Coupling of bone resorption and formation by RANKL reverse signalling.

Receptor activator of nuclear factor-kappa B (RANK) ligand (RANKL) binds RANK on the surface of osteoclast precursors to trigger osteoclastogenesis. Recent studies have indicated that osteocytic RANKL has an important role in osteoclastogenesis during bone remodelling; however, the role of osteoblastic RANKL remains unclear. Here we show that vesicular RANK, which is secreted from the maturing osteoclasts, binds osteoblastic RANKL and promotes bone formation by triggering RANKL reverse signalling, which activates Runt-related transcription factor 2 (Runx2). The proline-rich motif in the RANKL cytoplasmic tail is required for reverse signalling, and a RANKL(Pro29Ala) point mutation reduces activation of the reverse signalling pathway. The coupling of bone resorption and formation is disrupted in RANKL(Pro29Ala) mutant mice, indicating that osteoblastic RANKL functions as a coupling signal acceptor that recognizes vesicular RANK. RANKL reverse signalling is therefore a potential pharmacological target for avoiding the reduced bone formation associated with inhibition of osteoclastogenesis.

Comparison of Osteoconductive Ability of Two Types of Cholesterol-Bearing Pullulan (CHP) Nanogel-Hydrogels Impregnated with BMP-2 and RANKL-Binding Peptide: Bone Histomorphometric Study in a Murine Calvarial Defect Model.

The receptor activator of NF-κB ligand (RANKL)-binding peptide is known to accelerate bone morphogenetic protein (BMP)-2-induced bone formation. Cholesterol-bearing pullulan (CHP)-OA nanogel-crosslinked PEG gel (CHP-OA nanogel-hydrogel) was shown to release the RANKL-binding peptide sustainably; however, an appropriate scaffold for peptide-accelerated bone formation is not determined yet. This study compares the osteoconductivity of CHP-OA hydrogel and another CHP nanogel, CHP-A nanogel-crosslinked PEG gel (CHP-A nanogel-hydrogel), in the bone formation induced by BMP-2 and the peptide. A calvarial defect model was performed in 5-week-old male mice, and scaffolds were placed in the defect. In vivo µCT was performed every week. Radiological and histological analyses after 4 weeks of scaffold placement revealed that the calcified bone area and the bone formation activity at the defect site in the CHP-OA hydrogel were significantly lower than those in the CHP-A hydrogel when the scaffolds were impregnated with both BMP-2 and the RANKL-binding peptide. The amount of induced bone was similar in both CHP-A and CHP-OA hydrogels when impregnated with BMP-2 alone. In conclusion, CHP-A hydrogel could be an appropriate scaffold compared to the CHP-OA hydrogel when the local bone formation was induced by the combination of RANKL-binding peptide and BMP-2, but not by BMP-2 alone.

A missense mutation sheds light on a novel structure-function relationship of RANKL.

The tumor necrosis factor (TNF)-like core domain of receptor activator of nuclear factor-κB ligand (RANKL) is a functional domain critical for osteoclast differentiation. One of the missense mutations identified in patients with osteoclast-poor autosomal recessive osteopetrosis (ARO) is located in residue methionine 199 that is replaced with lysine (M199K) amid the TNF-like core domain. However, the structure-function relationship of this mutation is not clear. Sequence-based alignment revealed that the fragment containing human M199 is highly conserved and equivalent to M200 in rat. Using site-directed mutagenesis, we generated three recombinant RANKL mutants M200K/A/E (M200s) by replacing the methionine 200 with lysine (M200K), alanine (M200A), and glutamic acid (M200E), representative of distinct physical properties. TRAcP staining and bone pit assay showed that M200s failed to support osteoclast formation and bone resorption, accompanied by impaired osteoclast-related signal transduction. However, no antagonistic effect was found in M200s against wild-type rat RANKL. Analysis of the crystal structure of RANKL predicted that this methionine residue is located within the hydrophobic core of the protein, thus, likely to be crucial for protein folding and stability. Consistently, differential scanning fluorimetry analysis suggested that M200s were less stable. Western blot analysis analyses further revealed impaired RANKL trimerization by M200s. Furthermore, receptor-ligand binding assay displayed interrupted interaction of M200s to its intrinsic receptors. Collectively, our studies revealed the molecular basis of human M199-induced ARO and elucidated the indispensable role of rodent residue M200 (equivalent to human M199) for the RANKL function.

