Molecular Cell, Vol.

4, 563571, 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 OConnell,* 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 ligandreceptor 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 Apo2LDR4 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 LTTNFR1 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 ligandreceptor 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 1130) and Apo2L (residues 114281) 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 119131 and 144281 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 domaincontaining 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@

gene.com).

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Figure 1. The Apo2LDR5 Complex The Apo2L trimer is drawn as ribbon rendering in gradations of blue, and the three receptors are rendered as tubes in yellow and orange colors. The disordered loop (residues 132143) in Apo2L is rendered as small spheres. The bound zinc atom is colored green, and the bound chloride ion is pink. strands and relevant loops are labeled. (A) Side view. In this orientation, the membrane of the receptor-containing cell is at the bottom of the figure. (B) View down the 3-fold axis of the complex, perpendicular to (A).

residues 21128 of one copy and 21130 of the other two (Figure 1), together with a zinc ion, a chloride ion, and 284 water molecules (Table 1). Structure of Apo2L We have previously determined the structure of the D218A mutant of unbound Apo2L (Hymowitz et al., submitted). Like other TNF superfamily members, the Apo2L monomer consists of an all strand jelly roll composed of two flat sheets, which are formed by strands A, H, C, and F in one sheet and B, G, D, and E in the other (Figure 1A). Two small strands, A and B, are inserted between strands A and B. The core residues of the AHCF and BGDE sheets are well conserved in the four structurally characterized TNF superfamily ligands, TNF (Eck and Sprang, 1989; Jones et al., 1989), LT (Eck et al., 1992), CD40L (Karpusas et al., 1995), and Apo2L (Hymowitz et al., submitted), while the conformations of

the surface loops and side chains are extremely variable, especially near the bottom of the trimer. A unique zinc-binding site, first observed in the structure of unbound Apo2L (Hymowitz et al., submitted), is present in the structure of the Apo2LDR5 complex. 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); the zinc site is located on the 3-fold axis and buried in the trimer interface (Figure 1B). We have previously shown that this bound zinc ion is important for the structure and stability and, hence, the biological activity of Apo2L. Removal of the zinc ion decreases Apo2L apoptotic activity 90-fold and reduces the thermal stability of the trimer by 20C. The geometry of this site is identical in the complex, and the buried zinc ion does not interact with the receptor (Figure 1). While the overall structure of Apo2L bound to DR5 is

Crystal Structure of the Apo2LDR5 Complex 565

Table 1. 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, Rfree (F 0) Number of residues Number of solvent molecules Number of nonH atoms 2) Average B factor (A ) Rmsd bonds (A Rmsd angles () 2) Rmsd B (bonded atoms) (A
a b

SSRL 302.4 (2.492.40)a 0.056 (0.396)a 152,986 38,908 99.8 (99.7)a 302.4 38,850 0.222, 0.267 781 286 6577 47.6 0.013 1.7 2.4

303.5 (3.623.50)a 0.185 (0.398)a 51,527 12,459 99.8 (99.9)a

Numbers in parentheses refer to the highest resolution shell. Rsym |I I|/ I. I is the average intensity of symmetry related observations of a unique reflection. c R |Fo Fc|/Fo. Rfree is calculated as R, but for 10% of the reflections excluded from all refinement.

