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• W2022121540 abstract "The Wnt pathway inhibitors DKK1 and sclerostin (SOST) are important therapeutic targets in diseases involving bone loss or damage. It has been appreciated that Wnt coreceptors LRP5/6 are also important, as human missense mutations that result in bone overgrowth (bone mineral density, or BMD, mutations) cluster to the E1 propeller domain of LRP5. Here, we report a crystal structure of LRP6 E1 bound to an antibody, revealing that the E1 domain is a peptide recognition module. Remarkably, the consensus E1 binding sequence is a close match to a conserved tripeptide motif present in all Wnt inhibitors that bind LRP5/6. We show that this motif is important for DKK1 and SOST binding to LRP6 and for inhibitory function, providing a detailed structural explanation for the effect of the BMD mutations. The Wnt pathway inhibitors DKK1 and sclerostin (SOST) are important therapeutic targets in diseases involving bone loss or damage. It has been appreciated that Wnt coreceptors LRP5/6 are also important, as human missense mutations that result in bone overgrowth (bone mineral density, or BMD, mutations) cluster to the E1 propeller domain of LRP5. Here, we report a crystal structure of LRP6 E1 bound to an antibody, revealing that the E1 domain is a peptide recognition module. Remarkably, the consensus E1 binding sequence is a close match to a conserved tripeptide motif present in all Wnt inhibitors that bind LRP5/6. We show that this motif is important for DKK1 and SOST binding to LRP6 and for inhibitory function, providing a detailed structural explanation for the effect of the BMD mutations. First structure of any domain from Wnt coreceptors LRP5/6 (LRP6 E1 β-propeller) The E1 propeller binds a short peptide present in Wnt inhibitors SOST and DKK1 The LRP5/6 binding motif is required for DKK1 and SOST function Human bone mineral density mutations in LRP5 disrupt critical peptide contacts The Wnt/β-catenin pathway is essential for processes of embryonic development through those of tissue renewal and homeostasis in the adult. Signaling is initiated at the cell surface by binding of the secreted Wnt to its coreceptors Frizzled (Fz) (Bhanot et al., 1996Bhanot P. Brink M. Samos C.H. Hsieh J.C. Wang Y. Macke J.P. Andrew D. Nathans J. Nusse R. A new member of the frizzled family from Drosophila functions as a Wingless receptor.Nature. 1996; 382: 225-230Crossref PubMed Scopus (1223) Google Scholar) and low-density lipoprotein receptor-related protein (LRP5/6) (Pinson et al., 2000Pinson K.I. Brennan J. Monkley S. Avery B.J. Skarnes W.C. An LDL-receptor-related protein mediates Wnt signalling in mice.Nature. 2000; 407: 535-538Crossref PubMed Scopus (890) Google Scholar, Tamai et al., 2000Tamai K. Semenov M. Kato Y. Spokony R. Liu C. Katsuyama Y. Hess F. Saint-Jeannet J.P. He X. LDL-receptor-related proteins in Wnt signal transduction.Nature. 2000; 407: 530-535Crossref PubMed Scopus (1088) Google Scholar). Recruitment of the very closely related (69% identical) LRP5 or LRP6 into this ternary complex (Bourhis et al., 2010Bourhis E. Tam C. Franke Y. Bazan J.F. Ernst J. Hwang J. Costa M. Cochran A.G. Hannoush R.N. Reconstitution of a frizzled8.Wnt3a.LRP6 signaling complex reveals multiple Wnt and Dkk1 binding sites on LRP6.J. Biol. Chem. 2010; 285: 9172-9179Crossref PubMed Scopus (153) Google Scholar, Tamai et al., 2000Tamai K. Semenov M. Kato Y. Spokony R. Liu C. Katsuyama Y. Hess F. Saint-Jeannet J.P. He X. LDL-receptor-related proteins in Wnt signal transduction.Nature. 