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Molecular Cell, Vol.

4, 563–571, October, 1999, Copyright ©1999 by Cell Press

Triggering Cell Death: The Crystal Structure of Apo2L/TRAIL in a Complex with Death Receptor 5
Sarah G. Hymowitz,* Hans W. Christinger,* Germaine Fuh,* Mark Ultsch,* Mark O’Connell,* Robert F. Kelley,* Avi Ashkenazi,† and Abraham M. de Vos*‡ * Department of Protein Engineering † Department of Molecular Oncology Genentech, Inc. 1 DNA Way South San Francisco, California 94080 the nonsignaling decoy receptors DcR1 (Delgi-Esposti et al., 1997a; Pan et al., 1997b; Sheridan et al., 1997) and DcR2 (Delgi-Esposti et al., 1997b; Marsters et al., 1997), and osteoprotegerin (OPG) (Simonet et al., 1997; Emery et al., 1998). The apoptosis-inducing activity of TNF and FasL has prompted attempts to use these ligands as potential agents for treatment of cancer. Unfortunately, these molecules exert toxic side effects that limit their therapeutic utility. Unlike TNF and FasL, Apo2L appears to have a unique selectivity for triggering apoptosis in tumor cells while leaving normal tissues intact. Thus, soluble Apo2L administered to mice bearing human tumors results in tumor shrinkage, without discernible toxicity to normal tissues (Ashkenazi et al., 1999; Walczak et al., 1999). One mechanism that might contribute to the relative selectivity of Apo2L for tumor cells is the preferential expression of the decoy receptors in normal tissues compared to tumor cells (Pan et al., 1997b; Sheridan et al., 1997). However, the interaction between death and decoy receptors in modulating Apo2L activity is not well understood. To date, the structure of one ligand–receptor complex belonging to the TNF receptor superfamily has been determined, namely, lymphotoxin (LT) in complex with the extracellular region of the p55 TNF receptor (TNFR1) (Banner et al., 1993). However, the generally low sequence conservation in the superfamily, both among the ligands and the receptors, precludes meaningful prediction of interactions in complexes formed by the other members. For example, the sequence identity between Apo2L and LT is only 19% and between DR5 and TNFR1 is only 23%. It is therefore not surprising that in a recent model of an Apo2L–DR4 complex, based on a low-resolution structure of the ligand (Cha et al., 1999), the proposed details of the interface are not consistent with the experimental structure presented here. To search for unifying principles that may govern ligand– receptor interaction in the superfamily, we have determined the structure of the complex between Apo2L and DR5 and compared it to the LT–TNFR1 complex. The structure reveals the existence of two main interaction patches: one consists of a mostly hydrophobic motif that is likely to be conserved in ligand–receptor complexes throughout the superfamily, while the other contains specific features unique for each individual complex that appear to be used to control receptor selectivity and cross-reactivity. Results and Discussion The complex formed between the extracellular portions of DR5 (residues 1–130) and Apo2L (residues 114–281) crystallized readily and was found to contain three receptors and three ligands assembled as a hexameric complex in the asymmetric unit. Diffraction data were collected using synchrotron radiation, and the structure ˚ resolution and an R value of 22.2% was refined to 2.4 A (Rfree of 26.7%). The final model consists of Apo2L residues 119–131 and 144–281 in each monomer and DR5

Summary Formation of a complex between Apo2L (also called TRAIL) and its signaling receptors, DR4 and DR5, triggers apoptosis by inducing the oligomerization of intracellular death domains. We report the crystal structure of the complex between Apo2L and the ectodomain of DR5. The structure shows three elongated receptors snuggled into long crevices between pairs of monomers of the homotrimeric ligand. The interface is divided into two distinct patches, one near the bottom of the complex close to the receptor cell surface and one near the top. Both patches contain residues that are critical for high-affinity binding. A comparison to the structure of the lymphotoxin– receptor complex suggests general principles of binding and specificity for ligand recognition in the TNF receptor superfamily. Introduction Apoptosis is essential for proper development and homeostasis in metazoans (Jacobson et al., 1997). Several members of the mammalian tumor necrosis factor (TNF) gene superfamily, including TNF (or TNF␣), lymphotoxin (or TNF␤), FasL, and Apo2L (or TRAIL), trigger apoptosis by binding to related, cysteine-rich receptors that contain cytoplasmic death domains (Tartaglia et al., 1993; Nagata, 1997; Ashkenazi and Dixit, 1998). Oligomerization of these death domains, triggered through ligandinduced receptor trimerization, generates homophilic interaction surfaces for death domain–containing adaptor proteins such as FADD. These adaptor proteins in turn engage initiator caspases such as caspase 8, leading to subsequent activation of effector caspases that execute apoptotic death of the cell (Thornberry and Lazebnik, 1998). Apo2L is a homotrimeric, type II transmembrane protein (Wiley et al., 1995; Pitti et al., 1996). Like most TNF superfamily members, Apo2L can be cleaved at the cell surface to form a soluble molecule (Mariani et al., 1997). Apo2L binds to an unusually complex family of receptors: the apoptosis-signaling death receptors DR4 (Pan et al., 1997a) and DR5 (also called Apo2 or TRAILR) (Pan et al., 1997b; Sheridan et al., 1997; Walczak et al., 1997),
