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Structural and Functional Insights of RANKL −RANK Interaction and Signaling Changzhen Liu, Thomas S. Walter, Peng Huang, Shiqian Zhang, Xuekai Zhu, Ying Wu, Lucy R. Wedderburn, Peifu This information is current as Tang, Raymond J. Owens, David I. Stuart, Jingshan Ren and of October 1, 2021. Bin Gao J Immunol published online 14 May 2010 http://www.jimmunol.org/content/early/2010/05/14/jimmun ol.0904033 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published May 14, 2010, doi:10.4049/jimmunol.0904033 The Journal of Immunology

Structural and Functional Insights of RANKL–RANK Interaction and Signaling

Changzhen Liu,*,†,1 Thomas S. Walter,‡,1 Peng Huang,x Shiqian Zhang,{ Xuekai Zhu,*,† Ying Wu,*,† Lucy R. Wedderburn,‖ Peifu Tang,x Raymond J. Owens,‡ David I. Stuart,‡ Jingshan Ren,‡ and Bin Gao*,†,‖

Bone remodeling involves by and synthesis by and is tightly regulated by the activator of the NF-kB (RANKL)/receptor activator of the NF-kB (RANK)/ molecular triad. RANKL, a member of the TNF superfamily, induces differentiation, activation and survival upon interaction with its receptor RANK. The decoy receptor osteoprotegerin inhibits osteoclast formation by binding to RANKL. Imbalance in this molecular triad can result in diseases, including and . In this study, we report the crystal structures of unliganded RANK

and its complex with RANKL and elucidation of critical residues for the function of the receptor pair. RANK represents the longest Downloaded from TNFR with four full cysteine-rich domains (CRDs) in which the CRD4 is stabilized by a sodium ion and a rigid linkage with CRD3. On association, RANK moves via a hinge region between the CRD2 and CRD3 to make close contact with RANKL; a significant structural change previously unseen in the engagement of TNFR superfamily 1A with its ligand. The high-affinity interaction between RANK and RANKL, maintained by continuous contact between the pair rather than the patched interaction commonly observed, is necessary for the function because a slightly reduced affinity induced by mutation produces significant disruption of

osteoclast formation. The structures of RANK and RANKL–RANK complex and the biological data presented in the paper are http://www.jimmunol.org/ essential for not only our understanding of the specific nature of the signaling mechanism and of disease-related mutations found in patients but also structure based drug design. The Journal of Immunology, 2010, 184: 000–000.

he receptor activator of the NF-kB (RANK; TNFR super- and (1–5). Recently the pair was also found family [TNFRSF] 11A), and its cognate ligand, RANKL, to be important for thermoregulation (6), demonstrating them to T play a pivotal role in , immune function be one of the most versatile physiological modulators in the body. and development in conjunction with various RANK is a type I transmembrane , consisting of around 620 aas with ∼85% homology between mouse and human by guest on October 1, 2021 *The Center for Molecular Immunology and †China-Japan Joint Laboratory for homologs (7). The extracellular region (residues 30–194) is Molecular Immunology and Virology, Key Laboratory of Pathogenic Microbiology comprised of four tandem cysteine-rich pseudo-repeat domains and Immunology, Institute of Microbiology, Chinese Academy of Sciences; xDepart- ment of Orthopaedics, Chinese People’s Liberation Army General Hospital, Beijing; (CRDs) that are characteristic of the TNFRSF (8). The C- {Department of Orthopaedics, the First Clinical College of Harbin Medical Univer- terminal 383 residues form one of the largest cytoplasmic domains ‡ sity, Harbin, China Division of Structural , Oxford Protein Production Fa- in the TNFRSF. Like other members of the family this region of cility, The Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford; and ‖Unit of Rheumatology, University College London Institute of Child the protein lacks intrinsic enzymatic activity, therefore it trans- Health, London, United Kingdom duces intracellular signals by the recruitment of various adaptor 1C.L. and T.S.W. contributed equally to this work. , including TNFR-associated factors (TRAFs), leading to Received for publication December 15, 2009. Accepted for publication April 1, 2010. the activation of JNK, ERK, p38, NFATc1, AKT, and NF-kB This work was supported by the 973 Scheme of Ministry of Science and Technology signaling pathways (9–13). RANKL (TNF superfamily [TNFSF] (Grant 2006CB504306), the National Natural Science Foundation of China (Grant 11), the only ligand binding to the extracellular portion of RANK 30700749 and 30600623), the National S and T Major Project (Grant 2009ZX09503- 007), the Ministry of Science and Technology Science Exchange Program (Grant (14) was cloned respectively by four different groups (7, 15–17) 2007DFC30240), the Beijing Municipal Natural Science Foundation (Grant and identified as a member of the TNFSF. It is a type II trans- 7072072), and the United Kingdom Medical Research Council and Biotechnology , primarily expressed on the surface of activated Biological Sciences Research Council. T-cells, stromal cells, and osteoblasts. Soluble forms The coordinates and structure factors presented in this article for the mouse RANK and RANKL–RANK complex have been deposited in the with of RANKL that arise from either proteolytic processing or alter- access code 3ME4 and 3ME2, respectively, (www.rcsb.org/pdb/) for immediate re- native mRNA splicing have also been observed (18). Both the lease on publication. membrane-spanning and soluble forms of RANKL are assembled Address correspondence and reprint requests to Prof. Bin Gao and Prof. Jingshan Ren, into functional homotrimers like other members of the TNFSF. Institute of Microbiology, Chinese Academy of Sciences, 1 Beichn Xilu Road, Beijing 100101, China, or Division of Structural Bioloy, Oxford Protein Production Facility, The The binding of RANKL to RANK causes trimerisation of the Wellcome Trust Centre for Human Genetics, The Henry Wellcome Building for Genomic receptor, which activates the signaling pathway and results in Medicine, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, U.K.. E-mail ad- osteoclastogenesis from progenitor cells and the activation of ma- dresses: [email protected] and [email protected] ture osteoclasts (19–21). Abbreviations used in this paper: CRD, cysteine-rich domain; GST, glutathione S- transferase; M-CSF, -CSF; OPG, osteoprotegerin; RANK, receptor Osteoprotegerin (OPG, TNFRSF11B), a soluble homolog of activator of the NF-kB; RANKL, RANK ligand; rmsd, root mean square deviation; RANK primarily secreted by bone marrow stromal cells and TRAF, TNFR-associated factor; TNFRSF, TNFR superfamily; TNFSF, TNF super- osteoblasts, acts as a decoy receptor of RANKL to block the family; TRAP, tartrate-resistant acid phosphatase. binding of RANKL to RANK (22). The RANKL/OPG ratio is Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 differently regulated between physiological and pathological

