Crystal Structure of Human RANKL Complexed with Its Decoy Receptor Osteoprotegerin This information is current as Xudong Luan, Qingyu Lu, Yinan Jiang, Senyan Zhang, Qing of September 26, 2021. Wang, Huihui Yuan, Wenming Zhao, Jiawei Wang and Xinquan Wang J Immunol 2012; 189:245-252; Prepublished online 4 June 2012; doi: 10.4049/jimmunol.1103387 Downloaded from http://www.jimmunol.org/content/189/1/245 Supplementary http://www.jimmunol.org/content/suppl/2012/06/04/jimmunol.110338 Material 7.DC1 http://www.jimmunol.org/ References This article cites 45 articles, 13 of which you can access for free at: http://www.jimmunol.org/content/189/1/245.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision by guest on September 26, 2021 • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2012 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Crystal Structure of Human RANKL Complexed with Its Decoy Receptor Osteoprotegerin Xudong Luan,*,1 Qingyu Lu,*,1 Yinan Jiang,* Senyan Zhang,* Qing Wang,* Huihui Yuan,† Wenming Zhao,† Jiawei Wang,* and Xinquan Wang* Receptor activator of NF-kB 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-A˚ 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 Downloaded from 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. The Journal of Immunology, 2012, 189: 245–252. http://www.jimmunol.org/ one remodeling is a dynamic and balanced progress that creted by osteoblasts inhibits osteoclastogenesis by binding involves bone matrix formation mediated by osteoblasts RANKL to prevent RANKL/RANK interaction. In the bone mi- B and bone resorption mediated by osteoclasts. Osteoblasts croenvironment, the local ratio of RANKL and OPG determines are cells that arise from bone marrow stem cells, whereas osteo- the level of osteoclast activation. The normal osteoclast activation clasts are multinucleated cells derived from hematopoietic pre- results in physiological bone resorption in bone remodeling, cursors of the monocyte/macrophage lineage. Receptor activator whereas overactivated osteoclasts result in various bone diseases of NF-kB ligand (RANKL), its signaling receptor RANK, and its such as osteoporosis and rheumatoid arthritis (8, 9). Besides the decoy receptor osteoprotegerin (OPG) play key roles in regulating best-known critical roles in bone remodeling, RANKL also plays bone remodeling (1–3). Experiments using knockout mice have multiple roles in immune system (10), mammary gland develop- by guest on September 26, 2021 unanimously established their pivotal roles as central regulators of ment (11), cancer bone metastasis (12), hormone-derived breast osteoclast function (4–7). Specifically, the interaction of RANKL cancer development (13), and thermal regulation (14). Therefore, expressed by osteoblasts with RANK expressed on osteoclast the RANKL-RANK-OPG molecular triad is an attractive target precursors is essential for the differentiation, activation, and sur- to develop rational therapy for many osteopenic conditions and vival of osteoclasts. The decoy receptor OPG expressed and se- prevent bone destruction in osteoporosis and arthritis (15). RANKL is a member in the TNF superfamily (TNFSF), con- sisting of a membrane-anchor domain, a connecting stalk, and *Center for Structural Biology, School of Life Sciences, Ministry of Education Key Laboratory of Protein Science, Tsinghua University, Beijing 100084, People’s Re- a receptor-binding ectodomain. RANKL also exists in a soluble public of China; and †Department of Immunology, Capital Medical University, Bei- form from proteolytic cleavage of the transmembrane form or jing 100069, People’s Republic of China alternative mRNA splicing (16, 17). The ectodomain of RANKL 1 X.L. and Q.L. contributed equally to this work. adopts the characteristic jellyroll b-sandwichfoldinTNFSF Received for publication November 28, 2011. Accepted for publication March 30, cytokines and assembles into a homotrimer in either membrane- 2012. bound or soluble form (18, 19). The receptor RANK belongs to This work was supported by the Ministry of Science and Technology (2010CB912402, 2011CB910502, and 2010CB529106), the National Natural Science Foundation of the TNFR superfamily (TNFRSF), consisting of four extracellular China (30970573 and 31070667), and the Fok Ying Tung Education Foundation. cysteine-rich TNFR homologous domains (CRD1-CRD4), a trans- The coordinates and structure factors presented in this article for the human RANKL/ membrane helix, and a cytoplasmic region. RANKL/RANK in- OPG-CRD complex have been deposited in the Protein Data Bank (http://www.rcsb. teraction induces RANK receptor trimerization, triggering the org/pdb/) under access code 3URF for immediate release on publication. association of RANK cytoplasmic region with TNFR-associated Address correspondence and reprint request to Dr. Xinquan Wang, Medical Science Building C226, Tsinghua University, Beijing 100084, People’s Republic of China. factors and subsequent multiple signaling pathways (20, 21). The E-mail address: [email protected] decoy receptor OPG represents an atypical member in the TNFRSF The online version of this article contains supplemental material. because it lacks the transmembrane region and is secreted as a Abbreviations used in this article: CRD, cysteine-rich TNFR homologous domain; soluble protein. The OPG has four CRD domains in the N-ter- DR, death receptor; OPG, osteoprotegerin; OPG-CRD, N-terminal four CRDs of minal region that are necessary and sufficient for the inhibition of human OPG; RANK, receptor activator of NF-kB; RANKL, receptor activator of NF-kB ligand; SPR, surface plasmon resonance; TNFSF, TNF superfamily; TNFRSF, osteoclastogenesis (7, 22), and its C-terminal region contains two TNFR superfamily; TRAP, tartrate-resistant acid phosphatase; vWF, von Willebrand death domain homologous regions, a heparin-binding domain, and factor. a cysteine residue at the C-terminal end necessary for dimer for- Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 mation (7, 22, 23). www.jimmunol.org/cgi/doi/10.4049/jimmunol.1103387 246 COMPLEX STRUCTURE OF RANKL WITH OPG N-TERMINAL CRDs The crystal structure of mouse RANKL was determined 10 years SCALA from the CCP4 suite (29). The final statistics of data processing ago (18, 19), and residues in RANKL that are important for are listed in Table I. RANK recognition have also been proposed based on structural Structural determination and refinement modeling and mutational studies (18, 19, 24, 25). The crystal structure of the mouse RANKL/RANK complex was recently The position of human RANKL monomer in the asymmetric unit was solved independently by two groups, providing precise molecular determined by using molecular replacement program PHASER (30) with mouse RANKL monomer (Protein Data Bank code: 1IQA) as search details of RANKL/RANK interaction (26, 27). In this article, we model. Starting with initial phases provided by RANKL, the OPG-CRD report the complex structure of human RANKL ectodomain with model was built gradually by cycles of iterative manual model building N-terminal four CRD domains of human OPG (OPG-CRD). Key with program COOT (31) and structural refinement with program PHENIX residues in OPG-CRD for RANKL binding were identified by (32) refinement module. In the final model, 80.4, 16.4, 2.1, and 1.1% of the amino acids reside in the most favorable, additional allowed, generously structure-based mutagenesis of OPG-CRD and surface plasmon allowed, and disallowed regions of the Ramachandran plots, respectively. resonance (SPR) analyses, which was further collaborated by The final refinement statistics are listed in Table I. osteoclast differentiation assay. These results, together with pre- vious reported mouse RANKL/RANK complex