‡ To whom correspondence should be addressed (e-mail: devos@

and the three receptors are rendered as tubes in yellow and orange colors. and the buried zinc ion does not interact with the receptor (Figure 1). Like other TNF superfamily members. and F in one sheet and B. 1989). In this orientation. the biological activity of Apo2L. and E in the other (Figure 1A). D. Two small strands. hence. are inserted between strands A and B. perpendicular to (A). H.Molecular Cell 564 Figure 1. which are formed by strands A. and 284 water molecules (Table 1). (A) Side view. TNF (Eck and Sprang. the Apo2L monomer consists of an all ␤ strand jelly roll composed of two flat ␤ sheets.. submitted). C. Removal of the zinc ion decreases Apo2L apoptotic activity 90-fold and reduces the thermal stability of the trimer by 20ЊC. While the overall structure of Apo2L bound to DR5 is . The Apo2L–DR5 Complex The Apo2L trimer is drawn as ribbon rendering in gradations of blue. and the bound chloride ion is pink. residues 21–128 of one copy and 21–130 of the other two (Figure 1). 1989.. ␤ strands and relevant loops are labeled. the zinc site is located on the 3-fold axis and buried in the trimer interface (Figure 1B). and Apo2L (Hymowitz et al. submitted). CD40L (Karpusas et al. submitted)... Jones et al. first observed in the structure of unbound Apo2L (Hymowitz et al.. We have previously shown that this bound zinc ion is important for the structure and stability and. The disordered loop (residues 132–143) in Apo2L is rendered as small spheres. A unique zinc-binding site. LT (Eck et al. Structure of Apo2L We have previously determined the structure of the D218A mutant of unbound Apo2L (Hymowitz et al. a chloride ion. The geometry of this site is identical in the complex.. AЈ and BЈ. while the conformations of the surface loops and side chains are extremely variable. The tetrahedral metal-binding site is formed by the side chain of Cys-230 from each ligand monomer and completed by an interior solvent molecule (probably a chloride ion). 1992). G. The bound zinc atom is colored green. together with a zinc ion. the membrane of the receptor-containing cell is at the bottom of the figure. 1995). is present in the structure of the Apo2L–DR5 complex. especially near the “bottom” of the trimer. The core residues of the AHCF and BGDE sheets are well conserved in the four structurally characterized TNF superfamily ligands. (B) View down the 3-fold axis of the complex.

1993). Prior to the structure of the Apo2L–DR5 complex. c R ϭ ⌺ |Fo Ϫ Fc|/⌺Fo.50)a 0. with the exception of the C-terminal portion of CRD3 (residues 104–130). The Overall Structure of DR5 DR5 is a member of the TNF receptor superfamily. The two loops that form most of the contacts with the ligand (below) have very similar conformations in all three copies (Figure 2). These pseudorepeats.49–2.4 38.Crystal Structure of the Apo2L–DR5 Complex 565 Table 1.62–3. Park et al. In contrast. which exhibits a rigid-body variation in orientation (Figure 2). DR5 residues 1–42. 1996) and in complex with LT (Banner et al. The first disulfide bridge of DR5. Rfree (F Ͼ 0) Number of residues Number of solvent molecules Number of non–H atoms ˚ 2) Average B factor (A ˚) Rmsd bonds (A Rmsd angles (Њ) ˚ 2) Rmsd B (bonded atoms) (A a b SSRL 30–2.4 (2. where the C-terminal subdomain of the receptor was disordered. Thus. This allows us to inspect the geometry of the complex at the point where the receptors would enter the cell membrane. Rsym ϭ ⌺ |I Ϫ ϽIϾ|/⌺ I. each containing three disulfide bridges. but for 10% of the reflections excluded from all refinement. In our complex..8 (99.7)a 30–2. The 150s loop was in a crystal packing contact in the unbound structure.850 0... 43–85. Rfree is calculated as R. It is tethered into an elongated shape by a series of seven disulfide bridges. both unbound (Naismith et al. between residues 28 and 41. The Ligand–Receptor Interface The Apo2L–DR5 complex is composed of a wedgeshaped Apo2L homotrimer with three receptors bound diagonally along the crevices in the Apo2L monomer– monomer interfaces. and CRD3 of TNFR1. CRD2. the only structural information about these receptors was for TNFR1. Residue 130 is predicted to be the final extracellular residue before the putative single transmembrane helix connecting the receptor to its cytoplasmic death domain. More subtle changes between the structures are found around Tyr-216 and Pro-217.459 99. gle approximately 50 A this same spacing is also found for the receptor-binding sites on the TRAF-2 trimer. and 86–130 form analogous subdomains to CRD1.7 2. 1995. The 150s loop is in a significantly different conformation than in the free ligand (with an rmsd for ˚ ).9 A position of the buried side chain of Trp-154.527 12.267 781 286 6577 47. 0.5 (3.185 (0. respectively) that correspond structurally to the second and third CRDs of TNFR1. termed cysteine-rich domains (CRDs). six of which are found in subdomains of DR5 (residues 43–84 and 85–130.5 A ˚ shift in the the C␣ atoms of 1. Each of these three extensive inter˚ 2 of solvent-accessible action surfaces buries 2750 A .6 0.8 (99. Data Collection and Refinement Statistics In House Data Collection ˚) Resolution (A Rsymb Number of observations Unique reflections Completeness (%) Refinement ˚) Resolution (A Number of reflections Final Rc. corresponds to the last disulfide bridge (between Cys-33 and Cys-52) in CRD1 of TNFR1.986 38. while the first 20 residues of DR5 are disordered. 