www.jimmunol.org/cgi/doi/10.4049/jimmunol.0904033 2 STRUCTURAL INSIGHTS OF RANKL–RANK COMPLEX conditions. Various pathological conditions characterized as Materials and Methods deregulated bone remodeling are associated with an imbalance Cloning, protein expression, and purification between OPG and RANKL. Thus, RANKL, RANK, and OPG Oligonucleotides were prepared by Sangon Biotech (Shanghai, China). provide a ligand/receptor/ system for control- Restriction enzymes, T4 DNA ligase, and First Strand cDNA Synthesis ling bone homeostasis and other related biological processes. were purchased from Fermentas (Burlington, Ontario, Canada). Pfu DNA OPG-deficient mice exhibit a decrease in total bone density and polymerase was purchased from Tiangen Biotech (Beijing, China). Glu- develop osteoporosis (23). Mice with a genetic mutation of , tathione (reduced and oxidized) was purchased from Sigma-Aldrich phenotypically exactly like rankl2/2 knock-out mice (24), have (St. Louis, MO). The cDNA coding for the extracellular domain of murine RANK severely defective osteoclast development (25), which can be re- (residues 26–210) was obtained by RT-PCR from the mRNA of mouse stored by the reintroduction of rank cDNA into bone marrow RAW264.7 cells and cloned into pET28a vector (Novagen, Madison, WI). progenitor cells (26). In humans, mutations in the encoding The expression plasmid for GST–RANKL (encoding residues 159–361 of RANKL and RANK have been found to dramatically reduce the mouse RANKL as a fusion with Glutathione S-transferase) was a gift from Prof. Fremont (Washington University School of Medicine, St. Louis, number of osteoclasts and cause , a disease associ- MO). RANK was expressed with a His6 tag at each terminus of the protein. ated with a high density of bone, resulting in blindness, facial Site-directed mutagenesis of the rankl was performed using QuickChange paralysis, and deafness due to the increased pressure put on the Kit supplied by Stratagene (Agilent Technologies, Palo Alto, CA). The nerves by the extra bone (27, 28). Presumably the mutations dis- mutants were verified by DNA sequencing. Escherichia coli strain BL21- rupt the association of RANKL to its receptor (29). A human mAb Gold (DE3) was used to express the recombinant proteins. The recombinant RANK was produced as inclusion bodies that were to RANKL has been developed for treatments of post- dissolved by sonication in 6 M guanidine hydrochloride, 50 mM Tris (pH menopausal osteoporosis and rheumatoid arthritis (30, 31). 8.5), 1 mM EDTA, 150 mM NaCl, and 10 mM DTT to a protein concentration Members of the TNFSF, although structurally related, show sig- of ∼30 mg/ml at room temperature. The refolding of recombinant RANK Downloaded from nificant sequence diversity. Structures of several ligands, receptors, was performed at 4˚C by diluting the solubilized protein in 20 mM Na2HPO4 (pH 7.3), 1 M L-arginine, 20% glycerol, 10 mM reduced glutathione, and and ligand–receptor complexes have been resolved, including 1 mM oxidized glutathione, followed by sequential dialysis against 20 mM a TNF- (32), RANKL (33, 34), CD40L (35), CrmE (36), TNFRSF1A Na2HPO4 (pH 7.3), 0.5 M L-arginine, and 10% glycerol for 12 h, 20 mM (37), TNF-b–TNFRSF1A (38), and TRAIL-DR5 (39, 40) com- Na2HPO4 (pH 7.3), 0.2 M L-arginine, and 5% glycerol for 12 h, and finally plexes. Members of the TNFSF are homotrimeric with a core scaf- twice against 20 mM Na2HPO4 (pH 7.3) for 12 h. After centrifugation at fold of b-sandwich jellyroll topology, whereas members of the 20,000g for 10 min, the supernatant was further purified by size exclusion http://www.jimmunol.org/ chromatography (Superdex 200, GE Healthcare) and the correctly refolded TNFRSF consist of variable numbers of CRDs, the majority of RANK was collected and analyzed by SDS-PAGE. which comprise five irregular b-strands linked by three disulphides The soluble extracellular domain of mouse RANKL was expressed as (41). Receptor molecules bind to the clefts between the subunits of a GST fusion protein, purified by affinity purification with glutathione- the ligand trimer to form a heterohexamer. The published structures Sepharose fast flow 4B beads (GE Healthcare) according to the manufac- turer’s protocol and the tag cleaved with PreScission protease (GE health- of ligand–receptor complexes have provided detailed information care). The cleaved RANKL was further purified by size exclusion about receptor–ligand interactions and the functional mechanism chromatography (Superdex 200) in Tris pH 7.0. at atomic resolution for those pairs of molecules. Although the three-dimensional structure of mouse RANKL shows an overall fold Crystallization and data collection by guest on October 1, 2021 characteristic of TNFSF molecules, there are large structural and Purified RANKL and RANK were concentrated to 10 mg/mL in with 0.1 M conformational differences in the loops that form the receptor bind- Tris at pH 7.0. Crystallization screens of RANK and RANKL–RANK ing cleft (33, 34). In addition, the structures of TNFR molecules have complex were performed at a temperature of 294 K using nano-liter sitting drop vapor diffusion in the crystallization facility of the Oxford Protein shown great domain flexibility between the CRDs as well as struc- Production Facility (42). The best RANK crystals were grown in 10% tural flexibility within each CRD, and there is little sequence homol- polyethylene glycol 3350, 15% polyethylene glycol 5000, 0.1 M ammo- ogy among the members of the TNFRSF. The structures of RANK nium sulfate, 0.1 M sodium tartrate, and 0.05 M MES at pH 6.5. Crystals and the RANKL–RANK complex are therefore essential for our of RANKL–RANK complex were grown in 0.1 M sodium dihydrogen phosphate, 2 M sodium chloride, 0.1 M potassium dihydrogen phosphate, understanding of the basis of ligand-receptor specificity in this sys- and 0.1 M MES (pH 6.5), using a 1:1 molar ratio of RANKL and RANK tem and the mechanism of molecular signaling. (10 mg/ml). Details of protein purification and crystallization have been In this study, we report crystal structures of the extracellular published elsewhere (43). region of mouse RANK alone and in complex with the ectodomain X-ray diffraction data for RANK were collected at beamline BM14 at the of RANKL. The structure of RANK contains four full-length ESRF (Grenoble, France). A total of 180 images of 1.0˚ oscillation were collected from a single crystal at a wavelength of 0.954 A˚ . X-ray diffraction CRDs and folds into an elongated shape. There are distinct fea- data of the RANKL–RANK complex were collected at two ESRF beam- tures in CRD disulphide topology and domain connectivity. lines; 130 images of 1.0˚ oscillation from one crystal were collected at The structure of the RANKL–RANK complex, when compared beamline ID14-4 at a wavelength of 0.940 A˚ , and 180 images of 1.0˚ oscil- with the TNF-b–TNFRSF1A and TRAIL–DR5 complexes, lation were collected from two positions of a single crystal at ID23-2 oper- ated at a wavelength of 0.873 A˚ . In all cases, 25% glycerol was added to the reveals that both the position and orientation of the bound crystallization drops as cryoprotectant, and crystals were frozen and main- receptor differ significantly, and there is little conservation in tained at 100 K by a stream of nitrogen gas during data collections. Data the ligand–receptor interface contacts. A sodium ion bound images were indexed, integrated, and merged using HKL2000 (44). The betweenCRD3andCRD4ofRANKmaybecrucialformain- statistics for x-ray data are given in Table I. taining the structural integrity of the receptor and explains some Structure solution and refinement of the disease-related mutations. The affinity between RANKL and RANK has been determined using Biacore analysis, and the The space group of the RANK crystals is P212121 with unit dimensions of a = 39.8 A˚ , b = 94.3 A˚ , and c = 102.4 A˚ . The RANKL–RANK complex results (K up to 10211 M) indicate that the pair is bound strongly d crystals belong to a hexagonal space group of P63 with unit cell dimensions together. Structure-guided mutations of RANKL show that the of a and b = 121.2 A˚ and c = 94.7 A˚ . The structure of the complex was contribution of the individual residues tested to the binding of solved first using RANKL monomer (33) as a search model for molecular RANKL to RANK is directly related to RANKL signaling- replacement with MOLREP (45) of the CCP4 program suite (46). There is one RANKL subunit and one RANK molecule in the crystal asymmetric dependent osteoclast formation. A slight disruption of binding unit, giving a solvent content of 74%. The 3-fold axis of the heterohexa- between RANK and RANKL would have a dramatic effect on meric RANKL–RANK complex is aligned with the crystallographic 3-fold osteoclast formation. axis. The initial difference electron density map calculated from this partial The Journal of Immunology 3