very similar to the structure of the free ligand, there are alterations in the conformation of several surface loops that appear to be a consequence of receptor binding. Superposition of the bound and unbound Apo2L trimers based on the C atoms of residues 120128, 145154, 162194, and 203281 gives a root-mean-square devia . In contrast, substantial changes tion (rmsd) of 0.42 A occur in the conformation of the 190s loop (residues 195201) connecting strands C and D and in the 150s loop (residues 154162) linking strands A and A. It is noteworthy that these loops are in contact with the receptor (see below). The 190s loop is ordered in this structure but was disordered in the unbound Apo2L structure. The 150s loop is in a significantly different conformation than in the free ligand (with an rmsd for ), including a 0.5 A shift in the the C atoms of 1.9 A position of the buried side chain of Trp-154. The 150s loop was in a crystal packing contact in the unbound structure, which may have influenced its conformation. More subtle changes between the structures are found around Tyr-216 and Pro-217, which are at the heart of the Apo2LDR5 interface (below). The Overall Structure of DR5 DR5 is a member of the TNF receptor superfamily. Prior to the structure of the Apo2LDR5 complex, the only structural information about these receptors was for TNFR1, both unbound (Naismith et al., 1995, 1996) and in complex with LT (Banner et al., 1993). These studies showed that the extracellular region of TNFR1 is an extended structure composed of four pseudorepeats or subdomains, each containing three disulfide bridges. These pseudorepeats, termed cysteine-rich domains (CRDs), are approximately 40 residues long. DR5 resembles TNFR1 in overall structure with relatively little defined secondary structure (Figure 2). It is tethered into an elongated shape by a series of seven disulfide bridges, six of which are found in subdomains of DR5 (residues 4384 and 85130, respectively) that correspond structurally to the second and third CRDs of TNFR1.

The first disulfide bridge of DR5, between residues 28 and 41, corresponds to the last disulfide bridge (between Cys-33 and Cys-52) in CRD1 of TNFR1, while the first 20 residues of DR5 are disordered. Thus, DR5 residues 142, 4385, and 86130 form analogous subdomains to CRD1, CRD2, and CRD3 of TNFR1. The three copies of DR5 in the complex are very similar to each other, with the exception of the C-terminal portion of CRD3 (residues 104130), which exhibits a rigid-body variation in orientation (Figure 2). The two loops that form most of the contacts with the ligand (below) have very similar conformations in all three copies (Figure 2). Unlike the LTTNFR1 complex, where the C-terminal subdomain of the receptor was disordered, 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). Residue 130 is predicted to be the final extracellular residue before the putative single transmembrane helix connecting the receptor to its cytoplasmic death domain. This allows us to inspect the geometry of the complex at the point where the receptors would enter the cell membrane. In our complex, the C termini of the receptors form a trian on a side (Figure 1B). Intriguingly, gle approximately 50 A this same spacing is also found for the receptor-binding sites on the TRAF-2 trimer, which is known to interact with the intracellular portions of some TNFR superfamily members (albeit not with DR4 or DR5) (McWhirter et al., 1999; Park et al., 1999). 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. The LigandReceptor Interface The Apo2LDR5 complex is composed of a wedgeshaped Apo2L homotrimer with three receptors bound diagonally along the crevices in the Apo2L monomer monomer interfaces. Each of these three extensive inter 2 of solvent-accessible action surfaces buries 2750 A

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Figure 2. Superposition of the Three Copies of DR5, 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, and the 50s and 90s loops are highlighted in shades of gray. The approximate boundaries of CRD1, CRD2, and CRD3 are marked.