145–154. which are at the heart of the Apo2L–DR5 interface (below). which may have influenced its conformation. These studies showed that the extracellular region of TNFR1 is an extended structure composed of four pseudorepeats or subdomains. The three copies of DR5 in the complex are very similar to each other. are approximately 40 residues long. 1999). Unlike the LT–TNFR1 complex.908 99. substantial changes tion (rmsd) of 0. very similar to the structure of the free ligand.42 A occur in the conformation of the “190s loop” (residues 195–201) connecting strands C and D and in the “150s loop” (residues 154–162) linking strands A and AЈ. It is noteworthy that these loops are in contact with the receptor (see below). the C termini of the receptors form a trian˚ on a side (Figure 1B). the C-terminal residues of DR5 are well ordered up to residue 128 in one copy (the “R” chain) and up to residue 130 in the other two (the “S” and “T” chains). including a 0. Superposition of the bound and unbound Apo2L trimers based on the C␣ atoms of residues 120–128..396)a 152. and 203–281 gives a root-mean-square devia˚ . 1999.4 30–3. Intriguingly. which is known to interact with the intracellular portions of some TNFR superfamily members (albeit not with DR4 or DR5) (McWhirter et al.013 1. 162–194. The 190s loop is ordered in this structure but was disordered in the unbound Apo2L structure. there are alterations in the conformation of several surface loops that appear to be a consequence of receptor binding. DR5 resembles TNFR1 in overall structure with relatively little defined secondary structure (Figure 2).9)a Numbers in parentheses refer to the highest resolution shell.222. ϽIϾ is the average intensity of symmetry related observations of a unique reflection. This suggests that the observed extracellular geometry could be propagated through the rigid transmembrane helix to the death domains and that this spacing may be important for proper triggering of the intracellular apoptotic cascade.398)a 51.40)a 0.056 (0.

Of the other functionally important residues. Of the residues found to be important for activity. has four pseudorepeats of a cysteine-rich motif (a CRD or cysteine-rich subdomain) (Figure 5) (Banner et al. 1400 A from the ligand. 122. The structurally conserved and functionally important ˚ 2 from the receptor and 1350 A ˚2 surface area. Interestingly. Arg-149 forms a salt bridge to receptor residue Glu-94. and 4–5 (Figure 5. These peripheral interactions are not critical for high-affinity binding. Patch B involves general hydrophobic features of TNF-like ligands and appears to be important for binding in complexes throughout the superfamily. Lys201. 3–6. 1993). Tyr-216 had been identified by alanine scanning mutagenesis as a critical residue for bioactivity and receptor binding (Hymowitz et al. in contrast to the three subdomains believed to be present in Fas. the 50s loop of the receptor interacts with the 210s and the 150s loops of Apo2L (Figure 4B). the extracellular domains of DR5 and Fas are roughly the same size (Figure 5). DR5. has a different topology. CRD2. For instance. ˚ 2. which binds in a hydrophobic groove on the receptor surface formed by the side chains of DR5 residues His 53. Each of these subdomains contains six cysteines in three disulfide bridges. The CRD terminology is less precise when applied to other members of the superfamily. Lys-102. while the remaining 130 A ˚ 2 is a result of small contriA butions from receptor residues 65–69 and 108. Therefore. The interactions with the 150s loop are more peripheral and polar in nature and are primarily mediated through contacts between Arg-62 of the receptor with Apo2L residues Glu-155 and Ser-159. In 480 A this patch. Asn55. Naismith and Sprang.. and Asp-122 of DR5 (Figures 3 and 4A). DcR1. which hydrogen bonds to the backbone of receptor residues . Four of the five residues of Apo2L identified by alanine scanning as important for biological activity are located in this patch (Hymowitz et al. and Asn-199. This interaction is centered on Apo2L residue Tyr-216. The final subdomain. In patch A. Patch B buries a solvent-accessible area of 890 A ˚ 2 from the receptor and 410 A ˚ 2 from the ligand. CRD4. Crystal structures of this receptor have shown that the first three of these CRDs exhibit a disulfide-bonding pattern 1–2.. The Domain Structure in the TNF Receptor Superfamily The TNF receptor superfamily is remarkable for its structural preservation despite a minimum of sequence conservation. Bazan. 1998). submitted). Leu-58. The difference between these receptors is that subdomain 1 in DR5 has only a single disulfide bridge. DR5 has at least part of CRD1 and all of CRD2 and CRD3. but its substitution by alanine causes a substantial loss of DR5 binding. the prototype of the superfamily. DR4. with 1790 A ˚ 2 from the of total buried accessible surface area (880 A ˚ 2 from the ligand). Superposition of the Three Copies of DR5. while patch A may control the specificity and cross-reactivity among the different superfamily members (see below). The approximate boundaries of CRD1. and our structure shows that like Fas. and Asp-203 form polar interactions with Arg-101. The 90s loop of receptor and 910 A DR5 contributes 85% of the buried surface area (750 ˚ 2). 