Table I. X-ray data collection

Data collection details Data set RANK RANKL/RANK X-ray source BM14, ESRF ID23-2/ID14-4, ESRF Wavelength (A˚ ) 0.9537 0.8726/0.9395 Space group P212121 P63 Unit cell (a, b, c [A˚ ]) 39.82, 94.25, 102.42 121.23, 121.23, 94.67 Resolution rangea (A˚ ) 30.0–2.00 (2.07–2.00) 30.0–2.80 (2.90–2.80) Unique reflections 26,472 (2582) 19,756 (1970) Completeness (%) 99.6 (99.2) 100 (100) Redundancy 6.9 (6.7) 16.3 (13.8) Average I/sI 17.7 (2.3) 18.6 (2.9) Rmerge 0.096 (0.644) 0.191 (0.995) Refinement statistics Resolution range (A˚ ) 30.0–2.00 30.0–2.80 No. of reflections (working/test) 24896/1332 18581/995 b R factor (Rwork/Rfree) 0.207/0.237 0.182/0.212 No. of atoms (protein/water/others) 2433/221/47 2487/48/5 Rms bond length deviation (A˚ ) 0.007 0.007 Rms bond angle deviation (˚) 1.1 1.0 Mean B factorc (A˚ 2) 31/35/49 50/41/49

a Numbers in parentheses are for the highest resolution shell. Downloaded from b Rwork and Rfree are defined by R=Shkl||Fobs |2 |Fcalc|| / Shkl |Fobs|, where h, k, and l are the indices of the reflections (used in refinement for Rwork; 5%, not used in refinement for Rfree) and Fobs and Fcalc are the structure factors, deduced from measured intensities and calculated from the model, respectively. cMean B factors for protein, water, and others, including ions and glycerol molecules. model clearly showed the RANK polypeptide and was of sufficient quality Mouse bone marrow-derived monocytes were isolated from 7-wk-old a

to allow the RANK molecule of four CRDs to be built. The structure of BALB/c mice, cultured in -MEM containing 10% FCS, and plated in http://www.jimmunol.org/ RANK in the complex was then used to solve the crystal structure of RANK a 10-cm petri dish overnight (52). The following day, nonadherent cells alone. However, structure solution using molecular replacement was not were collected, washed, and seeded into a 24-well tissue culture plate straightforward because of the thin elongated shape and the flexibility of the (5 3 105/well) with 20 ng/ml macrophage-CSF (M-CSF) in the presence molecule. The correct solution was only found by using the central two or absence of 50 ng/ml RANKL or its mutants. From the fourth day, the CRDs with the program PHASER (47). The initial R factor was 0.54 for medium was changed daily with fresh a-MEM containing 10% FCS, data from 30 to 4.0 A˚ . There are two molecules in one crystal asymmetric 20 ng/ml M-CSF, and 50 ng/ml RANKL or its mutants. Cells were then unit related by a local 2-fold rotation axis with the N terminus of one fixed on the eighth day and stained using the TRAP staining kit as before. molecule interacting with the C terminus of the other. Superposition of the RANK model from the complex onto the molecular replacement solu- tion resulted in a large number of clashes between the first and fourth CRDs, Results indicating large conformational changes. Nevertheless, the first and fourth Structure determinations by guest on October 1, 2021 CRDs were built after a round of refinement using all data to 2.0 A˚ reso- lution. Both structures were refined with the crystallography and NMR The extracellular domain of RANK has been crystallized both system (48) using simulated annealing, conjugate gradient minimization, alone and in complex with RANKL. The crystals of RANK and the and individual isotropic B factor refinement, followed by model rebuilding RANK–RANKL complex diffracted to 2.0 A˚ and 2.8 A˚ , re- and solvent molecule addition with COOT (49). Because of the large con- spectively, using synchrotron radiation. The structure of the formational differences between the two molecules of the RANK, no non- crystallographic symmetry restraints were applied during refinement. The complex was determined first using molecular replacement with final refined structures of both RANK and RANKL–RANK complex have the published structure of RANKL as an initial model (33, 34). good crystallographic R factors and stereochemistry as shown in Table I. The crystallographic asymmetric unit contains one molecule of RANK (residues 35–199) and one subunit of RANKL (residues Surface plasmon resonance 161–316), which are assembled to form the biological hetero- The affinities of RANKL and its mutants for the receptor, RANK were hexameric complex through 3-fold crystallographic symmetry. measured using Biacore 3000 (GE Heathcare) according to the published The model has been refined to an R factor of 18.2% (R of protocol (50). Briefly, an NTA chip (GE Heathcare) was charged with 0.3 free 21.2%) with root mean square deviations (rmsds) from ideal val- M NiSO4, and then RANK (50 nM, 15 ml) was injected into the channel to load. Recombinant TNFRSF9 with two His-tags was injected into a differ- ues of 0.007 A˚ for bond lengths and 1.0˚ for bond angles (Table I). ent channel as a control. Different concentrations of RANKL or its mutants The unliganded structure of RANK was solved using the receptor (0, 1.88, 3.75, 7.5, 15, and 30 nM, 30 ml) were injected into both channels. from the complex as the search model and has been refined to an R All steps were performed at 25˚C, and signals were recorded as sensor- ˚ grams. Sensorgrams were fitted into the 1:1 binding model using BIA factor of 20.7% (Rfree of 23.7%) with rmsds of 0.007 A for bond evaluation software 4.1 (Biacore, GE Heathcare), and the equilibrium- lengths and 1.1˚ for bond angles. There are two RANK monomers dissociation constants (Kd) calculated. related by noncrystallographic 2-fold symmetry perpendicular to the long axis of the molecules in the asymmetric unit. The final Osteoclast formation and tartrate-resistant acid phosphatase model consists of residues 33–201 in one monomer and residues staining 36–176 and 186–194 in the second monomer (Fig. 1). The murine monocytic cell line RAW264.7 (American Type Culture Col- lection, Manassas, VA) was cultured in a humidified incubator (5% CO2 in The structure of RANK air) at 37˚C, and maintained in a-MEM containing 10% (v/v) heat- inactivated FCS. For osteoclastogenesis experiments (20), cells were seeded The extracellular regions of members of the TNFRSF adopt elon- into a 24-well tissue culture plate (2 3 103/well) in the presence or absence gated structures of variable numbers of pseudorepeats of CRDs. A of 50 ng/ml RANKL or its mutants for 4 d. The cells were then fixed and typical CRD, normally ∼40 residues, consists of five irregular stained using the Acid Phosphatase, Leukocyte (tartrate-resistant acid phos- b phatase [TRAP]) Kit (Sigma-Aldrich, 387A) according to the manufac- -strands linked typically by three interstrand disulphides and can turer’s instructions. The numbers of TRAP-positive, multinucleated (.3) be further divided into two structural modules of various types de- cells per well were counted under a microscope as described (51). fined by topology and number of disulphides (41). RANK contains 4 STRUCTURAL INSIGHTS OF RANKL–RANK COMPLEX