Irijimasides A-E, Macrolide Glycosides from an Okeania sp. Marine Cyanobacterium.

Irijimasides A-E (1-5), a series of new 14-membered macrolide glycosides, were isolated from a marine cyanobacterium collected in Okinawa. The gross structures of 1-5 were established by spectroscopic analysis, including 2D NMR, while absolute stereostructures were determined based on NOESY spectra, chemical derivatization, and ECD data. All five macrolides suppressed receptor activator of nuclear factor-κB ligand (RANKL)-induced tartrate-resistant acid phosphatase (TRAP) activity in mouse RAW264 macrophage cells, indicating that these compounds inhibit osteoclast formation.

Jatrophane Diterpenoids from Euphorbia esula as Inhibitors of RANKL-Induced Osteoclastogenesis.

Eighteen new jatrophane diterpenoids, euphoesulatins A-R (1-18), and three known diterpenoids (19-21) were isolated from Euphorbia esula. Compounds 1-7, 14, and 18 represent a rare type of jatrophane-type diterpenoid containing a nicotinoyloxy group. The absolute configuration of 1 was determined by X-ray crystallography. The compounds were assayed for their antiosteoporotic activity in a bone-marrow-derived macrophage cell line, and compounds 1, 8, and 10 significantly inhibited the formation of osteoclasts, with IC50 values of 1.2, 3.5, and 2.3 µM, respectively. These three compounds also dose-dependently reduced the activity of nuclear factor activated T-cell cytoplasmic 1. This study reveals the antiosteoporotic effects of jatrophane diterpenoids for the first time.

Betulinic Acid Inhibits RANKL-Induced Osteoclastogenesis via Attenuating Akt, NF-κB, and PLCγ2-Ca2+ Signaling and Prevents Inflammatory Bone Loss.

The increase of bone-resorbing osteoclast activity in bone remodeling is the major characteristic of various bone diseases. Thus, inhibiting osteoclastogenesis and bone-resorbing function may be an effective therapeutic target for bone diseases. Betulinic acid (BA), a natural plant-derived pentacyclic triterpenoid compound, is known to possess numerous pharmacological and biochemical properties including anti-inflammatory, anticancer, and antiadipogenic activity. However, the effect of BA on osteoclast differentiation and function in bone metabolism has not been demonstrated so far. In this study, we investigated whether BA could suppress RANKL-induced osteoclastogenesis and bone resorption. Interestingly, BA significantly suppressed osteoclastogenesis by decreasing the phosphorylation of Akt and IκB, as well as PLCγ2-Ca2+ signaling, in pathways involved in early osteoclastogenesis as well as through the subsequent suppression of c-Fos and NFATc1. The inhibition of these pathways by BA was once more confirmed by retrovirus infection of constitutively active (CA)-Akt and CA-Ikkß retrovirus and measurement of Ca2+ influx. BA also significantly inhibited the expression of osteoclastogenesis-specific marker genes. Moreover, we found that BA administration restored the bone loss induced through acute lipopolysaccharide injection in mice by a micro-CT and histological analysis. Our findings suggest that BA is a potential therapeutic candidate for bone diseases involving osteoclasts.

Evaluation of Isoflavones as Bone Resorption Inhibitors upon Interactions with Receptor Activator of Nuclear Factor-κB Ligand (RANKL).

Receptor activator of nuclear factor-κB ligand (RANKL) is a cytokine responsible for bone resorption. It binds its receptor RANK, which activates osteoporosis. High levels of osteoprotegerin (OPG) competitively binding RANKL limit formation of ligand-receptor complexes and enable bone mass maintenance. The new approach to prevent osteoporosis is searching for therapeutics that can bind RANKL and support OPG function. The aim of the study was to verify the hypothesis that isoflavones can form complexes with RANKL limiting binding of the cytokine to its receptor. Interactions of five isoflavones with RANKL were investigated by isothermal titration calorimetry (ITC), by in silico docking simulation and on Saos-2 cells. Daidzein and biochanin A showed the highest affinity for RANKL. Among studied isoflavones coumestrol, formononetin and biochanin A showed the highest potential for Saos-2 mineralization and were able to regulate the expression of RANKL and OPG at the mRNA levels, as well as osteogenic differentiation markers: alkaline phosphatase (ALP), collagen type 1, and Runt-related transcription factor 2 (Runx2). Comparison of the osteogenic activities of isoflavones showed that the use of physicochemical techniques such as ITC or in silico docking are good tools for the initial selection of substances showing a specific bioactivity.