2000; 407: 530-535Crossref PubMed Scopus (1088) Google Scholar) results in a series of events necessary for the recruitment of Axin to the plasma membrane (Bilic et al., 2007Bilic J. Huang Y.-L. Davidson G. Zimmermann T. Cruciat C.-M. Bienz M. Niehrs C. Wnt induces LRP6 signalosomes and promotes dishevelled-dependent LRP6 phosphorylation.Science. 2007; 316: 1619-1622Crossref PubMed Scopus (692) Google Scholar, Noordermeer et al., 1994Noordermeer J. Klingensmith J. Perrimon N. Nusse R. dishevelled and armadillo act in the wingless signalling pathway in Drosophila.Nature. 1994; 367: 80-83Crossref PubMed Scopus (319) Google Scholar, Tamai et al., 2004Tamai K. Zeng X. Liu C. Zhang X. Harada Y. Chang Z. He X. A mechanism for Wnt coreceptor activation.Mol. Cell. 2004; 13: 149-156Abstract Full Text Full Text PDF PubMed Scopus (440) Google Scholar, Zeng et al., 2008Zeng X. Huang H. Tamai K. Zhang X. Harada Y. Yokota C. Almeida K. Wang J. Doble B. Woodgett J. et al.Initiation of Wnt signaling: control of Wnt coreceptor Lrp6 phosphorylation/activation via frizzled, dishevelled and axin functions.Development. 2008; 135: 367-375Crossref PubMed Scopus (353) Google Scholar, Zeng et al., 2005Zeng X. Tamai K. Doble B. Li S. Huang H. Habas R. Okamura H. Woodgett J. He X. A dual-kinase mechanism for Wnt co-receptor phosphorylation and activation.Nature. 2005; 438: 873-877Crossref PubMed Scopus (653) Google Scholar), stabilization of β-catenin, and, ultimately, activation of Wnt target gene expression (MacDonald et al., 2009MacDonald B.T. Tamai K. He X. Wnt/beta-catenin signaling: components, mechanisms, and diseases.Dev. Cell. 2009; 17: 9-26Abstract Full Text Full Text PDF PubMed Scopus (4151) Google Scholar). Misregulated Wnt signaling is implicated in diseases ranging from osteoporosis to cancer (Clevers, 2006Clevers H. Wnt/beta-catenin signaling in development and disease.Cell. 2006; 127: 469-480Abstract Full Text Full Text PDF PubMed Scopus (4491) Google Scholar, MacDonald et al., 2009MacDonald B.T. Tamai K. He X. Wnt/beta-catenin signaling: components, mechanisms, and diseases.Dev. Cell. 2009; 17: 9-26Abstract Full Text Full Text PDF PubMed Scopus (4151) Google Scholar, Nusse, 2008Nusse R. Wnt signaling and stem cell control.Cell Res. 2008; 18: 523-527Crossref PubMed Scopus (444) Google Scholar, Polakis, 2007Polakis P. The many ways of Wnt in cancer.Curr. Opin. Genet. Dev. 2007; 17: 45-51Crossref PubMed Scopus (753) Google Scholar). Recently, this list has expanded to include metabolic disorders (Mani et al., 2007Mani A. Radhakrishnan J. Wang H. Mani A. Mani M.A. Nelson-Williams C. Carew K.S. Mane S. Najmabadi H. Wu D. Lifton R.P. LRP6 mutation in a family with early coronary disease and metabolic risk factors.Science. 2007; 315: 1278-1282Crossref PubMed Scopus (493) Google Scholar) and neurodegeneration (Caricasole et al., 2004Caricasole A. Copani A. Caraci F. Aronica E. Rozemuller A.J. Caruso A. Storto M. Gaviraghi G. Terstappen G.C. Nicoletti F. Induction of Dickkopf-1, a negative modulator of the Wnt pathway, is associated with neuronal degeneration in Alzheimer's brain.J. Neurosci. 