Functional Dissection of Osteoprotegerin and Its Interaction with Receptor Activator of NF-κB Ligand

Abstract

The receptor activator of NF-κB (RANK) belongs to the neuregulin/tumor necrosis factor (TNF) receptor superfamily and is activated by RANK ligand (RANK-L), a homotrimeric, TNF-like cytokine. RANK is present on the surface of osteoclast cell precursors, where its interaction with RANK-L induces their terminal differentiation into osteoclasts, thus increasing bone breakdown. The secreted, soluble receptor osteoprotegerin (OPG) interrupts this activation by binding directly to RANK-L. Therefore, osteoclast maturation (and bone homeostasis) is regulated in vivo by OPG levels of expression. We have studied the assembly state and affinity of OPG for RANK-L by sedimentation analyses and surface plasmon resonance (Biacore). Full-length, homodimeric OPG binds to RANK-L with a KD of 10 nm. OPG is also a member of the TNF receptor superfamily and contains four disulfide-rich ligand-binding domains, yet lacks a transmembrane region separating the ligand-binding region from the two death domains, as observed for other receptor family members. We showed that dimerization of OPG results from noncovalent interactions mediated by the death domains and to a lesser extent by a C-terminal heparin-binding region. In contrast, a C-terminal intermolecular disulfide bond does not contribute to the formation or stability of OPG dimers. A truncate of osteoprotegerin, containing the ligand-binding domains but lacking the dimerization domains, bound RANK-L with a KD of ∼3 μm, indicating that monomer oligomerization for the OPG provides an increase of 3 orders of magnitude in the affinity for RANK-L. Therefore, OPG dimer formation is required for the mechanism of inhibition of the RANK-L/RANK receptor interaction. The receptor activator of NF-κB (RANK) belongs to the neuregulin/tumor necrosis factor (TNF) receptor superfamily and is activated by RANK ligand (RANK-L), a homotrimeric, TNF-like cytokine. RANK is present on the surface of osteoclast cell precursors, where its interaction with RANK-L induces their terminal differentiation into osteoclasts, thus increasing bone breakdown. The secreted, soluble receptor osteoprotegerin (OPG) interrupts this activation by binding directly to RANK-L. Therefore, osteoclast maturation (and bone homeostasis) is regulated in vivo by OPG levels of expression. We have studied the assembly state and affinity of OPG for RANK-L by sedimentation analyses and surface plasmon resonance (Biacore). Full-length, homodimeric OPG binds to RANK-L with a KD of 10 nm. OPG is also a member of the TNF receptor superfamily and contains four disulfide-rich ligand-binding domains, yet lacks a transmembrane region separating the ligand-binding region from the two death domains, as observed for other receptor family members. We showed that dimerization of OPG results from noncovalent interactions mediated by the death domains and to a lesser extent by a C-terminal heparin-binding region. In contrast, a C-terminal intermolecular disulfide bond does not contribute to the formation or stability of OPG dimers. A truncate of osteoprotegerin, containing the ligand-binding domains but lacking the dimerization domains, bound RANK-L with a KD of ∼3 μm, indicating that monomer oligomerization for the OPG provides an increase of 3 orders of magnitude in the affinity for RANK-L. Therefore, OPG dimer formation is required for the mechanism of inhibition of the RANK-L/RANK receptor interaction. Bone remodeling, a coordinated equilibrium between bone synthesis and bone resorption, is a continuously occurring process that maintains skeletal integrity in vertebrates (1Suda T. Takahashi N. Udagawa N. Jimi E. Gillespie M.T. Martin T.J. Endocr. Rev. 1999; 20: 345-357Crossref PubMed Google Scholar). The cellular differentiation signals responsible for the regulation of bone remodeling processes are ultimately determined by binding interactions between proteins localized at the cell surface of osteoblasts and osteoclasts (1Suda T. Takahashi N. Udagawa N. Jimi E. Gillespie M.T. Martin T.J. Endocr. Rev. 1999; 20: 345-357Crossref PubMed Google Scholar). Osteoblasts are cells derived from bone marrow stem cells, responsible for bone formation (2Theill L.E. Boyle W.J. Penninger J.M. Annu. Rev. Immunol. 