Dimerization interface of osteoprotegerin revealed by hydrogen-deuterium exchange mass spectrometry.

Previous structural studies of osteoprotegerin (OPG), a crucial negative regulator of bone remodeling and osteoclastogenesis, were mostly limited to the N-terminal ligand-binding domains. It is now known that the three C-terminal domains of OPG also play essential roles in its function by mediating OPG dimerization, OPG-heparan sulfate (HS) interactions, and formation of the OPG-HS-receptor activator of nuclear factor κB ligand (RANKL) ternary complex. Employing hydrogen-deuterium exchange MS methods, here we investigated the structure of full-length OPG in complex with HS or RANKL in solution. Our data revealed two noteworthy aspects of the OPG structure. First, we found that the interconnection between the N- and C-terminal domains is much more rigid than previously thought, possibly because of hydrophobic interactions between the fourth cysteine-rich domain and the first death domain. Second, we observed that two hydrophobic clusters located in two separate C-terminal domains directly contribute to OPG dimerization, likely by forming a hydrophobic dimerization interface. Aided by site-directed mutagenesis, we further demonstrated that an intact dimerization interface is essential for the biological activity of OPG. Our study represents an important step toward deciphering the structure-function relationship of the full-length OPG protein.

A new vaccine targeting RANKL, prepared by incorporation of an unnatural Amino acid into RANKL, prevents OVX-induced bone loss in mice.

Bone homeostasis is maintained by a dynamic balance between osteoblastic bone formation and osteoclastic bone resorption. The receptor activator of nuclear-κB ligand (RANKL) is essential for the function of the bone-resorbing osteoclasts, and targeting RANKL has been proved highly successful in osteoporosis patients. This study aimed to design a novel vaccine targeting RANKL and evaluate its therapeutic effects in OVX-induced bone loss model. Anti-RANKL vaccine was generated by incorporating the unnatural amino acid p-nitrophenylalanine (pNO2Phe) into selected sites in the murine RANKL (mRANKL) molecule. Specifically, mutation of a single tyrosine residue Tyr234 (Y234) or Tyr240 (Y240) of mRANKL to pNO2Phe (thereafter named as Y234pNO2Phe or Y240pNO2Phe) induced a high titer antibody response in mice, whereas no significant antibody response was observed for the wild type mRANKL (WT mRANKL). The antiserum induced by Y234pNO2Phe or Y240pNO2Phe could efficiently prevent osteoclastogenesis in vitro. Moreover, immunization with Y234pNO2Phe or Y240pNO2Phe could also prevent OVX-induced bone loss in mice, suggesting that selected pNO2Phe-substituted mRANKL may pave the way for creating a novel vaccine to treat osteoporosis.

Heparan Sulfate Regulates the Structure and Function of Osteoprotegerin in Osteoclastogenesis.

Osteoprotegerin (OPG), a decoy receptor secreted by osteoblasts, is a major negative regulator of bone resorption. It functions by neutralizing the receptor activator of nuclear factor κB ligand (RANKL), which plays a central role in promoting osteoclastogenesis. OPG is known to be a high-affinity heparan sulfate (HS)-binding protein. Presumably, HS could regulate the function of OPG and affect how it inhibits RANKL. However, the molecular detail of HS-OPG interaction remains poorly understood, which hinders our understanding of how HS functions in osteoclastogenesis. Here we report mapping of the HS-binding site of OPG. The HS-binding site, identified by mutagenesis study, consists of eight basic residues that are located mostly at the junction of the second death domain and the C-terminal domain. We further show that heparin-derived dodecasaccharide is able to induce dimerization of OPG monomers with a stoichiometry of 1:1. Small-angle X-ray scattering analysis revealed that upon binding of HS, OPG undergoes a dramatic conformational change, resulting in a more compact and less flexible structure. Importantly, we present here three lines of evidence that HS, OPG, and RANKL form a stable ternary complex. Using a HS binding-deficient OPG mutant, we further show that in an osteoblast/bone marrow macrophage co-culture system, immobilization of OPG by HS at the osteoblast cell surface substantially lowers the inhibitory threshold of OPG toward RANKL. These discoveries strongly suggest that HS plays an active role in regulating OPG-RANKL interaction and osteoclastogenesis.

