressed tumor progression (22Walczak H. Miller R.E. Ariail K. Gliniak B. Griffith T.S. Kubin M. Chin W. Jones J. Woodward A. Le T. Smith C. Smolak P. Goodwin R.G. Rauch C.T. Schuh J.C. Lynch D.H. Nat. Med. 1999; 5: 157-163Crossref PubMed Scopus (2201) Google Scholar) and improved survival of the animal (23Ashkenazi A. Pai R.C. Fong S. Leung S. Lawrence D.A. Marsters S.A. Blackie C. Chang L. McMurtrey A.E. Hebert A. DeForge L. Koumenis I.L. Lewis D. Harris L. Bussiere J. Koeppen H. Shahrokh Z. Schwall R.H. J. Clin. Invest. 1999; 104: 155-162Crossref PubMed Scopus (1982) Google Scholar). Furthermore, repeated intravenous injections of TRAIL in nonhuman primates did not cause detectable toxicity to normal tissues and organs, including liver tissues (23Ashkenazi A. Pai R.C. Fong S. Leung S. Lawrence D.A. Marsters S.A. Blackie C. Chang L. McMurtrey A.E. Hebert A. DeForge L. Koumenis I.L. Lewis D. Harris L. Bussiere J. Koeppen H. Shahrokh Z. Schwall R.H. J. Clin. Invest. 1999; 104: 155-162Crossref PubMed Scopus (1982) Google Scholar). However, susceptibility of human normal hepatocytes to TRAIL was reported very recently (24Jo M. Kim T.H. Seol D.W. Esplen J.E. Dorko K. Billiar T.R. Strom S.C. Nat. Med. 2000; 6: 564-567Crossref PubMed Scopus (762) Google Scholar). A working hypothesis for the selective antitumoral activity of TRAIL is that the decoy receptors are preferentially expressed in normal cells compared with tumor cells and interfere with the TRAIL action (13Pan G. Ni J. Wei Y.F., Yu, G. Gentz R. Dixit V.M. Science. 1997; 277: 815-818Crossref PubMed Scopus (1369) Google Scholar, 14Sheridan J.P. Marsters S.A. Pitti R.M. Gurney A. Skubatch M. Baldwin D. Ramakrishnan L. Gray C.L. Baker K. Wood W.I. Goddard A.D. Godowski P. Ashkenazi A. Science. 1997; 277: 818-821Crossref PubMed Scopus (1512) Google Scholar, 17Marsters S.A. Sheridan J.P. Pitti R.M. Huang A. Skubatch M. Baldwin D. Yuan J. Gurney A. Goddard A.D. Godowski P. Ashkenazi A. Curr. Biol. 1997; 7: 1003-1006Abstract Full Text Full Text PDF PubMed Scopus (570) Google Scholar, 25Mongkolsapaya J. Cowper A.E. Xu X.N. Morris G. McMichael A.J. Bell J.I. Screaton G.R. J. Immunol. 1998; 160: 3-6PubMed Google Scholar). The structure of TNFβ in complex with the extracellular domain of TNF-R55 (sTNFR55) had been determined (10Banner D.W. D'Arcy A. Janes W. Gentz R. Schoenfeld H.J. Broger C. Loetscher H. Lesslauer W. Cell. 1993; 73: 431-445Abstract Full Text PDF PubMed Scopus (971) Google Scholar). The structure of TRAIL-receptor complex should provide valuable insights into molecular recognition between the two families that are rapidly expanding. Recently, two crystal structures of TRAIL in complex with the extracellular domain of DR5 (sDR5) were reported (26Hymowitz S.G. Christinger H.W. Fuh G. Ultsch M. O'Connell M. Kelley R.F. Ashkenazi A. de Vos A.M. Mol. Cell. 1999; 4: 563-571Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar, 27Mongkolsapaya J. Grimes J.M. Chen N. Xu X.-N. Stuart D.I. Jones E.Y. Screaton G.R. Nat. Struct. Biol. 1999; 6: 1048-1053Crossref PubMed Scopus (229) Google Scholar) and provided detailed views of the ligand-receptor interactions. However, neither of them sheds light on biological implication of the frame-insertion in the AA loop. In this study, we independently determined the 2.2 Å crystal structure of TRAIL-sDR5 complex. It reveals extensive interactions of the AA loop with the receptor, whereas previous structures show virtually no interaction between the two. The newly revealed binding mode accurately reflects previous and a new deletion analyses of the loop and enables us to correctly highlight molecular strategies for controlling specificity between members of the TNF and TNFR superfamilies. Active human TRAIL (residues 114–281) was expressed and purified as a soluble form fromEscherichia coli (9Cha S.-S. Kim M.-S. Choi Y.H. Sung B.-J. Shin N.K. Shin H.-C. Sung Y.C. Oh B.-H. Immunity. 