3–5. and the cysteine connectivity is 1–2. and the 50s and 90s loops are highlighted in shades of gray. Two receptor loops mediate most of the interactions. and CRD3 are marked. the decrease in activity of the alanine mutant may be solely due to deleterious effects on DR4 binding. Showing Its Overall Structure as well as Variation in the Relative Position of CRD3 The sulfur atoms of the disulfide bridges are shown as yellow spheres. since substitutions of these Apo2L residues with alanine had only marginal effects on binding or activity. only Ser-259 does not participate in the Apo2L–DR5 interface. corresponding to the last disulfide in CRD1 of TNFR1 (Figures 5 and 6). and 125. and DcR2 have been predicted to have two CRDs.. Naismith et al. submitted) and is conserved in many TNF homologs. the 90s loop interacts with a cluster of Apo2L residues around Gln-205 near the bottom of the trimer. while patch B is formed by the 50s loop of DR5 and Apo2L residues clustered around Tyr-216 near the top (Figure 3). Arg-104.Molecular Cell 566 Figure 2. Substitution of this residue by alanine reduced apoptotic activity of the resulting mutant by 700-fold. TNFR1. Leu-57. For instance. and Phe-59. The most important of these residues is Gln-205. ˚2 Patch A is the larger of the two patches. Other residues only moderately important for activity are found in the periphery of the patch but do generally interact with the receptor. only Glu-236 buries less than 50% of its accessible surface area in the interface. and 4–6. dividing the interface in two distinct patches (Figure 3): the “50s loop” (residues 51–65) and the “90s loop” (residues 91–104). alanine substitution of this residue affects binding against DR4 but not DR5. 98 and 99 (Figure 4A).. In fact. 111. 1996. 1993.

and OPGL.. A probe size of 1. However. light yellow.. This structural similarity is particularly pronounced in the central third of the receptor. the C-terminal subdomains are rotated with respect to each other. Tyr-216 is conserved in many of the TNF superfamily ligands (including TNF. ˚ was used to calculate accessible orange. The difference in the position of this subdomain correlates with the observation that the second two-thirds of it has virtually no contact with ligand in the LT–TNFR1 complex (Banner et al.. but not as extreme. All of the TNF receptor superfamily members are expected to have these central subdomains. . red). 25%–50%. 50%–75%. Schneider et al. while many other members have a similar large hydrophobic residue at this position. LT. does not appear to be important for ligand binding. A high-resolution structure of unliganded TNFR1 shows that this final CRD has different disulfide connectivity than the other subdomains (Naismith et al. while the other two receptors are shown as green tubes. Similar movement. The final TNFR1 subdomain. 1991. This difference also results in a 550 A contact interface with the ligand. which is also present in OPG but not in DR5 and Fas (Figure 5). Superimposing the C␣ atoms of residues 27–34 and 48–67. yellow. Comparison to the LT–TNFR1 Complex The 130-residue extracellular domain of DR5 shares only 16 noncysteine residues with TNFR1. 1994.7 A receptors are similar.4 A surface area. with their counterparts from TNFR1 results in an rmsd of only ˚ . which interacts extensively with the ligand in both structures. resulting in a 3–5 ˚ displacement of the structurally equivalent residues A (Figure 6). portion of these receptors is the actual ligand-binding motif found in CRD2 and CRD3 (corresponding to DR5 residues 43 to 130). FasL. Goh et al. Yamagishi et al. while the N-terminal portions of the 0. (by up to 10 A was seen in a comparison of bound and unbound TNFR1. including the disulfide bonding patterns that present the functionally important 50s and 90s loops. despite the shorter overall length of DR5 compared to the TNFR1 sequence. submitted). Hymowitz et al. these 16 residues in conjunction with seven identical disulfide bridges result in a remarkable structural congruence in the C␣ traces of the ligand-binding portions of the receptors (Figure 6). When the structures of the receptor complexes of Apo2L and LT are superimposed by aligning the structurally equivalent ␤ strands in the ligands.. Open Book View of the Apo2L–DR5 Interface Apo2L and one receptor are rendered as space-filling models. 1993).. A and B (labeled). 75%–100%. and Apo2L have all shown that this residue is critical for binding (Van Ostade et al. Residues in the interface are colored by percent of buried accessible surface area upon complex formation (1%–25%... The interface divides into two patches. Among the least well-conserved parts of the DR5 structure with respect to the TNF receptor structure is the 90s loop (Figures 6 and 7A). Mutagenesis studies on TNF. FasL. CRD4. the C-terminal portions of the receptors differ even more ˚ ). 1996). However. LT.Crystal Structure of the Apo2L–DR5 Complex 567 Figure 3. Figure 7B). Binding and Specificity in the TNF Receptor Superfamily The dominant characteristic of patch B in the Apo2L– DR5 interface is the interaction between Tyr-216 and the 50s loop of the receptor. The interactions of the tyrosine side chain are conserved between the Apo2L–DR5 and LT–TNFR1 complexes. corresponding to the C-terminal portion of CRD1 and the structurally identical residues in CRD2. it could have a role in transducing the TNF binding signal into the cell. 1990. 1997. while the C-terminal portion of DR5 is in intimate contact with the 200s loop of ˚ 2 larger Apo2L.