FIGURE 1. Overall structures of RANK and the RANKL–RANK complex. A, The structure of RANK alone shown as coils with CRDs 1–4 colored red, green, orange, and cyan, respectively. The disulphides are shown in yellow as ball and sticks. The magenta sphere represents the Na+ bound between CRD3 and CRD4. B, The heterodimer of RANKL–RANK complex in the crystal asymmetric unit with the receptor-free RANKL overlapped. RANK is drawn as in A, the backbone of RANKL in the complex is shown as blue ribbons and coils with selected side chains shown as cyan sticks. The backbone of receptor- free RANKL is colored in gray with regions that have large conformational changes because of receptor binding colored in red and side chains as gray sticks. C, The biological heterohexameric complex as viewed perpendicular to the 3-fold axis. Downloaded from four such CRDs spanning a length of 100 A˚ , the longest among the the key contacts with the ligand (corresponding to the 90S loop of structures of the TNFR family determined to date. RANK CRD1 DR5 and residues 103–108 of TNFRSF1A) possesses an intra- (residues 35–71) is comprised of so-called A1-B2 modules, strand disulphide formed by Cys125 and Cys127 (Fig. 2F). b2 whereas CRDs 2–4 (residues 72–114, 115–154, and 155–197, re- (residues 119–122) and b3 (residues127–130) form a regular an- spectively) are all made of A1-B1 modules, where A and B define tiparallel b-sheet linked by a four residue turn. Cys125, the third http://www.jimmunol.org/ the module topology and 1 and 2 the number of disulphides (41). Of residue of the turn, is so close to Cys127 that the plane of the turn the members of TNFR family with full-length CRD1, crystal struc- is almost perpendicular to the b-sheet. This unusual conformation tures of TNFRSF1A, OX40, and CrmE (53, 36, 54), either alone or is stabilized by hydrogen bonds from the side chain of Asn122 to in complex with ligand, have been determined. CRD1 is the most the amide groups of residues 124 and 125, and a p-stacking of structurally conserved region among these receptors; .90% of Ca Trp121 with one side of the b-sheet (Fig. 2F). atoms can be overlapped with rmsds ranging from 1.0 A˚ to 1.4 A˚ Both the number and positions of cysteine residues in the ex- despite the low sequence identity. Apart from the six conserved tracellular regions of mouse and human RANKs are conserved, and cysteines, Tyr41 and Gly54 of RANK are the only fully conserved it would be expected that all four CRDs in the human molecule are by guest on October 1, 2021 residues among the CRD1 domains of these four proteins. Tyr41, comprised of the same structural modules as found in mouse. The positioned in the middle of the second strand of the A1 module, CRD3 domain of human RANK has, however, been wrongly makes hydrophobic interactions with the first disulphide of the B2 predicted to contain A1-B1 modules lacking the 4–6 disulphides module as well as a hydrogen bond to the highly conserved Ser67 because of sequence misalignment (53), highlighting the limita- (threonines in the other three structures) from the fifth strand. tions of sequence alignment. Ser67, in turn, binds to the carboxyl group of Ser49 positioned There are significant conformational differences and rigid-body between the third and fourth cysteines in the third strand, indicating movements apparent when the three independent copies of RANK an important role of Tyr41 for stabilizing the relative position and are compared (two [A and B] from the unliganded crystal asym- orientation of the two modules. In contrast, Gly54 acts to strengthen metric unit and one [chain R] from the complex). The CXC motif the interactions between CRD1 and CRD2. It is the third residue of linking the ligand binding CRD2 and CRD3 in both TNFR1 and a tight turn linking strands 3 and 4 and makes both hydrophobic DR5 has previously been identified as a hinge region that allows interactions with the first disulphide and main-chain hydrogen the two CRDs to orientate and position themselves onto the binding bonds to the amide group of Cys72 and the carbonyl group of regions of the ligands (40). This motif is also conserved in RANK Leu78 from CRD2. These interactions are conserved between these and the hinge region appears to extend into the C-terminal half four receptors, and also observed in DR5 (40). of the CRD2 B1 module (Fig. 2A). The difference in relative The A1-B1 modules of CRDs 2–4 in RANK do not have the orientation between CRD2 and CRD3 is 20˚ between the two third and fifth cysteines (the 3–5 disulphides); in contrast to the unliganded copies, and these differ by 49˚ and 32˚ from the only previously experimentally observed A1-B1 module of CRD3 liganded molecule (Fig. 2A). In contrast, there is little rigid- in OX40 that lacks the 4–6 disulphides (53). The two missing body movement between CRD1 and CRD2, and between CRD3 cysteines are substituted by aromatic and glycine residues in and CRD4. It is interesting to note that the CRD1-CRD2 and CRD2 (His90 and Gly105) and CRD4 (Trp173 and Gly187), CRD3-CRD4 junctions both have a CXXC motif, one residue replacing the disulphide constraint by ring-stacking hydrophobic longer than the CRD2-CRD3 linker. The two cysteine residues and hydrogen bond interactions. As a result, the B1 modules in at the domain junctions are actually located in the same strand these two domains are structurally very similar to the B2 module (b5) positioned on one side of the b3b4 loop. The longer CXXC (Fig. 2). In contrast, the B1 module in CRD3 of RANK is similar linker enables the b2b3loopoftheseconddomaintocontactthe to the CRD3 B1 module of CrmE (36), in that the two cysteines othersideoftheb3 b4 loop and stabilize the two domains. In are not replaced by aromatic and glycine residues so that the contrast, the CRD3 and CRD4 of TNFRSF1A (like CRD2 and topological constraint by the disulphide is not compensated for, CRD3 in RANK) are linked by a CXC motif such that the b2b3 and the module adopts a much broader conformation (Fig. 2D). In loop is unable to closely interact with the b3b4loopofCRD3 addition, the b2b3 loop (residues 119–132) of CRD3 that makes (Fig. 2H,2I). The Journal of Immunology 5 Downloaded from