1999; 11: 253-261Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). sDR5 gene (encoding residues 1-130) was amplified by polymerase chain reaction from human fetal cDNA library. The gene was inserted into the downstream of the T7 promoter on the expression plasmid pET-21b(+), and the plasmid was introduced in E. coli strain BL21 (DE3). Cells were grown to an A 600 of approximately 0.4 in Luria-Bertani medium containing 0.1 mg/ml ampicillin at 37 °C, and the expression of sDR5 was induced by 0.5 mmisopropyl-β-d-thiogalactoside. After 7 h induction at 27 °C, cells were harvested and resuspended in a 20 mm sodium phosphate buffer (pH 7.5) and disrupted by sonication. After centrifugation, supernatants were consecutively loaded on nickel-nitrilotriacetic acid (Qiagen) and Hi-Trap Q (Amersham Pharmacia Biotech) columns. The purified TRAIL and sDR5 were mixed in 1:1 molar ratio. After 12 h of incubation at 4 °C, the mixture was loaded on Superdex 200 HR 10/30 sizing column (Amersham Pharmacia Biotech). The fractions containing the TRAIL-sDR5 complex were collected and used for crystallization. Crystals of the complex were obtained using a precipitant solution containing 16% polyethylene glycol 3000, 0.05 m sodium acetate (pH 4.5), 0.55m sodium acetate, and 0.6 m NaCl. The crystals belonged to the space group P21 with cell dimensions a = 68.63, b = 124.81,c = 128.37 Å, and β = 104.49°. The asymmetric unit contained two trimeric complexes of TRAIL and sDR5. A 2.2 Å data set was collected using x-ray beam (λ= 1.0 Å) from the BL6A beamline at the Photon Factory (Japan) and processed with the programs DENZO and SCALEPACK (32Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref Scopus (38253) Google Scholar) (see Table I). For data collection, the crystal was frozen at 100 K after being briefly immersed in a cryoprotectant solution containing 15% glycerol, 16% polyethylene glycol 3000, 0.05m sodium acetate (pH 4.5), 0.3 m sodium acetate, and 0.3 m NaCl. With the uncomplexed TRAIL structure (9Cha S.-S. Kim M.-S. Choi Y.H. Sung B.-J. Shin N.K. Shin H.-C. Sung Y.C. Oh B.-H. Immunity. 1999; 11: 253-261Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar) as a search model, two promising solutions were found using the CCP4 version of AMoRe. The favorable crystal packing of the two solutions with marginal space for sDR5 molecules confirmed the correctness of the solutions. The initial electron densities obtained by solvent flattening and 2-fold noncrystallographic symmetry density averaging using the CCP4 suite (33CCP4 Acta Crystallogr. D. 1994; 50: 760-763Crossref PubMed Scopus (19668) Google Scholar) allowed us to build a partial sDR5 model. As the manual fitting and structure refinement were in progress, weak but continuous electron densities extended from Arg-130 became stronger, obviously showing features of the side chains of residues 130–135 on the AA loop. Crystallographic refinements, iterative map calculations, and model building were done using the programs X-PLOR (34Brünger, A. T. (1992) X-PLOR, Version 3.843. Yale University Press, New Haven, CT.Google Scholar) and program O (35Jones T.A. Kjeldgaard M. O , version 5.9. Uppsala University, Uppsala, Sweden1993Google Scholar). The 2-fold noncrystallographic symmetry restraints were maintained only for TRAIL until the last refinement cycle. From the beginning of the refinement, 5% of the total reflections were set aside for monitoring R freevalue. The final crystallographic statistics are shown in Table I.Table ICrystal structure determination and refinement statisticsData collection and refinement (F > 1ς)Resolution (Å)2.20R sym (%)6.1Completeness (F > 1ς)93.1R factor21.2R free29.1Number of protein atoms12,837Water molecules162Metal ion2Average B-factor (Å2)21.81Rmsd bond lengths (Å)0.011Rmsd bond angles (degree)1.772Ramachandran plot (%) Most favored region77 Additionally allowed region23Disallowed region0R sym = Σ‖I obs −I avg‖/ΣI obs, and Rfactor = Σ‖F o −F c‖/ΣF o. TheR free was calculated with 5% of the data. Open table in a new tab R sym = Σ‖I obs −I avg‖/ΣI obs, and Rfactor = Σ‖F o −F c‖/ΣF o. TheR free was calculated with 5% of the data. TRAIL deletion mutant (Δ132–135) was constructed using the overlapping polymerase chain reaction method as reported (36Du Z. Regier D.A. Desrosiers R.C. BioTechniques. 