Ceylonins A-F, Spongian Diterpene Derivatives That Inhibit RANKL-Induced Formation of Multinuclear Osteoclasts, from the Marine Sponge Spongia ceylonensis.

Six new spongian diterpene derivatives, ceylonins A-F (1-6), were isolated from the Indonesian marine sponge Spongia ceylonensis along with spongia-13(16),14-dien-19-oic acid (7). They contained three additional carbons in ring D to supply an ether-bridged bicyclic ring system. Their structures were elucidated by analyzing NMR spectroscopic data and calculated ECD spectra in comparison to experimental ECD spectra. The bicyclic ring system may be derived from the major metabolite 7 and a C3 unit (an acrylic acid equivalent) through an intermolecular Diels-Alder reaction, which was experimentally supported by the formation of 1-6 from 7 and acrylic acid. The inhibitory effects of the isolated compounds on the RANKL-induced formation of multinuclear osteoclasts in RAW264 macrophages were examined.

Effects of different orthodontic retention protocols on the periodontal health of mandibular incisors.

OBJECTIVES: To test the following two hypotheses: 1) different types of retainers result in distinct levels of biomarkers in gingival crevicular fluid (GCF) and 2) the retainer bonded to all mandibular anterior teeth induces more detrimental outcomes to the periodontium. SETTING AND SAMPLE POPULATION: The Department of Orthodontics at the University of Florida. The population consisted of individuals in the retention phase of orthodontic treatment. MATERIAL AND METHODS: This was a cross-sectional study that enrolled 36 individuals. Subjects in group 1 had retainers bonded to the mandibular canines only. Group 2 consisted of individuals having retainers bonded to all mandibular anterior teeth. Group 3 included patients using mandibular removable retainers. After clinical examination, GCF was collected from the mandibular incisor and biomarker levels were compared between the groups. RESULTS: Plaque accumulation and gingivitis differed significantly among groups, with the highest median values in group 2 subjects. Pairwise comparison of the groups with respect to gingivitis showed significant differences between groups 1 and 2. Significant differences among groups were detected for RANKL, OPG, OPN, M-CSF, MMP-3, and MMP-9. The ratio RANKL/OPG was significantly higher in group 2 subjects, with pairwise comparisons indicating that groups 1 and 2 differed from group 3. CONCLUSION: An association was found between orthodontic retention groups and GCF biomarker levels, which should be further explored in longitudinal studies. The presence of retainers bonded to all anterior teeth seems to increase plaque accumulation and gingivitis.

A RANKL G278R mutation causing osteopetrosis identifies a functional amino acid essential for trimer assembly in RANKL and TNF.

Receptor activator of nuclear factor-κB ligand (RANKL), a trimeric tumor necrosis factor (TNF) superfamily member, is the central mediator of osteoclast formation and bone resorption. Functional mutations in RANKL lead to human autosomal recessive osteopetrosis (ARO), whereas RANKL overexpression has been implicated in the pathogenesis of bone degenerative diseases such as osteoporosis. Following a forward genetics approach using N-ethyl-N-nitrosourea (ENU)-mediated random mutagenesis, we generated a novel mouse model of ARO caused by a new loss-of-function allele of Rankl with a glycine-to-arginine mutation at codon 278 (G278R) at the extracellular inner hydrophobic F ß-strand of RANKL. Mutant mice develop severe osteopetrosis similar to Rankl-deficient mice, whereas exogenous administration of recombinant RANKL restores osteoclast formation in vivo. We show that RANKL(G278R) monomers fail to assemble into homotrimers, are unable to bind and activate the RANK receptor and interact with wild-type RANKL exerting a dominant-negative effect on its trimerization and function in vitro. Since G278 is highly conserved within the TNF superfamily, we identified that a similar substitution in TNF, G122R, also abrogated trimerization, binding to TNF receptor and consequently impaired TNF biological activity. Notably, SPD304, a potent small-molecule inhibitor of TNF trimerization that interacts with G122, also inhibited RANKL activity, suggesting analogous inhibitory mechanisms. Our results provide a new disease model for ARO and identify a functional amino acid in the TNF-like core domain essential for trimer formation both in RANKL and in TNF that could be considered a novel potential target for inhibiting their biological activities.