2004; 24: 6021-6027Crossref PubMed Scopus (323) Google Scholar, De Ferrari et al., 2007De Ferrari G.V. Papassotiropoulos A. Biechele T. Wavrant De-Vrieze F. Avila M.E. Major M.B. Myers A. Sáez K. Henríquez J.P. Zhao A. et al.Common genetic variation within the low-density lipoprotein receptor-related protein 6 and late-onset Alzheimer's disease.Proc. Natl. Acad. Sci. USA. 2007; 104: 9434-9439Crossref PubMed Scopus (232) Google Scholar). An especially clear link exists between mutations of the protein adenomatous polyposis coli (APC), which prevent effective regulation of β-catenin levels, and colorectal cancers (Kinzler et al., 1991Kinzler K.W. Nilbert M.C. Vogelstein B. Bryan T.M. Levy D.B. Smith K.J. Preisinger A.C. Hamilton S.R. Hedge P. Markham A. et al.Identification of a gene located at chromosome 5q21 that is mutated in colorectal cancers.Science. 1991; 251: 1366-1370Crossref PubMed Scopus (634) Google Scholar, Nishisho et al., 1991Nishisho I. Nakamura Y. Miyoshi Y. Miki Y. Ando H. Horii A. Koyama K. Utsunomiya J. Baba S. Hedge P. Mutations of chromosome 5q21 genes in FAP and colorectal cancer patients.Science. 1991; 253: 665-669Crossref PubMed Scopus (1619) Google Scholar, Polakis, 2007Polakis P. The many ways of Wnt in cancer.Curr. Opin. Genet. Dev. 2007; 17: 45-51Crossref PubMed Scopus (753) Google Scholar). Also of particular note is the strong genetic relationship between LRP5 and bone homeostasis. Loss-of-function mutations in LRP5 cause the autosomal recessive disorder osteoporosis-pseudoglioma syndrome (OPPG), characterized by low bone mass, ocular defects and a predisposition to fractures (Gong et al., 2001Gong Y. Slee R.B. Fukai N. Rawadi G. Roman-Roman S. Reginato A.M. Wang H. Cundy T. Glorieux F.H. Lev D. et al.LDL receptor-related protein 5 (LRP5) affects bone accrual and eye development.Cell. 2001; 107: 513-523Abstract Full Text Full Text PDF PubMed Scopus (1853) Google Scholar). Conversely, several families whose members exhibit unusually high bone-mass density were found to carry missense (BMD) mutations in LRP5 (Boyden et al., 2002Boyden L.M. Mao J. Belsky J. Mitzner L. Farhi A. Mitnick M.A. Wu D. Insogna K. Lifton R.P. High bone density due to a mutation in LDL-receptor-related protein 5.N. Engl. J. Med. 2002; 346: 1513-1521Crossref PubMed Scopus (1333) Google Scholar, Rickels et al., 2005Rickels M.R. Zhang X. Mumm S. Whyte M.P. Oropharyngeal skeletal disease accompanying high bone mass and novel LRP5 mutation.J. Bone Miner. Res. 2005; 20: 878-885Crossref PubMed Scopus (48) Google Scholar, Whyte et al., 2004Whyte M.P. Reinus W.H. Mumm S. High-bone-mass disease and LRP5.N. Engl. J. Med. 2004; 350 (author reply 2096–2099): 2096-2099Crossref PubMed Scopus (59) Google Scholar). At the cell surface, Wnt/β-catenin signaling is regulated by two groups of secreted proteins with distinct modes of action. The secreted Frizzled-related protein (sFRPs) (Dann et al., 2001Dann C.E. Hsieh J.C. Rattner A. Sharma D. Nathans J. Leahy D.J. Insights into Wnt binding and signalling from the structures of two Frizzled cysteine-rich domains.Nature. 