FIGURE 2. The structure of RANK. A, A diagram showing the structural differences and highlighting the flexibility of the three copies of RANK. The superposition was performed using CRD1 and CRD2 only. The RANK molecule in the complex is shown in red and the two copies in the crystal of RANK are shown in blue and yellow. B, The structure of TNFRSF1A CRD2 comprised of A1-B2 modules (pdb ID, 1ext), a typical CRD with five irregular http://www.jimmunol.org/ b-strands linked by three interstrand disulphides. The side chains of key residues for structure stability are drawn as sticks with the position of each cystine marked and numbered. The backbone direction is indicated by arrows. C–E, The structures of RANK CRDs 2–4, which are all comprised of A1-B1 modules lacking a 3–5 disulphides. F, Close-up view of the b2b3 loop of CRD3, shows how the loop is stabilized by an intrastrand disulphide and a network of hydrogen bonds (yellow broken lines). G, The coordination of the Na+ binding site. H and I, Comparison of the relative conformations between CRD3 and CRD4 in TNFRSF1A (H) and RANK (I). In TNFRSF1A, the CXC linker between the two domains positions, the b2b3 loop of CRD4 on the left side of the b3b4 loop of CRD4, allows little interaction between the two domains, whereas the CXXC linker in RANK enables the b2b3 loop of CRD4 to be positioned on the right side of the b3b4 loop of CRD3 and makes a number of stabilizing interactions.

Superposition of the first two RANK CRDs of chain Awith those involved in trimer formation by making intersubunit contacts. The by guest on October 1, 2021 of chain B results in an rsmd of 1.3 A˚ for 77 equivalent Ca atoms second b-sheet contains strands B’, B, G, D, and E and forms the (1.5 A˚ for 75 Ca atoms and 1.2 A˚ for 78 Ca atoms, respectively, outer surface. The two b-sheets form a core scaffold that is highly when the two chains are compared with the first two domains of structurally conserved, whereas the loops linking the b-strands are chain R). Large conformational differences occur in the b4b5 loop involved in receptor binding and highly variable within the TNF (residues 60–67) of CRD1 and the hinge region of the B1 module family. Superposition of the RANKL monomer from the complex (residues 90–114) of CRD2. Overlaps of CRDs 3–4 of chain R with with the previously published structure of RANKL gives an rmsd of the corresponding regions of chain A and B show rmsds of 0.9 A˚ for 0.5 A˚ for 145 Ca atoms of 156 residues. As expected, conforma- 75 Ca atoms (of 83) and 0.7 A˚ for 69 Ca atoms (of 73), re- tional changes are only observed in the surface loops of the mole- spectively. The rigidity between CRD3 and CRD4 is further en- cule, including the AA”, CD, DE, and EF loops, all of which are hanced by a metal ion bound between the two domains. This metal involved in interactions with the receptor (Fig. 1B). The largest ion exists in all three copies of RANK and has an octahedral co- conformational change observed is in the CD loop, where the Ca ordination provided by the carbonyl oxygen atoms of Cys134, atom and the ring center of Tyr234 are shifted by 3.3 A˚ and 6.5 A˚ , Ala135, and Phe138 from the b3b4 loop of CRD3, the hydroxyl respectively. group of Ser161, and the carbonyl oxygen of Val163 from the b2b3 The RANKL–RANK complex loop of CRD4 and a water molecule. The bond distances range from 2.35 to 3.00 A˚ (Fig. 2G). This ion is assigned to be a Na+ based on RANKLandRANKformaheterohexamericcomplexwithareceptor the average calcium bond-valence sum of 1.2 calculated from the molecule bound along each of the three clefts formed by neighboring averaged bond lengths from the three binding sites (expected val- monomers of the ligand homotrimer. Of the four CRDs of RANK, ues: 1.6 for Na+,K+ 0.6, Ca2+ 2.0, Mn2+ 3.2, and Mg2+ 4.2) (55). only the middle two are involved in direct contacts with the ligand. This assignment is supported by the anomalous difference map Superposition of RANKL–RANK with TNF-b–TNFRSF1A and calculated using data collected at a wavelength of 1.698 A˚ , which TRAIL–DR5 complexes reveal that, although the RANK CRD2 is shows electron density peaks for all ordered sulfur atoms but not for bound in a similar orientation as its counterparts in TNFRSF1A and this ion (f’’: S 0.7e2, Na 0.2e2, K 1.4e2, Mg 0.2e2, Mn 3.5e2, and DR5, there is a large difference in the orientation of CRD3, with Ca 1.6e2). It is expected that this Na+ is conserved in human RANK a tilt of some 45˚ and 11˚ away from the ligand, whereas the position based on the sequence similarity. of RANK as a whole is ∼2A˚ lower (Fig. 3A). Each of the three interfaces buries 2660 A˚ 2 solvent accessible The structure of RANKL surface area, 1290 A˚ 2 from the ligand, and 1370 A˚ 2 from the re- Each subunit of the trimeric RANKL adopts a typical b-sandwich ceptor. Of the surface area buried on RANKL by each receptor, 540 with jellyroll topology consisting of two five-stranded antiparallel A˚ 2 is on subunit A and 780 A˚ 2 on subunit B, whereas, of the area b-sheets. The first b-sheet, containing strands A”, A, H, C, and F, is buried on the receptor, 840 A˚ 2 is from CRD2 and 530 A˚ 2 from 6 STRUCTURAL INSIGHTS OF RANKL–RANK COMPLEX Downloaded from http://www.jimmunol.org/ FIGURE 3. The RANKL–RANK complex. A, A ribbon and coil diagram showing the positions and orientations of receptors relative to the ligands in RANKL–RANK (green), TNF-b–TNFRSF1A (red), and TRAIL-DR5 (blue) complexes by superimposing the ligands. B, Open book view of RANKL– RANK interface. Residues in the interface are colored by the percentage of surface area buried on complex formation (1–20%, blue; 20–40%, cyan; 40– 60%, green; 60–80%, orange; 80–100%, red). C–E, Close up of three key interface areas with residues in RANKL labeled in red and those in RANK labeled in black; (C) residues Asp124 and Glu126 from the b2b3 loop of the receptor form salt bridges with residues His179 and Lys180 from the AA” loop of the ligand. D, Hydrophobic interactions centered at Leu89 of the receptor. E, Interface area centered at His224 of the ligand, where the His224 is fully buried, Glu225 and Glu268 make two salt bridges to Arg129 and Arg130 of the receptor. On receptor binding the ring center of Tyr234 from the ligand has moved 6.5 A˚ to make stacking interactions with Arg129 (from red to blue). by guest on October 1, 2021