98 and 99 (Figure 4A). Substitution of this residue by alanine reduced apoptotic activity of the resulting mutant by 700-fold. Of the other functionally important residues, only Glu-236 buries less than 50% of its accessible surface area in the interface, but its substitution by alanine causes a substantial loss of DR5 binding. Other residues only moderately important for activity are found in the periphery of the patch but do generally interact with the receptor. For instance, Arg-149 forms a salt bridge to receptor residue Glu-94, and Asn-199, Lys201, and Asp-203 form polar interactions with Arg-101, Lys-102, Arg-104, and Asp-122 of DR5 (Figures 3 and 4A). Of the residues found to be important for activity, only Ser-259 does not participate in the Apo2LDR5 interface. Interestingly, alanine substitution of this residue affects binding against DR4 but not DR5. Therefore, the decrease in activity of the alanine mutant may be solely due to deleterious effects on DR4 binding. 2, Patch B buries a solvent-accessible area of 890 A 2 from the receptor and 410 A 2 from the ligand. In 480 A this patch, the 50s loop of the receptor interacts with the 210s and the 150s loops of Apo2L (Figure 4B). This interaction is centered on Apo2L residue Tyr-216, which binds in a hydrophobic groove on the receptor surface formed by the side chains of DR5 residues His 53, Asn55, Leu-57, Leu-58, and Phe-59. Tyr-216 had been identified by alanine scanning mutagenesis as a critical residue for bioactivity and receptor binding (Hymowitz et al., submitted) and is conserved in many TNF homologs. 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. These peripheral interactions are not critical for high-affinity binding, since substitutions of these Apo2L residues with alanine had only marginal effects on binding or activity. The Domain Structure in the TNF Receptor Superfamily The TNF receptor superfamily is remarkable for its structural preservation despite a minimum of sequence conservation. TNFR1, the prototype of the superfamily, has four pseudorepeats of a cysteine-rich motif (a CRD or cysteine-rich subdomain) (Figure 5) (Banner et al., 1993; Bazan, 1993). Each of these subdomains contains six cysteines in three disulfide bridges. Crystal structures of this receptor have shown that the first three of these CRDs exhibit a disulfide-bonding pattern 12, 35, and 46. The final subdomain, CRD4, has a different topology, and the cysteine connectivity is 12, 36, and 45 (Figure 5; Naismith et al., 1996; Naismith and Sprang, 1998). The CRD terminology is less precise when applied to other members of the superfamily. For instance, DR4, DR5, DcR1, and DcR2 have been predicted to have two CRDs, in contrast to the three subdomains believed to be present in Fas. In fact, the extracellular domains of DR5 and Fas are roughly the same size (Figure 5), and our structure shows that like Fas, DR5 has at least part of CRD1 and all of CRD2 and CRD3. The difference between these receptors is that subdomain 1 in DR5 has only a single disulfide bridge, corresponding to the last disulfide in CRD1 of TNFR1 (Figures 5 and 6). The structurally conserved and functionally important

2 from the receptor and 1350 A 2 surface area, 1400 A from the ligand. Two receptor loops mediate most of the interactions, dividing the interface in two distinct patches (Figure 3): the 50s loop (residues 5165) and the 90s loop (residues 91104). In patch A, 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). Patch B involves general hydrophobic features of TNF-like ligands and appears to be important for binding in complexes throughout the superfamily, while patch A may control the specificity and cross-reactivity among the different superfamily members (see below). 2 Patch A is the larger of the two patches, with 1790 A 2 from the of total buried accessible surface area (880 A 2 from the ligand). The 90s loop of receptor and 910 A DR5 contributes 85% of the buried surface area (750 2), while the remaining 130 A 2 is a result of small contriA butions from receptor residues 6569 and 108, 111, 122, and 125. Four of the five residues of Apo2L identified by alanine scanning as important for biological activity are located in this patch (Hymowitz et al., submitted). The most important of these residues is Gln-205, which hydrogen bonds to the backbone of receptor residues

Crystal Structure of the Apo2LDR5 Complex 567

Figure 3. Open Book View of the Apo2LDR5 Interface Apo2L and one receptor are rendered as space-filling models, while the other two receptors are shown as green tubes. Residues in the interface are colored by percent of buried accessible surface area upon complex formation (1%25%, light yellow; 25%50%, yellow; 50%75%, was used to calculate accessible orange; 75%100%, red). The interface divides into two patches, A and B (labeled). A probe size of 1.4 A surface area.

portion of these receptors is the actual ligand-binding motif found in CRD2 and CRD3 (corresponding to DR5 residues 43 to 130). All of the TNF receptor superfamily members are expected to have these central subdomains, including the disulfide bonding patterns that present the functionally important 50s and 90s loops. The final TNFR1 subdomain, CRD4, which is also present in OPG but not in DR5 and Fas (Figure 5), does not appear to be important for ligand binding; it could have a role in transducing the TNF binding signal into the cell. A high-resolution structure of unliganded TNFR1 shows that this final CRD has different disulfide connectivity than the other subdomains (Naismith et al., 1996). Comparison to the LTTNFR1 Complex The 130-residue extracellular domain of DR5 shares only 16 noncysteine residues with TNFR1. However, 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). This structural similarity is particularly pronounced in the central third of the receptor. Superimposing the C atoms of residues 2734 and 4867, corresponding to the C-terminal portion of CRD1 and the structurally identical residues in CRD2, with their counterparts from TNFR1 results in an rmsd of only . However, while the N-terminal portions of the 0.7 A receptors are similar, the C-terminal subdomains are rotated with respect to each other, resulting in a 35 displacement of the structurally equivalent residues A (Figure 6). When the structures of the receptor complexes of Apo2L and LT are superimposed by aligning