Crystal structure of human RANKL complexed with its decoy receptor osteoprotegerin.

Receptor activator of NF-κB ligand (RANKL), its signaling receptor RANK, and its decoy receptor osteoprotegerin (OPG) constitute a molecular triad that is critical in regulating bone remodeling, and also plays multiple roles in the immune system. OPG binds RANKL directly to block its interaction with RANK. In this article, we report the 2.7-Å crystal structure of human RANKL trimer in complex with the N-terminal fragment of human OPG containing four cysteine-rich TNFR homologous domains (OPG-CRD). The structure shows that RANKL trimer uses three equivalent grooves between two neighboring monomers to interact with three OPG-CRD monomers symmetrically. A loop from the CRD3 domain of OPG-CRD inserts into the shallow groove of RANKL, providing the major binding determinant that is further confirmed by affinity measurement and osteoclast differentiation assay. These results, together with a previously reported mouse RANKL/RANK complex structure, reveal that OPG exerts its decoy receptor function by directly blocking the accessibilities of important interacting residues of RANKL for RANK recognition. Structural comparison with TRAIL/death receptor 5 complex also reveals structural basis for the cross-reactivity of OPG to TRAIL.

Structure-based development of a receptor activator of nuclear factor-kappaB ligand (RANKL) inhibitor peptide and molecular basis for osteopetrosis.

The receptor activator of nuclear factor-κB (RANK) and its ligand RANKL, which belong to the tumor necrosis factor (TNF) receptor-ligand family, mediate osteoclastogenesis. The crystal structure of the RANKL ectodomain (eRANKL) in complex with the RANK ectodomain (eRANK) combined with biochemical assays of RANK mutants indicated that three RANK loops (Loop1, Loop2, and Loop3) bind to the interface of a trimeric eRANKL. Loop3 is particularly notable in that it is structurally distinctive from other TNF-family receptors and forms extensive contacts with RANKL. The disulfide bond (C125-C127) at the tip of Loop3 is important for determining the unique topology of Loop3, and docking E126 close to RANKL, which was supported by the inability of C127A or E126A mutants of RANK to bind to RANKL. Inhibitory activity of RANK mutants, which contain loops of osteoprotegerin (OPG), a soluble decoy receptor to RANKL, confirmed that OPG shares the similar binding mode with RANK and OPG. Loop3 plays a key role in RANKL binding. Peptide inhibitors designed to mimic Loop3 blocked the RANKL-induced differentiation of osteoclast precursors, suggesting that they could be developed as therapeutic agents for the treatment of osteoporosis and bone-related diseases. Furthermore, some of the RANK mutations associated with autosomal recessive osteopetrosis (ARO) resulted in reduced RANKL-binding activity and failure to induce osteoclastogenesis. These results, together with structural interpretation of eRANK-eRANKL interaction, provided molecular understanding for pathogenesis of ARO.

In vitro stoichiometry of complexes between the soluble RANK ligand and the monoclonal antibody denosumab.

The in vitro binding stoichiometry of denosumab, an IgG2 fully human monoclonal therapeutic antibody, to RANK ligand was determined by multiple complementary size separation techniques with mass measuring detectors, including two solution-based techniques (size-exclusion chromatography with static light scattering detection and sedimentation velocity analytical ultracentrifugation) and a gas-phase analysis by electrospray ionization time-of-flight mass spectrometry from aqueous nondenaturing solutions. The stoichiometry was determined under defined conditions ranging from small excess RANK ligand to large excess denosumab (up to 40:1). High concentrations of denosumab relative to RANK ligand were studied because of their physiological relevance; a large excess of denosumab is anticipated in circulation for extended periods relative to much lower concentrations of free soluble RANKL. The studies revealed that an assembly including 3 denosumab antibody molecules bound to 2 RANKL trimers (3D2R) is the most stable complex in DPBS at 37 °C. This differs from the 1:1 binding stoichiometry reported for RANKL and osteoprotegerin (OPG), a soluble homodimeric decoy receptor which binds RANKL with high affinity. Denosumab and RANKL also formed smaller assemblies including 1 denosumab and 2 RANKL trimer molecules (1D2R) under conditions of excess RANKL, 3 denosumab molecules and 1 RANKL trimer (3D1R) under conditions of excess denosumab, and larger assemblies, but these intermediate species were only present at lower temperatures (4 °C), shortly after mixing denosumab and RANKL, and converted over time to the more stable 3D2R assembly.