Hydrogen bonds made by Gln-205 to the backbone of receptor residues 98 and 99 are shown as dashed lines. with the ligand-binding domains in red. The potential role of this loop is further highlighted by the observed crossreactivity between Apo2L and OPG (Emery et al. relevant side chains are shown. including the two central residues Glu-98 and Met-99 (Figure 7A). (A) The specificity patch (patch A) centered on Gln-205 of Apo2L interacting with the receptor 90s loop. The CRDs are shown as oval symbols and labeled. and DcR2 have seven identical residues in that loop. is at the bottom. Moreover.. 1998). DcR1.35 A Figure 5.. atoms of residues 51 to 62) (Figure 6). is virtually identical between DR5 and the ˚ between the equivalent C␣ TNFR1 (rmsd of only 0. 1997). DR4. submitted) and FasL (Schneider et al. DR5. Both Apo2L and OPGL have a glutamine at this position. while FasL has a proline (Figure 7B). which connects to the transmembrane helix. which forms the binding pocket for the side chain. accompanied by the sequence of the receptor 90s loop. In contrast to the conserved interactions in patch B. Apo2L binds to OPG but not to Fas. patch A near the bottom of the interface (Figure 3) involves interactions made by the 90s loop on CRD3 of DR5. The backbone atoms are represented as a C␣ trace. The structure of the Apo2L– DR5 complex shows that this glutamine side chain interacts with the backbone of DR5 residues 98 and 99 (Figure 4A). (B) The hydrophobic binding patch (patch B) between the receptor 50s loop and ligand residue Tyr-216. Additionally. We suggest that this loop functions as a general hydrophobic binding patch interacting with conserved hydrophobic features on the ligand. Residue 205 is known to be important for high-affinity binding by both Apo2L (Hymowitz et al. this interaction may help properly orient the upper part of the receptor for more specific contacts mediated by CRD3. and DR5. OPG. and receptor residues are colored orange. TNFR1. receptors that can bind to the same ligand have conserved features in their 90s loops. despite the fact that the OPG . Schematic Depiction of the TNFR Family Subdomain Structures Schematic depiction of the sudomain structures of Fas. which has a completely different conformation than the corresponding loop in the TNFR1 (Figure 6). Wall-Eyed Stereo View Showing Details of the Apo2L–DR5 Interface The Apo2L monomers are colored gray and dark pink. Interestingly. indicating receptor size and the relative location and number of disulfide bonds. The N terminus of the receptor is at the top of the figure and the C terminus. the backbone conformation of the 50s loop of the receptor. the length of this loop is conserved among the different receptor superfamily members.Molecular Cell 568 Figure 4. For instance. Given that the 90s loop of DR5 is generally similar to that of OPG.. The explanation for this may lie in the identity of the residue at position 205 of the ligand.

The fraction containing the Apo2L–DR5 complex was further purified by anion exchange chromatography (MonoQ. The crystals belonged to space group P212121 and had unit cell dimensions a ϭ ˚ . and DR5 was eluted with 2 M KSCN in 50 mM Tris (pH 8. Pharmacia) in 100 mM NaCl. (A) The 50s and 90s loops in DR5. DcR2) binding patches are boxed. respectively.4 A ˚ ). Experimental Procedures Expression and Purification Apo2L (residues 114–281) was expressed in E. DR4.1 M HEPES [pH 7.1 M NaCl. These are likely to form an additional disulfide bridge. In TNFR1.0) was added to purified DR5 in approximately equimolar concentrations.7 mg/mL and buffered with 20 mM Tris-HCl (pH 8. Sequence Alignments of the Main Interface Regions The residues in DR5.5 A was collected on a MAR imaging plate system using a Rigaku rotating anode generator with CuK␣ radiation. Pharmacia) and eluted with a 0–1 M NaCl gradient in 20 mM Tris (pH 8. and TNFR1. The complex was then concentrated to approximately 3. A 3. 0. 8% ethylene glycol. Residues of the ligands and receptors at the center of the observed (Apo2–DR5. Cysteines are highlighted in yellow. it also has a completely different conformation.0). DcR1. DR5. and the protein was eluted with a 0–1 M NaCl gradient in 20 mM Tris-HCl (pH 8. DR5 was further purified by size exclusion chromatography (S-200. which would drastically alter the conformation of the 90s loop.2 M ammonium sulfate. a crystal structure is not available. Apo2L in 20 mM Tris-HCl (pH 8. as noted above. 0. this interaction is probably conserved in the OPGL–OPG complex and also when Apo2L binds to OPG.0 A ˚ . The crystals were transferred briefly to a droplet containing reservoir solution with 20% glycerol before flash freezing in liquid nitrogen. Figure 6. The asymmetric unit contained 66. The supernatant was run over a Q-Sepharose (Pharmacia) column.5) and grew to a size of 0. coli and purified as described (Ashkenazi et al. DR4. Pharmacia).8 A ˚ data set one Apo2L trimer and three copies of the receptor.0).0). A subsequent data set to ˚ resolution was collected from a single crystal at beam line 2. c ϭ 130. and LT that bury more than 50% of their accessible surface area in the interface are colored red. The crystals used for data collection were grown by mixing 2 ␮L of protein solution with 2 ␮L of reservoir consisting of 15% PEG 8000. TNFR1.08 A . the loop is longer and has no sequence identity with DR5 other than the cysteines. 