the structurally equivalent strands in the ligands, the C-terminal portions of the receptors differ even more ). Similar movement, but not as extreme, (by up to 10 A was seen in a comparison of bound and unbound TNFR1. 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 LTTNFR1 complex (Banner et al., 1993), while the C-terminal portion of DR5 is in intimate contact with the 200s loop of 2 larger Apo2L. This difference also results in a 550 A contact interface with the ligand, despite the shorter overall length of DR5 compared to the TNFR1 sequence. 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), which interacts extensively with the ligand in both structures.

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. Tyr-216 is conserved in many of the TNF superfamily ligands (including TNF, LT, FasL, and OPGL; Figure 7B), while many other members have a similar large hydrophobic residue at this position. Mutagenesis studies on TNF, LT, FasL, and Apo2L have all shown that this residue is critical for binding (Van Ostade et al., 1994; Yamagishi et al., 1990; Goh et al., 1991; Schneider et al., 1997; Hymowitz et al., submitted). The interactions of the tyrosine side chain are conserved between the Apo2LDR5 and LTTNFR1 complexes.

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Figure 4. Wall-Eyed Stereo View Showing Details of the Apo2LDR5 Interface The Apo2L monomers are colored gray and dark pink, and receptor residues are colored orange. The backbone atoms are represented as a C trace; relevant side chains are shown. (A) The specificity patch (patch A) centered on Gln-205 of Apo2L interacting with the receptor 90s loop. Hydrogen bonds made by Gln-205 to the backbone of receptor residues 98 and 99 are shown as dashed lines. (B) The hydrophobic binding patch (patch B) between the receptor 50s loop and ligand residue Tyr-216.

Moreover, the backbone conformation of the 50s loop of the receptor, which forms the binding pocket for the side chain, is virtually identical between DR5 and the between the equivalent C TNFR1 (rmsd of only 0.35 A

Figure 5. Schematic Depiction of the TNFR Family Subdomain Structures Schematic depiction of the sudomain structures of Fas, OPG, TNFR1, and DR5, 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, which connects to the transmembrane helix, is at the bottom. The CRDs are shown as oval symbols and labeled, with the ligand-binding domains in red.

atoms of residues 51 to 62) (Figure 6). Additionally, the length of this loop is conserved among the different receptor superfamily members. We suggest that this loop functions as a general hydrophobic binding patch interacting with conserved hydrophobic features on the ligand; this interaction may help properly orient the upper part of the receptor for more specific contacts mediated by CRD3. In contrast to the conserved interactions in patch B, patch A near the bottom of the interface (Figure 3) involves interactions made by the 90s loop on CRD3 of DR5, which has a completely different conformation than the corresponding loop in the TNFR1 (Figure 6). Interestingly, receptors that can bind to the same ligand have conserved features in their 90s loops. For instance, DR5, DR4, DcR1, and DcR2 have seven identical residues in that loop, including the two central residues Glu-98 and Met-99 (Figure 7A). The potential role of this loop is further highlighted by the observed crossreactivity between Apo2L and OPG (Emery et al., 1998). Apo2L binds to OPG but not to Fas. The explanation for this may lie in the identity of the residue at position 205 of the ligand, accompanied by the sequence of the receptor 90s loop. Residue 205 is known to be important for high-affinity binding by both Apo2L (Hymowitz et al., submitted) and FasL (Schneider et al., 1997). Both Apo2L and OPGL have a glutamine at this position, while FasL has a proline (Figure 7B). 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). Given that the 90s loop of DR5 is generally similar to that of OPG, despite the fact that the OPG

Crystal Structure of the Apo2LDR5 Complex 569

Figure 7. Sequence Alignments of the Main Interface Regions The residues in DR5, TNFR1, Apo2L, and LT that bury more than 50% of their accessible surface area in the interface are colored red. Cysteines are highlighted in yellow. Residues of the ligands and receptors at the center of the observed (Apo2DR5, LTTNFR1) or predicted (FasFasL, OPGOPGL, TNFTNFR1, DR4, DR5, DcR1, DcR2) binding patches are boxed. (A) The 50s and 90s loops in DR5, DR4, DcR1, DcR2, OPG, Fas, and TNFR1. (B) The segment around residues 205 and 216 of Apo2L for Apo2L, OPGL, FasL, TNF or TNF, and LT or TNF.