2002; 20: 795-823Crossref PubMed Scopus (678) Google Scholar, 3Katagiri T. Takahashi N. Oral Dis. 2002; 8: 147-159Crossref PubMed Scopus (467) Google Scholar). Osteoclasts mature from cells of the innate immune system and cause bone resorption (4Kimbrell D.A. Beutler B. Nat. Rev. Genet. 2001; 2: 256-267Crossref PubMed Scopus (500) Google Scholar, 5Fuller K. Wong B. Fox S. Choi Y. Chambers T.J. J. Exp. Med. 1998; 188: 997-1001Crossref PubMed Scopus (508) Google Scholar). Receptor activator of NF-κB ligand (RANK-L) 3The abbreviations used are: RANK-Lreceptor activator of NF-κB ligandRANKreceptor activator of NF-κBOPGosteoprotegerinOPG-FcOPG Fc-fusionOPG-HOPG His6 fusionOPGC400A-HOPG cysteine 400 to alanine-His6 fusionOPGΔB-HOPG basic domain truncated construct-His6 fusionOPGΔDDB-HOPG basic and two death domains truncated construct-His6 fusionOPGΔCRD-HOPG cysteine-rich domains truncated construct-His6 fusionDDdeath domainCRDcysteine-rich domainsTNFtumor necrosis factorTNFRTNF receptorNTAnitrilotriacetic acidRUresponse units. is a TNF ligand superfamily member (6Wong B.R. Rho J. Arron J. Robinson E. Orlinick J. Chao M. Kalachikov S. Cayani E. Bartlett F.S. II I Frankel W.N. Lee S.Y. Choi Y. J. Biol. Chem. 1997; 272: 25190-25194Abstract Full Text Full Text PDF PubMed Scopus (917) Google Scholar, 7Lacey D.L. Timms E. Tan H.L. T. S. J. N. E. S. J. J. Boyle W.J. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). RANK-L present on the surface of osteoblasts binds to its on the surface of osteoclasts, their thus the bone K. Wong B. Fox S. Choi Y. Chambers T.J. J. Exp. Med. 1998; 188: 997-1001Crossref PubMed Scopus (508) Google Scholar). interaction or in osteoblasts and osteoclasts T. Takahashi N. Oral Dis. 2002; 8: 147-159Crossref PubMed Scopus (467) Google Scholar). RANK-L is also an factor for the of mature osteoclasts T. Takahashi N. Oral Dis. 2002; 8: 147-159Crossref PubMed Scopus (467) Google Scholar). the the of bone resorption of the of RANK-L and its two a receptor that osteoclast and and a soluble The regulation of the between bone and is to a extent by the secreted, soluble receptor OPG D.L. Timms E. Tan H.L. T. S. J. N. E. S. J. J. Boyle W.J. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, D.L. M. S. T. M. Tan H.L. J. E. N. S. J. Lee Boyle W.J. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). by binding to OPG RANK-L to osteoclast differentiation and K. Wong B. Fox S. Choi Y. Chambers T.J. J. Exp. Med. 1998; 188: 997-1001Crossref PubMed Scopus (508) Google Scholar, 7Lacey D.L. Timms E. Tan H.L. T. S. J. N. E. S. J. J. Boyle W.J. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, N. N. K. M. S. K. M. E. T. K. Udagawa N. Takahashi N. T. S. 1998; PubMed Scopus Google Scholar). a for bone remodeling and in of in M. J. Bone 2001; PubMed Scopus Google Scholar, 2001; Google Scholar, J. 2001; PubMed Scopus Google by the of RANK-L or its J. 1998; Google Scholar, S. D.L. Boyle W.J. J. Bone PubMed Scopus Google and where results in Boyle W.J. Penninger J.M. Immunol. Biol. 1999; PubMed Scopus Google Scholar, E. Gillespie M.T. Martin T.J. 2002; PubMed Scopus Google Scholar). receptor activator of NF-κB ligand receptor activator of NF-κB osteoprotegerin OPG OPG His6 OPG cysteine 400 to alanine-His6 OPG basic domain truncated construct-His6 OPG basic and two death domains truncated construct-His6 OPG cysteine-rich domains truncated construct-His6 death domain cysteine-rich domains necrosis factor TNF receptor units. RANK-L is as an on the surface of but is also The of this is not yet (6Wong B.R. Rho J. Arron J. Robinson E. Orlinick J. Chao M. Kalachikov S. Cayani E. Bartlett F.S. II I Frankel W.N. Lee S.Y. Choi Y. J. Biol. Chem. 1997; 272: 25190-25194Abstract Full Text Full Text PDF PubMed Scopus (917) Google Scholar, 7Lacey D.L. Timms E. Tan H.L. T. S. J. N. E. S. J. J. Boyle W.J. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, J. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). The or domain of RANK-L to the region for binding to the soluble receptor OPG and for activation of binding to RANK D.L. Timms E. Tan H.L. T. S. J. N. E. S. J. J. Boyle W.J. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, N. N. K. M. S. K. M. E. T. K. Udagawa N. Takahashi N. T. S. 1998; PubMed Scopus Google Scholar). The domain of is in RANK-L is a with equilibrium for of W.J. K. J. T. E. S. J. B. 20: Scopus Google but is to as this affinity is at the of for an that by sedimentation equilibrium The of the RANK-L by and to have a with other TNF-like S. K. S. T. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). In to other receptor superfamily the domain of RANK is by four C-terminal cysteine-rich domains domains are by a transmembrane and a domain containing J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). OPG is also a family secreted, soluble homodimeric receptor of four by two to the death domains of the that are OPG lacks a transmembrane domain E. PubMed Scopus Google Scholar). on other TNF receptor superfamily are to the Full Text Full Text PDF PubMed Scopus Google Scholar). is a C-terminal basic region in in a cysteine to the C-terminal to a disulfide bond in the homodimeric of OPG M. N. S. T. E. K. 1998; PubMed Scopus Google Scholar, J. Lee PubMed Scopus Google Scholar). superfamily also soluble but are by of the OPG is the soluble receptor in this superfamily with from the of a transmembrane domain Full Text Full Text PDF PubMed Scopus Google Scholar). The of the and the C-terminal basic region of OPG is OPG observed in vivo as a intermolecular disulfide dimer and as a lacking the intermolecular disulfide bond of C-terminal M. N. S. T. E. K. 1998; PubMed Scopus Google Scholar, K. E. N. M. K. K. Y. J. Bone 1999; PubMed Scopus Google Scholar, K. K. T. E. 2001; PubMed Scopus Google Scholar, T. T. K. E. N. Chem. 2001; PubMed Scopus Google Scholar). the truncated monomer and the dimer to have in inhibition M. N. S. T. E. K. 1998; PubMed Scopus Google Scholar). of the of between RANK and OPG for with RANK-L. The binding of RANK-L to the RANK receptor a soluble RANK-L and osteoclast as a of RANK D.L. Timms E. Tan H.L. T. S. S. Boyle W.J. S. 1999; PubMed Scopus Google Scholar). of RANK-L to the soluble OPG receptor observed an in In this the of osteoclast maturation by RANK-L by the of and cellular as in a in a by OPG K. Wong B. Fox S. Choi Y. Chambers T.J. J. Exp. Med. 1998; 188: 997-1001Crossref PubMed Scopus (508) Google Scholar, M. N. S. T. E. K. 1998; PubMed Scopus Google Scholar). binding of RANK-L to a domain also and to a KD of surface plasmon resonance W.J. K. J. T. E. S. J. B. 20: Scopus Google Scholar). of the cellular and affinity of binding of OPG to is the mechanism of formation and the inhibition of In the affinity interaction between OPG and RANK-L an for their interaction. We that the domain of OPG is for the oligomerization and of binding In this the and of the and the of OPG its to RANK-L. We also the of domains of OPG to its binding Therefore, results for the a for analyses of the of OPG on osteoclast of used the of a at W.J. K. J. T. E. S. J. B. 20: Scopus Google Scholar). to the and to of the of the D.L. Timms E. Tan H.L. T. S. J. N. E. S. J. J. Boyle W.J. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, N. N. K. M. S. K. M. E. T. K. Udagawa N. Takahashi N. T. S. 1998; PubMed Scopus Google Scholar). truncated is RANK-L to regulated from the cell surface in a to this J. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). of and the The truncate a from and its and W.J. K. J. T. E. S. J. B. 20: Scopus Google Scholar). The a from and its to that for the W.J. K. J. T. E. S. J. B. 20: Scopus Google Scholar). The RANK-L with an cysteine for surface to the The OPG are in and are the as of K. M. M. E. T. K. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google that A containing the into the and of a to a in the of by a at with a as by the and the His6 The by the and the His6 between the and of to a truncate at The of the and into the four of the death domain into the and of to from by of cells with and cells by of to as Lee J. PubMed Google Scholar). and an T. cells with at a of of at for for and for other and and with and at and at A used to in the and to and with RANK-L with at T. and proteins as W.J. K. J. T. E. S. J. B. 20: Scopus Google and as a from of proteins at sedimentation of and its in an an or sedimentation of other and and for sedimentation equilibrium in an a in 10 The of the from the a and the and from the of the of the T. of and J. with at for at on to PubMed Scopus Google Scholar, II I 1997; 8: PubMed Scopus Google and the of by of J. Full Text Full Text PDF PubMed Scopus Google Scholar). equilibrium with at and for and for and for and for and for and for or and for and at and equilibrium by of of and an in the of for where is the at is the at is the is the in is the of the is the is the is the and is the of the sedimentation the and the from sedimentation equilibrium is in in the The with from an for OPG and the of RANK-L with the of of observed in the and N. 1997; PubMed Scopus Google Scholar). The used for the and as for the of the basic to J. Biol. PubMed Scopus Google and thus as to have to the of OPG used to the of the OPG The by the to the of provides a of of the proteins and with a indicating at an from to in a and an of The to the of and as by at nm. on a or a to binding to RANK-L used a with at of a at 10 for RANK-L at for or at by 10 of The surface to the of with a of and a of of at the KD a that for the of and and M. PubMed Scopus Google Scholar). for binding of the OPG and its a with RANK-L its cysteine by the from The to interactions between OPG or its are and the on the surface OPG binding in the of RANK-L to the surface at with of OPG as a by a at by 10 of The surface a of of at the KD a for the of RANK-L to on the equilibrium binding of W.J. K. J. T. E. S. J. B. 20: Scopus Google and other of OPG and the affinity of this interaction is We to the mechanism of this We on the of W.J. K. J. T. E. S. J. B. 20: Scopus Google to of the interaction surface plasmon The of RANK-L binding to on a surface a of magnitude of RANK-L. a dimer in an on the RANK-L binds to and a The binding in to a binding The to a that in the the binding interaction of OPG and RANK-L is also that the surface by the OPG is for binding to the RANK-L The is a equilibrium binding of nm. determined from the from a binding and The to not have a of that of RANK-L bound to the surface at The KD is with equilibrium binding results W.J. K. J. T. E. S. J. B. 20: Scopus Google Scholar). the for binding observed and the of OPG and RANK-L that a in this interaction. the of this that have studied by sedimentation are in are used the sedimentation to the observed the of sedimentation for or in The sedimentation of and RANK-L A and and in are with a for RANK-L and a dimer for results are by the sedimentation equilibrium in does have a of that is to of the in a and observed by sedimentation in to of binding as by the to sedimentation in the of RANK-L the formation of a between and with a of is in and in The of this by sedimentation equilibrium for binding RANK-L. 3 3 3 3 3 A to RANK-L in the of but a to RANK-L does not in the of RANK-L. to are of proteins indicating an are not in this for and sedimentation for in the of the by for other in this by for other in this by for other in this sedimentation for in the of the by for other in this in a equilibrium of proteins The basic domain and the death domains contribute to OPG cysteine 400 The abbreviations used are as not monomer and dimer does not is to to monomer is the is or is determined by the to an for in and or a interaction for and is the is or is determined by the to an for in and or a interaction for and are from by of are from does not is to to monomer is the is or is determined by the to an for in and or a interaction for and are from by of are from PubMed Scopus Google in a results for OPG and binding RANK-L are in this and are in and of or the to a indicating a OPG to RANK-L The abbreviations used are as is is is that 3 for sedimentation for in the of the the the not the the not sedimentation for in the of the the the not in a the stability of the dimer to containing an of also by sedimentation of as in does not the but as a at S. that an by two RANK-L and a dimer is not is not observed by sedimentation at a of RANK-L does the from to a with RANK-L and two this does is not as as the is by the of a at to dimer The for of the of the of in the that the affinity of the dimer binding to RANK-L is to the for the a of 3 orders of magnitude for the of binding of the to to the affinity is of the of two of the RANK-L for binding the a to interaction for the does not from the to interaction is 3 orders of magnitude the to interaction. the observed in its interaction with the in and in sedimentation of the of the proteins and the and the of and the domain to the dimer a at and a to RANK-L at in the of an of The observed are RANK-L or of of domains contribute to OPG dimerization and their on the RANK-L a of OPG in the of the for the are in and the are in to in the for K. M. M. E. T. K. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). We studied the of and by equilibrium sedimentation the equilibrium a of the stability of the for the and in to does disulfide by its in the on the state or stability of the OPG the of by is in the basic region. Therefore, for C-terminal truncated lacking the basic region the is also the the is the The truncate basic region or C-terminal a in its dimer stability to OPG A observed with the truncate death domains, basic or C-terminal to into the the truncate ligand-binding cysteine-rich a with the We also determined the of OPG with RANK-L by sedimentation the studied in and their are in and and with the in to and showed to RANK-L to OPG dimer of but a with the and and at an OPG of μm, the dimerization of its OPG in of its interaction with RANK-L. is that that does not have the present for the of cysteine 400 to a dimer to for to the for binding RANK-L. 3 3 3 3 3 A to RANK-L is the in the of but a to RANK-L does not in the of RANK-L. to are results for OPG and binding RANK-L are in this and are in The abbreviations used are as is is is is and is RANK-L. and a OPG RANK-L in the of does does not a with sedimentation for in the of the that for the the not the the not sedimentation for in the of the that for the the not in a also with RANK-L and is at by sedimentation The observed a and with RANK-L bound to of is observed an of to RANK-L. is with a interaction between and with an equilibrium binding in the not with RANK-L by sedimentation analyses and for binding RANK-L. between and RANK-L to are the binding for the of the OPG with The by RANK-L of the OPG the not on a surface the of the and the His6 affinity a on the dimerization state of Therefore, of the and the truncated at surface this a cysteine the of the Therefore, the of the RANK-L is to a surface to bond of RANK-L. on the of the cysteine in the RANK-L monomer is the of the Therefore, is not for disulfide to the or to to the The of RANK-L is the cell of the RANK-L D.L. Timms E. Tan H.L. T. S. J. N. E. S. J. J. Boyle W.J. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, S. K. S. T. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). Therefore, the of RANK-L on the surface is to its on the surface of We determined that the binding of the RANK-L to is to that of binding to at and not that the RANK-L the and that is on the surface that RANK-L surface used to the of OPG to with to and affinity results for OPG binding RANK-L 400 a to RANK-L binding the basic domain and the death domains are to RANK-L binding the of the as as as as as as as not determined to binding of to the of the not determined to binding of to surface in a The affinity of for RANK-L to is of and are in the in affinity and oligomerization are determined by to The bound to RANK-L with an affinity that of as in of The truncate showed a to of of the affinity homodimeric at at or its dimerization of binding to at at or its dimerization of not of binding The results that does not RANK-L. that the observed in OPG dimerization and RANK-L binding stem from by the on the of In to the of OPG and its their by The that and to also a to that of that the of the region the death domains, basic and C-terminal disulfide is Therefore, the of this truncate to to RANK-L is of the of the ligand-binding domain and not of the of to RANK-L at and thus a its lacks the for of as and the of the of is to of a of in by an of disulfide as observed for the TNF receptor ligand-binding domain D.L. Timms E. Tan H.L. T. S. J. N. E. S. J. J. Boyle W.J. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). OPG and RANK-L are the and of the are not Therefore, the used for the sedimentation have for the to the this as as a of OPG that at sedimentation results of a of the interaction and its with to and not a and of proteins and that for sedimentation and equilibrium to the and of OPG The sedimentation and the used to the of as a The used to the for or The by the to the of in the of The this the the or to the of the proteins from and the of but the for the from that and the basic domain the of OPG as by the increase in in the of The binding of OPG to RANK-L a in indicating that RANK-L binding the of OPG is an and and its binding to RANK-L its and of proteins and OPG OPG is a and in binds RANK-L. used to RANK-L does of the RANK-L truncate that is not in The in 3 the from the for or sedimentation for in of of is a derived from the by the sedimentation for in of is a derived from the by the in a The of OPG provides a of the of OPG in bone We that homodimeric osteoprotegerin 10 with RANK-L to to a in the formation of as by two sedimentation not in containing a of RANK-L that OPG monomer in the a RANK-L OPG present at a RANK-L. the OPG dimer a of affinity The with results is that affinity binding of OPG to RANK-L on the two OPG dimer to two of the in the RANK-L monomer in the OPG is to with the (and RANK-L is the of OPG to that ultimately its affinity for RANK-L. is the of its to with the RANK receptor for this the that RANK as by the is for OPG binds to at two of the is to RANK oligomerization and thus osteoclast and surface plasmon resonance of the and that OPG a for affinity binding to RANK-L. The affinity of for RANK-L is 3 orders of magnitude the observed with OPG 3 that the to the interaction between OPG and RANK-L is for OPG is OPG at the basic at the C-terminal and the death domains of OPG are for dimerization in the the of on a for the C-terminal disulfide bond in the dimer formation of the cysteine does contribute to the stability of the OPG dimer at in of its and does have a on the affinity of for RANK-L with is to In contrast, the of the basic region the of OPG to by the KD of dimerization to and thus its affinity for RANK-L. The of the C-terminal the two death domains and the basic dimerization The cysteine-rich domains of the with a KD of Therefore, the C-terminal of OPG is for dimerization and thus the affinity binding to RANK-L. is that the death domains in other family are in Full Text Full Text PDF PubMed Scopus Google but in OPG to the receptor and the interaction with RANK-L. The cysteine-rich domains are for OPG binding to the ligand-binding binding of the is not of the of in have with in the of OPG to RANK-L activation of osteoclast in showed that of the dimerization domain the of OPG to osteoclast maturation K. M. M. E. T. K. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). also that cysteine 400 responsible for used and to the and as to the results that by the and dimer observed by K. M. M. E. T. K. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google and M. N. S. T. E. K. 1998; PubMed Scopus Google have Therefore, the by and M. N. S. T. E. K. 1998; PubMed Scopus Google that the monomer and dimer have the are two is a dimer and thus the as is an The ligand binding and dimerization domains of OPG to as not but also of the of OPG to by that the ligand binding of OPG at a with to the region of the is the TNF receptor to of a of in a disulfide bond D.L. Timms E. Tan H.L. T. S. J. N. E. S. J. J. Boyle W.J. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). the of the dimerization domains basic for the observed in that region of the The of the sedimentation for at that for this of the results in the with a of sedimentation results at in the and also with the in the at a interaction between OPG and is not by the KD observed at not the OPG used in the of and also increasing the of for the observed The OPG are not to of the of observed and thus their interaction with RANK-L not In of the domains of OPG are for affinity with RANK-L. The death domains and basic region of OPG the of the ligand-binding domains of two to a binding that RANK-L with that the of OPG as for in OPG or to the receptor binding and dimerization of the homodimeric OPG of of the mechanism of RANK for that to We and for and We also and for with for this with