2001; 412: 86-90Crossref PubMed Scopus (372) Google Scholar, Hoang et al., 1996Hoang B. Moos Jr., M. Vukicevic S. Luyten F.P. Primary structure and tissue distribution of FRZB, a novel protein related to Drosophila frizzled, suggest a role in skeletal morphogenesis.J. Biol. Chem. 1996; 271: 26131-26137Crossref PubMed Scopus (209) Google Scholar) and Wnt inhibitory factor (WIF) (Hsieh et al., 1999Hsieh J.C. Kodjabachian L. Rebbert M.L. Rattner A. Smallwood P.M. Samos C.H. Nusse R. Dawid I.B. Nathans J. A new secreted protein that binds to Wnt proteins and inhibits their activities.Nature. 1999; 398: 431-436Crossref PubMed Scopus (592) Google Scholar) inhibit the Wnt/β-catenin pathway by directly binding to the Wnt proteins. The second class of Wnt inhibitors is composed of the structurally unrelated Dickkopf (DKK) (Glinka et al., 1998Glinka A. Wu W. Delius H. Monaghan A.P. Blumenstock C. Niehrs C. Dickkopf-1 is a member of a new family of secreted proteins and functions in head induction.Nature. 1998; 391: 357-362Crossref PubMed Scopus (1349) Google Scholar, Semënov et al., 2001Semënov M.V. Tamai K. Brott B.K. Kühl M. Sokol S. He X. Head inducer Dickkopf-1 is a ligand for Wnt coreceptor LRP6.Curr. Biol. 2001; 11: 951-961Abstract Full Text Full Text PDF PubMed Scopus (592) Google Scholar) and WISE (SOSTDC1)/sclerostin (SOST) (Itasaki et al., 2003Itasaki N. Jones C.M. Mercurio S. Rowe A. Domingos P.M. Smith J.C. Krumlauf R. Wise, a context-dependent activator and inhibitor of Wnt signalling.Development. 2003; 130: 4295-4305Crossref PubMed Scopus (271) Google Scholar, Li et al., 2005bLi X. Zhang Y. Kang H. Liu W. Liu P. Zhang J. Harris S.E. Wu D. Sclerostin binds to LRP5/6 and antagonizes canonical Wnt signaling.J. Biol. Chem. 2005; 280: 19883-19887Crossref PubMed Scopus (1042) Google Scholar, Semënov et al., 2005Semënov M. Tamai K. He X. SOST is a ligand for LRP5/LRP6 and a Wnt signaling inhibitor.J. Biol. Chem. 2005; 280: 26770-26775Crossref PubMed Scopus (605) Google Scholar) families of proteins. These proteins inhibit the Wnt/β-catenin signaling pathway by directly competing with Wnt for binding to LRP5 and LRP6 (Bafico et al., 2001Bafico A. Liu G. Yaniv A. Gazit A. Aaronson S.A. Novel mechanism of Wnt signalling inhibition mediated by Dickkopf-1 interaction with LRP6/Arrow.Nat. Cell Biol. 2001; 3: 683-686Crossref PubMed Scopus (662) Google Scholar, Semënov et al., 2001Semënov M.V. Tamai K. Brott B.K. Kühl M. Sokol S. He X. Head inducer Dickkopf-1 is a ligand for Wnt coreceptor LRP6.Curr. Biol. 2001; 11: 951-961Abstract Full Text Full Text PDF PubMed Scopus (592) Google Scholar). Both DKK1 and SOST have been shown to be directly involved in bone growth regulation. In particular, complete or partial loss of SOST function is responsible for sclerosteosis or Van Buchem diseases, respectively (Balemans et al., 2001Balemans W. Ebeling M. Patel N. Van Hul E. Olson P. Dioszegi M. Lacza C. Wuyts W. Van Den Ende J. Willems P. et al.Increased bone density in sclerosteosis is due to the deficiency of a novel secreted protein (SOST).Hum. Mol. Genet. 2001; 10: 537-543Crossref PubMed Scopus (909) Google Scholar, Balemans et al., 2002Balemans W. Patel N. Ebeling M. Van Hul E. Wuyts W. Lacza C. Dioszegi M. Dikkers F.G. Hildering P. Willems P.J. et al.Identification of a 52 kb deletion downstream of the SOST gene in patients with van Buchem disease.J. Med. Genet. 