Figure 6. Superposition of DR5 with TNFR1 TNFR1 (in lavender and gray) is taken from the lymphotoxin complex (Banner et al., 1993) and superimposed on DR5 (in red and black) based on the shared C atoms of CRD2. The sulfur atoms of the disulfide bonds in DR5 and TNFR1 are shown as yellow and orange spheres, respectively, and the 90s and 50s loops are highlighted in gray and black. The three CRDs are labeled. CRD2 and CRD3 mediate all contacts to the ligand.

loop is three residues shorter, this interaction is probably conserved in the OPGLOPG complex and also when Apo2L binds to OPG. In contrast, the 90s loop is considerably different in TNFR1 and in Fas, which do not crossreact with each others ligands or with Apo2L. In TNFR1, the loop is longer and has no sequence identity with DR5 other than the cysteines; as noted above, it also has a completely different conformation. For Fas, a crystal structure is not available, but cysteine residues are present at positions 92 and 97 (Figure 7). These are likely to form an additional disulfide bridge, which would drastically alter the conformation of the 90s loop. While further structural and mutagensis experiments are necessary to confirm this analysis, 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, 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.
Experimental Procedures Expression and Purification Apo2L (residues 114281) was expressed in E. coli and purified as described (Ashkenazi et al., 1999). DR5 (residues 1130) was expressed in Hi5 insect cells with a baculovirus transfer vector under

the control of a polyhedron promoter. Protein was secreted from cells grown at 27C over 72 hr, and the DR5-containing medium was separated from the cells by centrifugation. The supernatant was run over a Q-Sepharose (Pharmacia) column, and the protein was eluted with a 01 M NaCl gradient in 20 mM Tris-HCl (pH 8.0). The fractions containing DR5 were pooled and loaded onto a CNBrApo2L affinity column. The column was washed with 0.5 M NaCl in 20 mM Tris-HCl (pH 8.0), and DR5 was eluted with 2 M KSCN in 50 mM Tris (pH 8.0). DR5 was further purified by size exclusion chromatography (S-200, Pharmacia). Apo2L in 20 mM Tris-HCl (pH 8.0) was added to purified DR5 in approximately equimolar concentrations, and the complex was purified by size exclusion chromatography (S-75, Pharmacia) in 100 mM NaCl, 20 mM Tris-HCl (pH 8.0). The fraction containing the Apo2LDR5 complex was further purified by anion exchange chromatography (MonoQ, Pharmacia) and eluted with a 01 M NaCl gradient in 20 mM Tris (pH 8.0). The complex was then concentrated to approximately 3.7 mg/mL and buffered with 20 mM Tris-HCl (pH 8.0), 0.1 M NaCl. Crystallization and Data Collection Crystals of the Apo2LDR5 complex were grown by vapor diffusion at 19C using the hanging drop method. The initial crystals were grown in condition 37 of the Hampton Crystal Screen II (10% PEG 8000, 8% ethylene glycol, 0.1 M HEPES [pH 7.5]). 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, 10% ethylene glycol, 0.2 M ammonium sulfate, 0.1 M Tris-HCl (pH 7.5) and grew to a size of 0.3 mm 0.15 mm 0.1 mm. The crystals were transferred briefly to a droplet containing reservoir solution with 20% glycerol before flash freezing in liquid nitrogen. The crystals belonged to space group P212121 and had unit cell dimensions a , b 112.0 A , c 130.8 A . The asymmetric unit contained 66.8 A data set one Apo2L trimer and three copies of the receptor. A 3.5 A was collected on a MAR imaging plate system using a Rigaku rotating anode generator with CuK radiation. A subsequent data set to resolution was collected from a single crystal at beam line 2.4 A ). 71 of the Stanford Synchrotron Radiation Laboratory ( 1.08 A