1995; 18: 376-378PubMed Google Scholar). The correct construction and expression were verified by nucleotide sequencing, mass spectrometry, and circular dichroism spectroscopy. Measurement of the apparent K D values between sDR5 and wild type and Δ132–135 TRAIL was carried out using a BIAcore 2000 biosensor (Biosensor, Sweden). sDR5 (30 μg/ml in 10 mm sodium acetate, pH 4.6) was covalently bound to the carboxylated dextran matrix at a concentration of 998 response units by an amine coupling method as suggested by the manufacturer. A flow path involving two cells was employed to simultaneously measure kinetic parameters from one flow cell containing sDR5-immobilized sensor chip and the other flow cell containing an underivatized chip. For kinetic measurements, TRAIL samples, ranging from 300 to 900 nm were prepared by dilution with the HBS buffer containing 150 mm NaCl, 3 mm EDTA, 0.005% polysorbate, and 10 mm HEPES (pH 7.4). Injection of each of 80 μl of TRAIL solution into the flow cells (association phase) was followed by the flow of the HBS buffer (dissociation phase), both at 30 μl/min. Between cycles, the immobilized ligand was regenerated by injecting 30 μl of 10 mm sodium phosphate buffer (pH 7.5) containing 1.0m NaCl at 10 μl/min. All experiments were performed at 25 °C. The kinetic parameters were determined by nonlinear regression analysis according to 1:1 binding model using the BIAevaluation version 2.1 software provided by the manufacturer. The cytotoxic activity of TRAIL variants were measured on human SK-HEP-1 hepatoma cells (American Type Culture Collection HTB-52). Briefly, a total of 50 μl of the cells in 10% fetal bovine serum and Dulbecco's modified Eagle's medium was transferred to individual wells of 96-well microplates at a density of 1 × 104 cells/well. Wild type or the mutant TRAIL diluted in the 50 μl of the same medium was added to the wells at various concentrations (0–1 μg/ml), and the cells were incubated for 24 h at 37 °C. Cell viability was determined by measuring the cellular metabolic activity with 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (thiazoyl blue, Sigma-Aldrich) added to the medium at a concentration of 0.5 mg/ml. Absorbance was measured at 540 nm. We obtained the crystals of the TRAIL-sDR5 complex whose crystal packing is totally different from those of the other two crystals of the complex reported earlier (26Hymowitz S.G. Christinger H.W. Fuh G. Ultsch M. O'Connell M. Kelley R.F. Ashkenazi A. de Vos A.M. Mol. Cell. 1999; 4: 563-571Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar, 27Mongkolsapaya J. Grimes J.M. Chen N. Xu X.-N. Stuart D.I. Jones E.Y. Screaton G.R. Nat. Struct. Biol. 1999; 6: 1048-1053Crossref PubMed Scopus (229) Google Scholar). The crystals contained six molecules of TRAIL and sDR5 (two trimeric complexes) in the asymmetric unit and provided six independent views. The structure of the complex was determined by molecular replacement (TableI) and refined to 2.2 Å resolution. The final model consists of TRAIL residues 120–135 and 146–281 in each monomer, sDR5 residues 21–128 of two copies, and residues 21–102 and 116–123 of the other four copies, two metal ions, and 162 water molecules. Residues 1–20 of sDR5 are completely disordered. In this section, we mainly describe the new findings obtained from this study and their biological implications. However, we refrain from describing structural features common in the three complex structures such as the interactions between TRAIL and sDR5 at the lower and upper contact regions (see below). The overall structure of TRAIL in a complex with sDR5 is virtually the same as that of the uncomplexed TRAIL structure we reported earlier (9Cha S.-S. Kim M.-S. Choi Y.H. Sung B.-J. Shin N.K. Shin H.-C. Sung Y.C. Oh B.-H. Immunity. 