CRD3 (Fig. 3B). The areas involved in the interactions in both Lys180, whose side chain, in turn, forms a salt bridge with Glu126 receptor and ligand are significantly different from the two pub- and a hydrogen bond to the carbonyl oxygen of Ser123. The lished structures of TNF family complexes. In the TRAIL–DR5 interactions from the CD loop are mainly van der Waals contacts; complex, the buried area on TRAIL is contributed not only equally Tyr240 is fully buried, largely by Glu126 of the receptor. Tyr234 by the two repeats of the receptor, but also shared equally between undergoes the largest conformational change on receptor binding, the two ligand subunits. In the case of TNF-b–TNFRSF1A making side chain stacking interactions with Arg129, which, in complex, the CRD2 and CRD3 of TNFRSF1A contribute in turn, is stabilized by a salt bridge with Glu268 from the EF loop of a similar ratio to their counterparts of RANK, but they are made subunit B. The unique intrastrand disulphide Cys125-Cys127 of approximately equally to the two ligand subunits. A unique feature the receptor is solvent inaccessible, contacting both the CD loop of the RANKL–RANK complex is that the solvent accessible areas of subunit A and the EF loop of subunit B. The AA” loops of buried on the receptor and on subunit B of the ligand are TNFSF and the b2b3 loops of TNFRSF members are both struc- continuous, contrasting with the TNF-b–TNFRSF1A and turally the most divergent, having the greatest number of amino TRAIL–DR5 complexes, where contact regions are discontinuous acid deletions and insertions. The AA” loop in RANKL folds and form two distinct patches on both ligands and receptors. toward the top third of the molecule and is positioned above the All solvent accessible loops bridging the b-strands in RANKL, b2b3 loop of the receptor. In contrast, the much longer AA” loop apart from A”B’, B’B, and BC, are involved in receptor binding. of TRAIL runs across the middle surface of the ligand and lies These loops are structurally unique in terms of length and confor- below the b2b3 loop to make a salt bridge from Arg149 to Glu147 mation and there is little sequence conservation compared with of DR5, whereas the same loop in TNF-b is very short and does other members of the TNF family. Receptor residues from the not make any interaction with TNFRSF1A (38). Deletion mutation b2b3 loop, the b3 strand of CRD2 and the b2b3 loop of CRD3 of the AA” loop in both RANKL and TRAIL completely abolishes contribute most of the interactions. Residues involved in the li- biological activity (33, 40). The structural diversity between the gand–receptor interactions are mainly hydrophilic in nature (34 of members of the TNF family, charged interactions, and 50), forming 9 hydrogen bonds and 12 salt bridges as well as the mutagenesis all suggest that the AA” loop confers specificity. majority of hydrophobic contacts. The contact area on the lower part of RANKL subunit A is The top patch of RANKL subunit A interacts with the receptor mediated by residues Ile248 and His252 of the DE loop to Glu84 and through the AA” and CD loops to the b2b3 loop of CRD3 (Fig. Leu88 of the b2b3 loop of CRD2. Ile248 corresponds to Tyr108 in 3C). The interactions between the AA” loop and the b2b3 loop TNF-b and Tyr214 in TRAIL and has been predicted to make are predominantly hydrophilic; Asp124 in the b2b3 loop makes strong hydrophobic interactions with the receptor analogous to a salt bridge to His179 and a hydrogen bond to the main chain of those in the TNF-b–TNFRSF1A and TRAIL–DR5 complexes The Journal of Immunology 7

(38–40). Tyr108 in TNF-b is fully buried in a hydrophobic osteoclast formation, the following RANKL mutants were made depression formed by the b2b3 loop consisting of residues 60–71 according to the buried area on complex formation (Fig. 3B): of TNFRSF1A, and Tyr214 in TRAIL makes similar interactions Asn266Ala, 1–20%; Glu225Ala, 40–60%; Arg222Ala, 60–80%, with the b2b3 loop (residues 50–61, the 50S loop) of DR5; the only and Asp299Ala 80–100%. All four of these residues interact with conserved ligand–receptor interactions in the two complexes. The the receptor via either hydrogen bonds or salt bridges. The binding importance of this tyrosine has been shown by mutagenesis in affinities of RANKL mutants for immobilized RANK were mea- TNF-a, TNF-b, FasL, and TRAIL that abolishes receptor binding sured as before. The binding affinities of Glu225Ala, Arg222Ala, (39, 40, 56–58). An Ile248 Asp mutation in mouse RANKL, and Asp299Ala RANKL mutants for RANK are dramatically however, showed only an 8-fold decrease in activity (33). Ile248 decreased by .100-fold (Fig. 5A) and these mutants have com- has direct contact with a charged residue (Glu84) of RANK, at an pletely lost their ability to promote functional osteoclast formation equivalent position to Leu67 of TNFRSF1A and Leu57 of DR5, and (Fig. 5B). In contrast, amino acid Asn266 (marked in blue on Fig. the reduction in activity is likely due to the introduction of an 3B) contributes moderately to binding with ,20% buried area. Its electrostatic repulsive force. The DE loop is one of the regions that mutant Asn266Ala only marginally affects its binding to RANK 211 211 has the highest B factors and the side chains of Lys247 and Ile248 (Kd 8.8 3 10 M compared with a Kd of 6.8 3 10 M for wild do not have well-defined electron density. It is likely that in type). Interestingly, this slightly reduced affinity between RANKL RANKL, unlike other members of the TNFSF, the DE loop is not and RANK significantly affects the ability of RANKL to promote critical for receptor binding. Arg283 of the FG loop in RANKL osteoclast formation (Fig. 5B,5C), demonstrating that a strong forms a salt bridge with Asp85 that is at an equivalent position to association between RANK and RANKL is prerequisite for proper Arg68 of TNFRSF1A and Leu58 of DR5 (Fig. 4). The FG loop is RANK signaling and subsequent osteoclast formation. not involved in receptor binding in either TNF-b or TRAIL. Downloaded from There are two key interface areas between subunit B and the Discussion receptor. One, at the lower part of the interface, is mediated by the Members of the TNFSF adopt the same trimeric structural scaffold b1b2 loop and b3 of CRD2 of the receptor that interacts with the with each receptor-binding cleft formed between two neighboring GH loop and the N and C termini of the AA” loop from the ligand. ligand subunits. Receptor binding is mediated predominantly by sur- The interaction is centered on Leu89 that nests in a hydrophobic face loops with little and much structural diver- pocket formed by Tyr187, Arg190, and Gln302 of RANKL (Fig. gence between family members. The multidomain TNFRs possess http://www.jimmunol.org/ 3D). This interface area, together with the two salt bridges formed two ligand-binding CRDs with a similar overall fold (but differing from Asp94 and Lys97 of b3b4 loop at the C-terminal part of disulphide topology) and a flexible CXC domain junction. Thus, CRD2 to residues Arg222 and Asp229, appears to be the deter- for a given TNF ligand-receptor pair, the structural diversity of minant for the position and orientation of CRD2. The second key the ligand surface loops is coupled to structural variations and do- interface area is centered on His224 of the CD loop that is located main flexibility of the receptor, leading to a distinct, specific, binding in a pocket formed between the two ligand-binding repeats of the mode. It is therefore unsurprising that ligand–receptor interactions receptor (Fig. 3E). Residues lining the pocket include Ala98 from in the RANKL–RANK complex are significantly different to the the b3b4 loop of CRD2, Tyr119 from the b1b2 loop, the first b