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The data sets were processed using the programs in the HKL package (Otwinowski and Minor, 1997). Structure Determination and Refinement The Apo2LDR5 structure was determined by molecular replace resolution structure of Apo2L alone (Hymowitz ment using the 1.3 A et al., submitted) as the search model in combination with 3-fold noncrystallographic symmetry (NCS) map averaging. Using all data to 4 A , the program AMoRe (CCP4, 1994) gave a clear from 8 A rotation solution with a correlation coefficient of 30.4% (the highest incorrect peak had a correlation coefficient of 7.3%). The solution to the translation function had an initial Rfree (Bru nger, 1992a) of and 42.4% following rigid-body fitting using all data between 8 A . Similar searches using the structure of Apo2L bound to a 3.5 A homology model of DR5 based on the LTTNFR1 complex resulted in worse molecular replacement statistics and were abandoned. A 3fold NCS-averaged and solvent-flattened map (program DM [CCP4, 1994]) phased using the solution for the Apo2L trimer revealed partial density for the receptors. Three cycles of model building, NCS mask refinement, and density averaging allowed building of receptor residues 22130. This model was refined with X-PLOR 98.1 (Bru nger, 1992b) as modified by Molecular Simulations, Inc., using a maximum likelihood target function, NCS constraints, positional refinement, simulated annealing, and grouped B factors until Rfree reached 33.5%. This partially refined model was then further refined against data set, using programs X-PLOR and REFMAC (CCP4, the 2.4 A 1994). Examination of sigmaa weighted 2Fo-Fc and Fo-Fc maps revealed that differences existed among the three receptor copies, especially in residues 100 to 130. In subsequent refinement, the Apo2L trimer was subject to tight NCS restraints, while weak restraints were applied to the most similar regions of the receptors. An overall anisotropic B factor correction was applied to the data as was a real-space bulk-solvent correction (Bru nger, 1992b). The final model consists of residues 119 to 131 and 144 to 281 of each Apo2L monomer (A, B, and D), receptor chains R (residues 21 to 128) and S and T (residues 21 to 130), a zinc and a chloride ion, and 284 water molecules. Of all nonglycine residues, 87% are in the most favored (Laskowski et al., 1993) region of the Ramachandran plot, two residues are in the generously allowed regions, and no residues are in the disallowed regions. Refinement and model statistics are shown in Table 1. Molscript (Kraulis, 1991) and Raster3D (Merrit and Murphy, 1994) were used to make figures. Acknowledgments We thank Roger Pai and Susan Leung for providing purified Apo2L, Christian Wiesmann, Felix Vajdos, Charles Eigenbrot, and the staff at SSRL beam line 71 for assistance with data collection, Jenny Stamos for help with and advice on insect cell culture, as well as our colleagues in mass spectrometry and protein sequencing. Received August 26, 1999; revised September 13, 1999. References Ashkenazi, A., and Dixit, V.M. (1998). Death receptors: signaling and modulation. Science 281, 13051308. Ashkenazi, A., Pai, R.C., Fong, S., Leung, S., Lawrence, D.A., Marsters, S.A., Blackie, C., Chang, L., McMurtrey, A.E., Hebert, A., et al. (1999). Safety and anti-tumor activity of recombinant soluble Apo2 ligand. J. Clin. Invest. 104, 155162. Banner, D.W., DArcy, A., Janes, W., Gentz, R., Schoenfeld, H.J., Broger, C., Loetscher, H., and Lesslauer, W. (1993). Crystal structure of the soluble human 55 kd TNF receptorhuman TNF beta complex: implications for TNF receptor activation. Cell 73, 431445. Bazan, J.F. (1993). Emerging families of cytokines and receptors. Curr. Biol. 3, 603606. Bru nger, A.T. (1992a). Free R value: a novel statistical quantity for assessing the accuracy of crystal structures. Nature 355, 472475. Bru nger, A.T. (1992b). X-PLOR Manual, Version 3.1 (New Haven, Connecticut: Yale University). Cha, S.-S., Kim, M.-S., Choi, Y.H., Sung, B.-J., Shin, N.K., Shin,

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