7–1 of the Stanford Synchrotron Radiation Laboratory (␭ ϭ 1. and the DR5-containing medium was separated from the cells by centrifugation. OPG. For Fas. (B) The segment around residues 205 and 216 of Apo2L for Apo2L. The sulfur atoms of the disulfide bonds in DR5 and TNFR1 are shown as yellow and orange spheres. and the complex was purified by size exclusion chromatography (S-75. Crystallization and Data Collection Crystals of the Apo2L–DR5 complex were grown by vapor diffusion at 19ЊC using the hanging drop method.0).5]). In contrast. and LT or TNF␤. Fas. and the 90s and 50s loops are highlighted in gray and black. b ϭ 112. OPGL. 1999). Apo2L. TNF–TNFR1.. which do not crossreact with each other’s ligands or with Apo2L. DcR2. the 90s loop is considerably different in TNFR1 and in Fas.1 M Tris-HCl (pH 7. but cysteine residues are present at positions 92 and 97 (Figure 7)..0). TNF or TNF␣. 20 mM Tris-HCl (pH 8. DcR1. Superposition of DR5 with TNFR1 TNFR1 (in lavender and gray) is taken from the lymphotoxin complex (Banner et al. LT–TNFR1) or predicted (Fas–FasL. The three CRDs are labeled. While further structural and mutagensis experiments are necessary to confirm this analysis.0).3 mm ϫ 0. DR5 (residues 1–130) was expressed in Hi5 insect cells with a baculovirus transfer vector under the control of a polyhedron promoter. FasL. whereas in patch A the interactions between the receptor 90s loop and the ligand residue at or near position 205 control the specificity and cross-reactivity among the superfamily members. The initial crystals were grown in condition 37 of the Hampton Crystal Screen II (10% PEG 8000.Crystal Structure of the Apo2L–DR5 Complex 569 Figure 7. we conclude that in patch B the 50s loop of the receptor and ligand residue 216 provide a hydrophobic patch generally important for binding in the superfamily. 0. The fractions containing DR5 were pooled and loaded onto a CNBrApo2L affinity column. OPG–OPGL. 10% ethylene glycol. CRD2 and CRD3 mediate all contacts to the ligand. loop is three residues shorter. Protein was secreted from cells grown at 27ЊC over 72 hr. 1993) and superimposed on DR5 (in red and black) based on the shared C␣ atoms of CRD2.1 mm. The column was washed with 0.5 M NaCl in 20 mM Tris-HCl (pH 8. 0.15 mm ϫ 0.8 A ˚ .

2 A extracellular fragment of human CD40 ligand. 13303–13307. and Dixit. 1993) region of the Ramachandran plot. Devine. Blackie.4% (the highest incorrect peak had a correlation coefficient of 7. E. Dubose.9-A Emery. Similar searches using the structure of Apo2L bound to a 3.A. Chem.. Janes. Dougall. (1999).A. Dul. especially in residues 100 to 130. S. This model was refined with X-PLOR 98.. Bru ¨ nger. D. Kim. (1998).. Smolak.. 1992b) as modified by Molecular Simulations.A.-S.-J. Clin... and Ashkenazi. Three cycles of model building.. Cryst. and Porter. 1999. 14363–14367. and Murphy. and Thomas. A. Osteoprotegerin is a receptor for the cytotoxic ligand TRAIL. R. Hsu.M. D 50... NCS constraints. (1997). T. N. C. The final model consists of residues 119 to 131 and 144 to 281 of each Apo2L monomer (A. Nature 338. Gentz. M.S. Interleukin 1 beta-converting enzyme related proteases/caspases are involved in TRAIL-induced apoptosis of myeloma and leukemia cells. The novel receptor TRAIL-R4 induces NF-␬B and protects against TRAIL-mediated apoptosis..A.P.P.. Biol.H.. J.-H. A. Shin. the program AMoRe (CCP4.J. Gurney.. 2. A 3fold NCS-averaged and solvent-flattened map (program DM [CCP4. S.R. 760–763.H. J..Q. Thompson. B. E. and Minor. using a maximum likelihood target function. and density averaging allowed building of receptor residues 22–130. 273. and Sprang. Yu. C.A. T.F. Science 277. Ni.M... (1997). D.. S. and Dixit. Pullen.. Acta Crystallogr.. J.. Delgi-Esposti. P..C. a zinc and a chloride ion. Nagata... Karpusas.. Pai. Cell Biol. Exp. A. Cell 88. et al.. de Vos. A. J. (1993). B. Appl. Goodwin. 347–354. References Ashkenazi. S. P. S. Acad.. Sheridan.A. and Sprang. C.J. Immunity 7. S. Waugh... M. The structure of human lymphotoxin (tumor necrosis ˚ resolution. Baldwin. a program for photorealistic molecular graphics. Safety and anti-tumor activity of recombinant soluble Apo2 ligand. (1998). J.W. 4. 23.. R.R. Chang. J.. 431–445. (1992). H. 7. Sci.H. Moss.5%. a novel member of the emerging TRAIL receptor family. 785–791. Jacobson. Structure 3. Mariani. Devine. Jones. Natl.-M. A. Structure Determination and Refinement The Apo2L–DR5 structure was determined by molecular replace˚ resolution structure of Apo2L alone (Hymowitz ment using the 1. Version 3. Emerging families of cytokines and receptors. M. B. J. USA 96. 137. 17595–17605. Appelbaum. Otwinowski. factor-␤) at 1. M.. C. Kehry. P. 221–229. Connecticut: Yale University). M. P. Choi. In subsequent refinement.. 1997). 307–326... S.