structurally known complexes of TNF- –TNFRSF1A and TRAIL- by guest on October 1, 2021 disulphide Cys115-Cys128, and Cys128 of CRD3. The hydropho- DR5. bic interactions centered on His224 are sandwiched by two clus- The majority of residues involved in complex formation are hy- ters of charged interactions: the two salt bridges mentioned drophilic in nature, achieving both surface and electrostatic com- previously, and two additional salt bridges made from Glu225 of plementary. Of the three key interface areas identified in the the CD loop and Glu268 of the EF loop to Arg129 and Arg130 of RANKL–RANK complex (the AA” loop mediated interactions with the b3 strand of CRD3. The CD and AA” loops, located opposite the b2b3 loop of CRD3, the area centered on His224 of the ligand, each other across the receptor binding cleft, act as two anchor and the hydrophobic contacts centered on Leu89 of the receptor) points for the CRD3 of the receptor. The interactions centered none is conserved in the other two complexes, giving an indication on His224 are not observed in the TNF-b–TNFRSF1A or in the of the complexity of ligand–receptor binding in the superfamily. Our TRAIL–DR5 complexes, because of the different orientations of observations are in agreement with the notion that the interactions of the receptors. the b2b3 loop of CRD3 with the ligand may have an important role in controlling the specificity and cross-reactivity among the super- High-affinity binding is critical for functional osteoclast for- family members, but do not support the proposal that the hydropho- mation bic interaction between the DE loop of the ligand and the b2b3loop The affinity of RANKL and RANK was measured by Biacore with of CRD2 of the receptor, as observed in the complexes of TNF-b– RANK immobilized in a channel of a chelating NTA sensor chip TNFRSF1A and TRAIL-DR5, is a general feature important for and RANKL as the mobile phase. Recombinant TNFRSF9, as binding in the superfamily (39). a nonspecific protein control, was immobilized in a different The decoy receptor OPG is a soluble protein containing four N- channel in the same chip. As seen in Fig. 5A, the affinity between terminal CRDs, followed by two death domains and a C-terminal 211 RANK and RANKL is very high with a Kd of 6.8 3 10 M. basic domain. It has been shown that in vivo the protein exists in To elucidate the critical residues responsible for this very tight two states: as a homodimer cross-linked via C-terminal cysteines or binding, and the contribution of the binding affinity to functional as a C-terminal truncated monomer, both of which appear to have

FIGURE 4. Sequence alignment of the interface regions. The mouse RANK (mRANK) is aligned with its human equivalent (hRANK), the decoy receptors of both species (mOPG and hOPG) and human TNFRSF1A and DR5 (hTNR1, hDR5). Residues known to be directly involved in inter- actions are shown in red. 8 STRUCTURAL INSIGHTS OF RANKL–RANK COMPLEX Downloaded from http://www.jimmunol.org/