1999; 11: 253-261Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). A TRAIL monomer contains two antiparallel β-pleated sheets that form a β-sandwich as a core scaffold and interacts with the adjacent subunits in a head-to-tail fashion to form a bell-shaped homotrimer. Structural variation between the uncomplexed and complexed TRAIL structures is concentrated in the loop regions, with dramatic differences observed in the AA, CD, and EF loops (Fig. 1). The CD and EF loops that are disordered in the uncomplexed structure become ordered on binding to sDR5. The AA loop (residues 130–160) displays the most remarkable structural changes. Although the conformation of residues 146–160, which interact heavily with the core scaffold, is well maintained, the flexible segment composed of residues 130–145 (9Cha S.-S. Kim M.-S. Choi Y.H. Sung B.-J. Shin N.K. Shin H.-C. Sung Y.C. Oh B.-H. Immunity. 1999; 11: 253-261Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar) undergoes a drastic positional change (Fig. 1). The initial part of the segment penetrates into the receptor-binding site and interacts with sDR5 (Fig.1 B). This translocation is manifested by the strong electron densities observed for residues 130–135 of all the six TRAIL molecules in the asymmetric unit. A TRAIL monomer contains a single cysteine, Cys-230. The cysteine residues from the three monomers are close to each other and form a triangular geometry with the three cysteinyl sulfur atoms as apexes. This triangular arrangement is perpendicular to the molecular 3-fold axis. A strong electron density observed near the cysteinyl sulfur atoms at the first cycle of the structure refinements indicated a metal binding. This is different from the structure of uncomplexed TRAIL (9Cha S.-S. Kim M.-S. Choi Y.H. Sung B.-J. Shin N.K. Shin H.-C. Sung Y.C. Oh B.-H. Immunity. 1999; 11: 253-261Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar) and that of TRAIL-sDR5 complex (Protein Data Bank code 1D4V) of Mongkolsapaya et al. (27Mongkolsapaya J. Grimes J.M. Chen N. Xu X.-N. Stuart D.I. Jones E.Y. Screaton G.R. Nat. Struct. Biol. 1999; 6: 1048-1053Crossref PubMed Scopus (229) Google Scholar), both of which were determined with refolded TRAIL. It is, however, consistent with the structure of TRAIL-sDR5 complex (Protein Data Bank code 1D0G) of Hymowitz et al. (26Hymowitz S.G. Christinger H.W. Fuh G. Ultsch M. O'Connell M. Kelley R.F. Ashkenazi A. de Vos A.M. Mol. Cell. 1999; 4: 563-571Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar) in which the three cysteines chelate Zn2+ with the fourth coordination arm, Cl−. Removal of the Zn2+ by chelating agents was shown to result in a 90-fold decrease in apoptotic activity and a marked decrease in melting temperature (28Hymowitz S.G. O'Connell M.P. Ultsch M.H. Hurst A. Totpal K. Ashkenazi A. de Vos A.M. Kelley R.F. Biochemistry. 2000; 39: 633-640Crossref PubMed Scopus (226) Google Scholar). Previously, we reported that refolded Zn2+-unbound TRAIL slowly converted into an inactive monomeric or dimeric form (9Cha S.-S. Kim M.-S. Choi Y.H. Sung B.-J. Shin N.K. Shin H.-C. Sung Y.C. Oh B.-H. Immunity. 1999; 11: 253-261Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). Although the zinc site is buried in the trimeric interface (Fig. 1), and thus Zn2+binding cannot directly influence the binding affinity of TRAIL for the receptors, it must be critical for maintaining the active trimeric structure of the protein (26Hymowitz S.G. Christinger H.W. Fuh G. Ultsch M. O'Connell M. Kelley R.F. Ashkenazi A. de Vos A.M. Mol. Cell. 1999; 4: 563-571Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar). The TRAIL receptors contain two repeats of extracellular CRDs, whereas other TNFR family members contain three or more CRDs (29Smith C.A. Farrah T. Goodwin R.G. Cell. 1994; 76: 959-962Abstract Full Text PDF PubMed Scopus (1829) Google Scholar). sTNFR55 is composed of four CRDs; two central CRDs corresponding to the CRDs of TRAIL receptors, the N-terminal and the C-terminal CRDs. The basic repeating structural unit of the extracellular