-H. R. Eck. E. (1997a). Free R value: a novel statistical quantity for assessing the accuracy of crystal structures. Cloning and characterization of TRAIL-R3. W.M. 104.. the Apo2L trimer was subject to tight NCS restraints..3%). A.. A. G. Molscript (Kraulis. L... A..4 A 1994). Naismith. Smith.K. Felix Vajdos. and Oh. A.C.. J. Silverman. Marsters.D.. Lyn. using programs X-PLOR and REFMAC (CCP4. Huang. P. Kraulis. S... (1989).. submitted) as the search model in combination with 3-fold noncrystallographic symmetry (NCS) map averaging.R. J. Gentz.. Lederman..G.3 A et al. Merrit. M.G.. R. Walczak. D.1 (Bru ¨ nger. E. NCS mask refinement.. M. (1991). 155–162. J.C. Ultsch. S. Naismith.. Modularity in the TNF-receptor family. and Alber.J.G. M. Y.H.B. Curr.E.Molecular Cell 570 The data sets were processed using the programs in the HKL package (Otwinowski and Minor. (1997).R. W.S. (1989). Christian Wiesmann.. An overall anisotropic B factor correction was applied to the data as was a real-space bulk-solvent correction (Bru ¨ nger. B. Structure of tumour necrosis factor. M..C. C. 8408–8413.. (1997b).. Shin.J. Y. (1995). Immunity 11.. 1031– 1039. 1999. Charles Eigenbrot. Y. Loetscher. C.. J.. M. H. (1993). (1999). Yuan.F. (1998). Raster3D version 2. McWhirter..4% following rigid-body fitting using all data between 8 A ˚ .H.T. J. ˚ crystal structure of an S. two residues are in the “generously allowed” regions. R. Rinderknecht.. M. McMurtrey. E. S. 1003–1006. C. Inc.. as well as our colleagues in mass spectrometry and protein sequencing. Delgi-Esposti. 1165–1170. Sung. Cell 73. 603–606. J. Nature 355..R. Chem. Aspartic acid 50 and tyrosine 108 are essential for receptor binding and cytotoxic activity of tumour necrosis factor-␤ (lymphotoxin). Matiba.. Ashkenazi.. R. W. MOLSCRIPT: a program to produce both detailed and schematic plots of protein structures. J.Y. and Sprang. Holten.A. Broger. 24. A.P. Using all data ˚ to 4 A ˚ . D. a cytokine with selective antitumor activity.. T.A.M. Huang. Science 281. 355–356. J..J. (1992a).M.Y..A. 1992a) of ˚ and 42. The solution to the translation function had an initial Rfree (Bru ¨ nger. C. X-PLOR Manual.. 186.. and grouped B factors until Rfree reached 33. Skubatch. 253–261.M. P. Weil. 869–873. DiPrinzio. Invest. (1993).. J. Godowski... K. 264. ˚ resolution crystal H. 176. Z. and Sprang.. A.J. Pan. and Smith. (1995). Biol.. Deen.5 A homology model of DR5 based on the LT–TNFR1 complex resulted in worse molecular replacement statistics and were abandoned. J. Crystallographic evidence for dimerization of unliganded tumor necrosis factor receptor. J. D’Arcy. (1996). Schoenfeld. and Walker. A. S.C. R. 472–475.D.. Procheck: a program to check the stereochemical quality of protein structures. T.A. Biol. positional refinement. 87% are in the “most favored” (Laskowski et al.. Protein Eng. Appl. Curr.. Brandhuber.-C. A novel receptor for Apo2L/TRAIL contains a truncated death domain. Jenny Stamos for help with and advice on insect cell culture. Sci..F.. McDonnell.. (1997a). Chem. J.M. Leung.0.1 (New Haven. V.R.S. Kohono. Bru ¨ nger.6 A Biol. Structure 4. M. An antagonist decoy receptor and a death domain-containing receptor for TRAIL..W. Apoptosis by death factor. 813–820. 1992b).. M. 225–228. Wang. Loh. and Raff. (1991). Cryst. R.C. and Sprang. J.M..8 A structure of human TRAIL. factor-␣ at 2.. Biol. revised September 13. Of all nonglycine residues. (1997). Goh. J. 26. Goddard. Eck. Med. and 284 water molecules. M. 267. N. H. Structures of the extracellular domain of the type I tumor necrosis factor receptor. 283–291. (1997). Crystal structure of the soluble human 55 kd TNF receptor–human TNF beta complex: implications for TNF receptor activation. Lawrence.R. L.. This partially refined model was then further refined against ˚ data set. Biol. Wei. (1994). A.E. Chess..G. receptor chains R (residues 21 to 128) and S and T (residues 21 to 130). J. Trends Biochem. Crute.. Fong. Eichman. 3. S. V. 1994]) phased using the solution for the Apo2L trimer revealed partial density for the receptors. Waugh..J. Acta Crystallogr. Laskowski. Chem. Refinement and model statistics are shown in Table 1. Bazan. 74–79. and Goodwin. M.. Armandola. MacArthur. C. Proc. (1992b). Pitti. Cell 88. while weak restraints were applied to the most similar regions of the receptors. yet retains an incomplete death domain. and no residues are in the “disallowed” regions..Q. . 815–818. Acknowledgments We thank Roger Pai and Susan Leung for providing purified Apo2L. D 50. J.. S.R. Death receptors: signaling and modulation.P. Burke. R. (1999). 1991) and Raster3D (Merrit and Murphy. Naismith. S. 2119–2122. CCP4 (1994). Processing of X-ray diffraction data collected in oscillation mode.I. and Krammer. Smolak.. E... and Thornton... Hebert. Cha. Methods Enzymol. B.. Programmed cell death in animal development. Stuart. the 2.J. Crystallographic analysis of CD40 recognition and signaling by human TRAF2. G. 1251–1262. and the staff at SSRL beam line 7–1 for assistance with data collection. The structure of tumor necrosis ˚ resolution: implications for receptor binding. 1305–1308. D.T. 1994) were used to make figures.-S. 1994) gave a clear from 8 A rotation solution with a correlation coefficient of 30. Sung. and D). 270... D. J. et al. 946–950. Received August 26. Banner. W... Marsters. The CCP4 suite: programs for protein crystallography... Examination of sigmaa weighted 2Fo-Fc and Fo-Fc maps revealed that differences existed among the three receptor copies. and Lesslauer. simulated annealing. Y. S.