2002; 39: 91-97Crossref PubMed Scopus (574) Google Scholar); the unusually dense and strong bone observed in these conditions resembles the BMD phenotype caused by LRP5 gain-of-function mutations. DKK1 mutations causing comparable effects have not been reported, although the function of DKK1 in murine bone development is comparable to that of SOST (Li et al., 2005aLi X. Liu P. Liu W. Maye P. Zhang J. Zhang Y. Hurley M. Guo C. Boskey A. Sun L. et al.Dkk2 has a role in terminal osteoblast differentiation and mineralized matrix formation.Nat. Genet. 2005; 37: 945-952Crossref PubMed Scopus (262) Google Scholar). Several aspects of the DKK1 and SOST function remain unresolved. In particular, it is not fully understood how these inhibitors recognize LRP5/6, and accordingly, how mutations in LRP5 affect their function. The LRP coreceptors have large extracellular regions with a modular domain structure. Each module (designated below as an E domain) consists of a YWTD-class β-propeller followed by an EGF-like domain (Springer, 1998Springer T.A. An extracellular beta-propeller module predicted in lipoprotein and scavenger receptors, tyrosine kinases, epidermal growth factor precursor, and extracellular matrix components.J. Mol. Biol. 1998; 283: 837-862Crossref PubMed Scopus (167) Google Scholar). The four modules (E1–E4, or E1E4) are followed by three LDL type A repeats (LDLa) before the transmembrane segment. Recently, we reported that LRP6 is modular not only in structure but in its central biochemical function, binding to Wnt ligands (Bourhis et al., 2010Bourhis E. Tam C. Franke Y. Bazan J.F. Ernst J. Hwang J. Costa M. Cochran A.G. Hannoush R.N. Reconstitution of a frizzled8.Wnt3a.LRP6 signaling complex reveals multiple Wnt and Dkk1 binding sites on LRP6.J. Biol. Chem. 2010; 285: 9172-9179Crossref PubMed Scopus (153) Google Scholar, Gong et al., 2010Gong Y. Bourhis E. Chiu C. Stawicki S. DeAlmeida V.I. Liu B.Y. Phamluong K. Cao T.C. Carano R.A.D. Ernst J.A. et al.Wnt isoform-specific interactions with coreceptor specify inhibition or potentiation of signaling by LRP6 antibodies.PLoS ONE. 2010; 5: e12682Crossref PubMed Scopus (96) Google Scholar). Rather than having a single “Wnt site,” LRP6 has at least two. One of these is in the N-terminal region E1E2, which binds, for example, to WNT9B, while the other is in E3E4 and binds to WNT3A (Bourhis et al., 2010Bourhis E. Tam C. Franke Y. Bazan J.F. Ernst J. Hwang J. Costa M. Cochran A.G. Hannoush R.N. Reconstitution of a frizzled8.Wnt3a.LRP6 signaling complex reveals multiple Wnt and Dkk1 binding sites on LRP6.J. Biol. Chem. 2010; 285: 9172-9179Crossref PubMed Scopus (153) Google Scholar). Based on patterns of Wnt antagonism by anti-LRP6 antibodies, Wnts may be divided into three groups; the largest of these includes WNTs 1, 2, 2B, 6, 8A, 9A, 9B, and 10B (Gong et al., 2010Gong Y. Bourhis E. Chiu C. Stawicki S. DeAlmeida V.I. Liu B.Y. Phamluong K. Cao T.C. Carano R.A.D. Ernst J.A. et al.Wnt isoform-specific interactions with coreceptor specify inhibition or potentiation of signaling by LRP6 antibodies.PLoS ONE. 2010; 5: e12682Crossref PubMed Scopus (96) Google Scholar). Signaling in response to these Wnts is inhibited by the antibody YW210.09, originally raised against the antigen E1E2. Like the Wnt ligands, DKK1 binds to at least two sites on LRP5/6 (Bourhis et al., 2010Bourhis E. Tam C. Franke Y. Bazan J.F. Ernst J. Hwang J. Costa M. Cochran A.G. Hannoush R.N. Reconstitution of a frizzled8.Wnt3a.LRP6 