FIGURE 5. The affinity of RANKL–RANK interaction and the key residues involved in binding and osteoclast formation. A, The affinities of RANKL and its mutants for binding to RANK were measured by Biacore analyses using a chelating NTA sensor chip. Recombinant TNFRSF9 was used as a negative control. Different concentrations of RANKL or its mutants (0, 1.88, 3.75, 7.5, 15, and 30 nM) were injected into channels and signals recorded as sensorgrams. The equilibrium-dissociation constants were calculated using BIA evaluation software 4.1. B, TRAP staining of bone marrow-derived monocytes cells. Bone marrow-derived cells were cultured with M-CSF and in the presence or absence of 25 ng/ml RANKL or its mutants for 8 d and TRAP stained. Scale bar, 200 mm. C, RAW264.7 cells were cultured in the presence or absence of 50 ng/ml RANKL or its mutants for 4 d. The numbers of matured osteoclastic cells (TRAP-positive, multinucleated) per well were scored and expressed as means 6 SD of four wells. The experiments were repeated three times in each group. by guest on October 1, 2021 similar specific activities in the inhibition of osteoclastogenesis The last ordered residue at the C terminus of RANK in the (59). However, in a more recent report it has been shown that complex is Met199, 15 residues away from the start of the the dimerization of OPG is a result of noncovalent interactions transmembrane helix. Thus, the apex of RANKL is probably more mediated by the two death domains, and the dimer binds RANKL than 50 A˚ from the target cell surface, in contrast to the ∼6A˚ and with an affinity of three orders of magnitude tighter than the 25 A˚ for TRAIL and TNF-b. The distance between the C termini monomer lacking the death domains. One dimer interacts with of two neighboring RANK molecules is 76 A˚ , much larger than one RANKL trimer by occupying two of the three binding sites the ∼50 A˚ spacing of the DR5 transmembrane helices and the on the ligand (60). Nevertheless, aligning the OPG sequence with intracellular receptor binding sites on the trimeric TRAF our structure of RANKL–RANK complex suggests that OPG molecules, which initiate downstream signaling (61, 62). A would bind RANKL via its CRD2-3 in a similar mode to RANK greater separation between the RANK transmembrane helices is (Fig. 4). The CRD2 of OPG has the same disulphide connectivity to be expected because the RANK intracellular domain is much as the first three CRDs of TNFRSF1A, comprising A1-B2 struc- larger than that of DR5 and the spacing of the receptor binding tural modules; whereas OPG CRD3 is made of A1-B1 modules. sites on the trimeric TRAF molecules is similar. It has been The Asn131 and Leu144 at the third and fifth cystine positions of suggested that extracellular ligand-receptor binding may trigger RANK CRD3 are however substituted by a histidine and a glycine the signal cascade by dictating a precise positioning of the three in OPG; replacing the disulphide constraint by stacking and hy- transmembrane helices (40). This is in agreement with the obser- drogen bond interactions. The CRD3 of OPG is thus expected to vation of the rigid domain connection between CRD3 and CRD4 be structurally similar to the CRD2 of RANK (Fig. 2C). Most of of RANK. The sodium ion bound between these two CRDs may the key structural features observed in the RANKL–RANK com- play an important role in the RANKL–RANK signaling by main- plex are expected to be conserved in the RANKL–OPG complex, taining the structural integrity of these two domains. despite the two receptors having only 30% sequence identity. The Bone remodeling is a dynamically equilibrated process regulated b2b3 loop of CRD3 in OPG is two residues shorter and does not by the RANKL/RANK/OPG system. Perturbation of the process contain the intrastrand disulphide and so would adopt a more by mutations in genes of the molecular system results in various extended conformation to make similar interactions to the AA” bone diseases. The structure of the RANKL–RANK complex is loop of the ligand as observed for RANK (Fig. 3C). A tyrosine essential for our understanding of the structural mechanism of residue in OPG that substitutes Leu89 could fill up the extra space these disease related mutations. Four autosomal-recessive osteo- of the leucine binding pocket and make ring-stacking interactions petrosis-related mutations in the extracellular region of RANK with Tyr187 of RANKL (Fig. 3D). have been reported recently: Gly53Arg, Arg129Cys, Arg170Gly, The Journal of Immunology 9 and Cys175Arg (27), which correspond to residues of Gly54, differentiation factor osteoprotegerin-ligand is essential for mammary gland de- velopment. Cell 103: 41–50. Arg130, Lys171, and Cys176, respectively, in the mouse RANK. 5. Loser, K., A. Mehling, S. Loeser, J. Apelt, A. Kuhn, S. Grabbe, T. Schwarz, Lys171 forms a salt bridge with the conserved Asp162, which lies J. M. Penninger, and S. Beissert. 2006. Epidermal RANKL controls regulatory between the Na+ binding residues Ser161 and Val163 (Fig. 2G). A T-cell numbers via activation of dendritic cells. Nat. Med. 12: 1372–1379. + 6. Hanada, R., A. Leibbrandt, T. Hanada, S. Kitaoka, T. Furuyashiki, H. Fujihara, mutation to glycine is likely to disturb the Na binding. Cys176 J. Trichereau, M. Paolino, F. Qadri, R. Plehm, et al. 2009. Central control of forms the 4–6 disulphide of CRD4 with Cys195; removing the fever and female body temperature by RANKL/RANK. Nature 462: 505–509. disulphide constraint by an arginine mutation will result in con- 7. Anderson, D. M., E. Maraskovsky, W. L. Billingsley, W. C. Dougall, M. E. Tometsko, E. R. Roux, M. C. Teepe, R. F. DuBose, D. Cosman, and formational flexibility in the second structural module of CRD4, L. Galibert. 1997. A homologue of the TNF receptor and its ligand enhance especially the b5 strand leading to the transmembrane helix. T-cell growth and dendritic-cell function. Nature 390: 175–179. Gly54 is highly conserved in the TNFRSF, and its structural role 8. Marsters, S. A., A. D. Frutkin, N. J. Simpson, B. M. Fendly, and A. Ashkenazi. 1992. Identification of cysteine-rich domains of the type 1 has been discussed earlier. The Gly54Arg mutation is likely to receptor involved in ligand binding. J. Biol. Chem. 267: 5747–5750. cause conformational changes of the b1b2 loop of CRD2 and 9. Boyle, W. J., W. S. Simonet, and D. L. Lacey. 2003. Osteoclast differentiation and activation. Nature 423: 337–342. disrupt the interactions between Leu89 and the ligand. Arg130 10. Chung, J. Y., Y. C. Park, H. Ye, and H. Wu. 2002. All TRAFs are not created is directly involved in ligand binding, forming a salt bridge with equal: common and distinct molecular mechanisms of TRAF-mediated signal Glu225 and a hydrogen bond to Asn266. An Arg130Cys mutation transduction. J. Cell Sci. 115: 679–688. 11. Feng, X. 2005. Regulatory roles and molecular signaling of TNF family mem- would abolish these interactions. bers in osteoclasts. 350: 1–13. Three RANKL mutations, an N-terminal deletion of residues 12. Liu, W., D. Xu, H. Yang, H. Xu, Z. Shi, X. Cao, S. Takeshita, J. Liu, M. Teale, 145–177, a C-terminal truncation starting at residue 277, and and X. Feng. 2004. Functional identification of three receptor activator of NF- kappa B cytoplasmic motifs mediating osteoclast differentiation and function. a Met199Lys substitution, have been identified in patients with J. Biol. Chem. 279: 54759–54769. Downloaded from autosomal-recessive osteopetrosis (28). The deletion of 145–177 13. Takayanagi, H., S. Kim, K. Matsuo, H. Suzuki, T. Suzuki, K. Sato, T. Yokochi, results in loss of bA and half of the AA” loop. The loop interacts H. Oda, K. Nakamura, N. Ida, et al. 2002. RANKL maintains bone homeostasis through c-Fos-dependent induction of -beta. Nature 416: 744–749. with the CRD3 b2b3 loop and is crucial for receptor binding (33). 14. Xing, L., E. M. Schwarz, and B. F. Boyce. 2005. Osteoclast precursors, RANKL/ The C-terminal deletion mutation causes loss of two central strands RANK, and immunology. Immunol. Rev. 208: 19–29. 15. Wong, B. R., J. Rho, J. Arron, E. Robinson, J. Orlinick, M. Chao, S. Kalachikov, of the jellyroll scaffold, likely resulting in an unfolded ligand. The E. Cayani, F. S. Bartlett, III, W. N. Frankel, et al. 1997. TRANCE is a novel side chain of Met199 nests in a hydrophobic core between the two ligand of the tumor necrosis factor receptor family that activates c-Jun N- b-sheets and has direct contact with the backbone of Phe165 that terminal kinase in T cells. J. Biol. Chem. 272: 25190–25194. http://www.jimmunol.org/ 16. Lacey, D. L., E. Timms, H. L. Tan, M. J. Kelley, C. R. Dunstan, T. Burgess, stacks against Phe213 and Phe280 from a neighboring subunit. R. Elliott, A. Colombero, G. Elliott, S. Scully, et al. 1998. Osteoprotegerin ligand Mutation of Met199 to a charged residue in a hydrophobic envi- is a that regulates osteoclast differentiation and activation. Cell 93: ronment is expected to cause local conformational changes and 165–176. 17. Yasuda, H., N. Shima, N. Nakagawa, K. Yamaguchi, M. Kinosaki, S. Mochizuki, disturb the trimer interface. A. Tomoyasu, K. Yano, M. Goto, A. Murakami, et al. 1998. Osteoclast differentiation In summary, we have elucidated at atomic resolution the factor is a ligand for osteoprotegerin/osteoclastogenesis-inhibitory factor and is iden- structures of the extracellular domain of mouse RANK and of the tical to TRANCE/RANKL. Proc. Natl. Acad. Sci. USA 95: 3597–3602. 18. Dougall, W. C., and M. Chaisson. 2006. The RANK/RANKL/OPG triad in RANK–RANKL complex. Our data show that although the com- -induced bone diseases. Cancer Metastasis Rev. 25: 541–549. plex between RANK and its cognate ligand RANKL is similar in 19. Burgess, T. L., Y. Qian, S. Kaufman, B. D. Ring, G. Van, C. Capparelli, by guest on October 1, 2021 overall architecture to that observed for other members of the M. Kelley, H. Hsu, W. J. Boyle, C. R. Dunstan, et al. 1999. The ligand for osteoprotegerin (OPGL) directly activates mature osteoclasts. J. 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