J.L. (1993). Donahue.S.G. S. H.A. A. 818–821. P.-S.. T. Kelley... 845–853.Q. Gerhart. J.. Induction of apoptosis by Apo-2 ligand.. Skubatch. Miller.. and Lazebnik. J. M... S. Smith. 5. Pitti. L. 272. N.L. Wiley.. K.. Yamagishi. K. a new member of the tumor necrosis factor cytokine family. D. Characterization of Fas (Apo-1. and Yamada. D. J. R. Griffith.-P. R. Bennett.. V. C. W. and Dixit. Structural basis for self-association and receptor recognition of human TRAF-2. Ebner. Marsters. R. Tumoricidal activity of tumor necrosis factor-related apoptosis-inducing ligand in vivo. Control of TRAIL-induced apoptosis by a family of signaling and decoy receptors.. et al.. Timour. and Goodwin.. R. 1312–1316... N. P. R. Nguyen. Nicholl.. R. Jones. Burkitt. J. Mutational analysis of structure-activity relationships in human tumor necrosis factor-␣. B..H... Baker.M. M. J. (1998). C. G. C. Chang. 309–319.Y.. V. Smith.. C. Nakano... Van Ostade. 12687–12690. Lacey. W. Chem. Structure-activity studies of human tumour necrosis factors.. P. K. Terskikh. M. Smolak.. Science 281.. J. Villa. Y. Y. A. 271. W. Din.. Nature 398. 18827–18833.M. J.J. EMBO J. J.M. Protein Eng. H. Kawashima. A.. Walczak. Identification and characterization of a new member of the TNF family that induces apoptosis. Gentz.. Boiani.. Schooley. Scuderi. Yamayoshi. Sheridan. Bodmer.R..A..A.. D. T.C. (1994). C. Fukui. M. H. Pitti. W.A. A.P.. K..V. T. Ayes.S. Science 276. Dunstan. 533–538. Protein Data Bank ID Code The coordinates have been deposited in the Protein Data Bank for immediate release (access code 1D0G). T. 16. .. Schneider. T.J. (1996). 3... Furuta. Tartaglia. Med. O’Rourke. A novel domain within the 55 kd TNF receptor signals cell death. (1995). A. G. Moore..R.. (1999).. Ni. Park.. Smith... J.. 5–22.S. Simonet. Le. TRAIL-R2: a novel apoptosis-mediating receptor for TRAIL. Lu ¨ thy. Chinnaiyan.. P. Protein Eng. M. Gurney.. Ariall. Kubin.. Tavernier. Nat.Crystal Structure of the Apo2L–DR5 Complex 571 Pan. N. 7. H. and Tschopp..E. L.. M. Thornberry. (1997b)..R. Boone. M. et al. Cell 74. Walczak. W. Wood. Gray. Baldwin. K.. Matsuo. Chin. S.C..R.. Kotani.S. (1997). H.. Woodward.D.. Delgi-Esposti. 5386–5397. (1999).. T. Science 277. and Fiers.. M.M. Rauch..A. Biol. C. 673–682. M. 713–719. Tong.A. Ohue.. et al. et al. Mattmann...... Huang. L.A.. and Goeddel. Wong. A. Sutherland. K.K. Ruppert. (1997). J. Wooden.A.. Holler. Marsters. The receptor for the cytotoxic ligand TRAIL. H. (1990).-L. Schooley.M. R. Immunity 3. 111–113.. X. C. N. Gliniak. C. Caspases: enemies within.S. M.. Smolak. R. and Ashkenazi.. Osteoprotegerin: a novel secreted protein involved in the regulation of bone density.J.. R.. Ramakrishnan. CD95)–Fas ligand interaction.... S. Waugh. and Wu... 157–163. (1997)... L... M.I. G. J. Chem... Cell 89.. Peitsch.. A. S. (1997). Biol.. Johnson.