domain is a set of smaller modules that are the building blocks of CRDs (5Bazan J.F. Curr. Biol. 1993; 3: 603-606Abstract Full Text PDF PubMed Scopus (94) Google Scholar, 30Naismith J.H. Sprang S.R. Trends Biochem. Sci. 1998; 23: 74-79Abstract Full Text PDF PubMed Scopus (187) Google Scholar). sDR5 has modular composition of N1 (residues 28–41), A1+B2 (CRD1; residues 44–84), and A1+B2 (CRD2; residues 86–125) (Fig. 2 A). A1 and B2 modules are composed of 12–17 and 21–24 amino acids, respectively. They are defined by the consensus sequence Cys1-Xaa2-Gly-Xaa-Tyr (or Phe)-Xaa-Xaa4–9-Cys2 and Cys1-Xaa2-Cys2-Xaa3–6-Xaa5-Cys3-Thr-Xaa2–5-Asn-Thr-Val-Cys4, respectively (30Naismith J.H. Sprang S.R. Trends Biochem. Sci. 1998; 23: 74-79Abstract Full Text PDF PubMed Scopus (187) Google Scholar), where n in Xaan is the number of intervening amino acids. The A1 modules exhibit a single disulfide bond between Cys1 and Cys2, while the B2 module has two disulfide bonds (Cys1:Cys3 and Cys2:Cys4). The N1 module has only one disulfide bond but is structurally related to the B2 module (Fig. 2 B). Interestingly, in place of a N1 module in sDR5, a B2 module is located at the corresponding position in sTNFR55. Residues 28–34 of the N1 module, which is structurally homologous to the B2 module (Fig.2 B), are involved in tight backbone interactions with the A1 module in CRD1 of sDR5 (figure not shown), and these interactions are conserved in sTNFR55. Consequently, the domain conformation and the arrangement of CRD1 relative to the N1 module in sDR5 are virtually the same as those of the corresponding CRD relative to the B2 module in sTNFR55. A superposition of the structures of TRAIL-sDR5 and TNFβ-sTNFR55 discloses that the N1 module and CRD1 in sDR5 and the corresponding B2 module and CRD in sTNFR55 adopt virtually the same geometry on complex formation (Fig.3 A). The structurally similar B2 and N1 modules appear to be functionally replaceable without affecting receptor-binding mode.Figure 3Superposition of the ligand-receptor complexes and that of sDR5 molecules. A, the TRAIL-sDR5 and TNFβ−sTNFR55 complexes are superposed using only the structurally conserved Cα atoms on the β-sheets of the ligand molecules. Cα atoms are shown with the omission of TNFβ. sDR5 and sTNFR55 are shown in green and violet, respectively. TRAIL is in light green. B, the six sDR5 molecules with the ordered B2 modules in three TRAIL-sDR5 complex structures are superposed, showing the variation in the relative position of the B2 module in CDR2.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The domain structures of TNFR family members are very flexible, as was first observed for sTNFR55 that adapts its structure to changes in solvent conditions or upon binding to TNFβ (31Naismith J.H. Devine T.Q. Kohno T. Sprang S.R. Structure. 1996; 4: 1251-1262Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). A superposition of the TRAIL-sDR5 and TNFβ-sTNFR55 structures shows a clear difference in the relative orientation between CRD2 and the corresponding CRD of sTNFR55 (Fig. 3 A). A torsional angle change of ∼30° occurs at the connection (Gln-85) between CRD1 and CRD2 of sDR5 compared with sTNFR55 bound to TNFβ (Fig. 3 A). Apparently, the domain movement complements the geometric differences in the receptor-binding surfaces of the two ligands for specific interactions between pairs of residues. It probably is a common feature of the receptor family. The six sDR5 molecules in the asymmetric unit exhibit a large structural disparity in the B2 module of CRD2. Although the B2 module and the three successive residues at the C terminus in two sDR5 molecules are well ordered, those in the other four molecules are highly disordered. Because of different crys" @default.
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• W2017086893 title "Crystal Structure of TRAIL-DR5 Complex Identifies a Critical Role of the Unique Frame Insertion in Conferring Recognition Specificity" @default.
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