Structural and functional insights of RANKL-RANK interaction and signaling.

Bone remodeling involves bone resorption by osteoclasts and synthesis by osteoblasts and is tightly regulated by the receptor activator of the NF-kappaB ligand (RANKL)/receptor activator of the NF-kappaB (RANK)/osteoprotegerin molecular triad. RANKL, a member of the TNF superfamily, induces osteoclast 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 osteoporosis and rheumatoid arthritis. 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 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 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.

Mutations within the TNF-like core domain of RANKL impair osteoclast differentiation and activation.

Receptor activator of nuclear factor-kappaB ligand (RANKL) is a key factor necessary for osteoclast differentiation and activation. Mutations within the TNF-like core domain of RANKL have been recently reported in patients with osteoclast-poor autosomal recessive osteopetrosis. However, the functional consequence owing to RANKL mutations has not been well characterized. Here we describe the functional propensity of RANKL mutants in osteoclast differentiation and their impact on RANKL-mediated signaling cascades. Recombinant RANKL (rRANKL) mutants within the TNF-like core domain exhibited diminished osteoclastogenic potential as compared with wild-type rRANKL1 encoding the full TNF-like core domain [amino acids (aa) 160-318]. Consistent with the insufficient activities on osteoclastogenesis, rRANKL mutants showed reduced activation of nuclear factor-kappaB, IkappaBalpha degradation, and ERK phosphorylation. In addition, we found that rRANKL mutants interfered with wild-type rRANKL-induced osteoclastogenesis with deletion mutant rRANKL5 (aa 246-318) exhibiting the greatest inhibitory effect. The same mutant also significantly reduced wild-type rRANKL1 (aa 160-318)-induced osteoclastic bone resorption in vitro. BIAcore assays demonstrated that rRANKL5 alone, lacking the AA'' and CD loops, weakly binds to receptor activator of nuclear factor-kappaB (RANK). Intriguingly, preincubation of mutant rRANKL5 with rRANKL1 before exposure to RANK enhanced the maximal binding level to RANK, indicating that rRANKL5 forms hybrid trimeric complexes with rRANKL1. Furthermore, RANKL mutant mimicking human RANKL V277 mutation in patients, impairs osteoclast differentiation and signaling. Taken together, these data lend support to the notion that the TNF-like core domain of RANKL contains structural determinants that are crucial for osteoclast differentiation and activation, thus providing a possible mechanistic explanation for the observed phenotype in osteopetrotic patients harboring RANKL mutations.

The affinity of human RANK binding to its ligand RANKL.

Receptor activator of nuclear factor-kappa B (RANK) and its ligand, RANKL play critical roles in bone re-modeling, immune function, vascular disease and mammary gland development. To study the interaction of RANK and RANKL, we have expressed both extracellular domain of RANK and ectodomain of RANKL using Escherichia coli expression system. RANK was expressed as an inclusion body first which properly refolded later, while RANKL was initially produced as a GST fusion protein, after which the GST was removed by enzyme digestion. Soluble RANK existed as a monomer while RANKL was seen as a trimer in solution, demonstrated by gel filtration chromatography and cross-linking experiment. The recombinant RANK and RANKL could bind to each other and the binding affinity of RANKL for RANK was measured with surface plasmon resonance technology and K(D) value is about 1.09 x 10(-10) M.

Crystallization and preliminary X-ray analysis of mouse RANK and its complex with RANKL.

The interaction between the TNF-family molecule receptor activator of NF-kappaB ligand (RANKL) and its receptor RANK induces osteoclast formation, activation and survival in the process of bone remodelling. RANKL-RANK also plays critical roles in T-cell/dendritic cell communication and lymph-node formation and in a variety of pathologic conditions such as tumour-cell migration and bone metastasis. Both the ectodomain of mouse RANKL and the extracellular domain of mouse RANK have been cloned, expressed and purified. Crystals of RANK alone and of RANK in complex with RANKL have been obtained that are suitable for structure determination.