signaling complex reveals multiple Wnt and Dkk1 binding sites on LRP6.J. Biol. Chem. 2010; 285: 9172-9179Crossref PubMed Scopus (153) Google Scholar, Li et al., 2005bLi X. Zhang Y. Kang H. Liu W. Liu P. Zhang J. Harris S.E. Wu D. Sclerostin binds to LRP5/6 and antagonizes canonical Wnt signaling.J. Biol. Chem. 2005; 280: 19883-19887Crossref PubMed Scopus (1042) Google Scholar), explaining, at least conceptually, its broad spectrum of Wnt inhibition. We report here the first structures of a YWTD propeller from the LRP receptor superfamily other than the prototype low-density lipoprotein receptor (LDLr) (Jeon et al., 2001Jeon H. Meng W. Takagi J. Eck M.J. Springer T.A. Blacklow S.C. Implications for familial hypercholesterolemia from the structure of the LDL receptor YWTD-EGF domain pair.Nat. Struct. Biol. 2001; 8: 499-504Crossref PubMed Scopus (183) Google Scholar). The structures reveal the E1 domain of LRP6 to recognize a short linear peptide sequence, and we find not only that this sequence is present in structurally diverse Wnt pathway inhibitors that bind to LRP5/6 but also that it is important for their function. The E1 binding motif in DKK1 defines a new DKK1 interaction domain (in addition to the previously characterized CRD2 domain), and on this basis we propose a two-site model for DKK1 recognition of LRP5/6 and inhibition of Wnt signaling. Finally, our results show that human BMD mutations involve LRP5 residues that make direct contacts with peptide ligand (or are important for structural integrity of the peptide binding pocket), explaining in detail how these mutations disrupt inhibition of Wnt signaling by SOST and DKK1. To better understand the mechanism of Wnt signaling inhibition by the YW210.09 antibody, we attempted to cocrystallize the Fab and the first two tandem β-propeller-EFG-like domains of LRP6 (E1E2). After an extended incubation (and degradation of E2), we obtained crystals of the E1 domain only in complex with the Fab. The structure was determined by molecular replacement and refined to 1.9 Å resolution (R/Rfree of 0.175/0.220; Table 1). The architecture of the LRP6 E1 domain closely resembles that of LDLr (Jeon et al., 2001Jeon H. Meng W. Takagi J. Eck M.J. Springer T.A. Blacklow S.C. Implications for familial hypercholesterolemia from the structure of the LDL receptor YWTD-EGF domain pair.Nat. Struct. Biol. 2001; 8: 499-504Crossref PubMed Scopus (183) Google Scholar). Indeed, the structures superimpose with an rmsd of 0.83 Å over 245 Cα atoms (of 304 total), despite a sequence identity of only 36%. The interaction between the EGF-like domain and the β-propeller is extensive, exhibiting features similar to those observed in LDLr structures (Jeon et al., 2001Jeon H. Meng W. Takagi J. Eck M.J. Springer T.A. Blacklow S.C. Implications for familial hypercholesterolemia from the structure of the LDL receptor YWTD-EGF domain pair.Nat. Struct. Biol. 2001; 8: 499-504Crossref PubMed Scopus (183) Google Scholar, Rudenko et al., 2002Rudenko G. Henry L. Henderson K. Ichtchenko K. Brown M.S. Goldstein J.L. Deisenhofer J. Structure of the LDL receptor extracellular domain at endosomal pH.Science. 2002; 298: 2353-2358Crossref PubMed Scopus (376) Google Scholar).Table 1Structural and Refinement Statistics for LRP6 E1 Domain ComplexesComplexLRP6E1-FabLRP6E1-DKK1pepLRP6E1-SOSTpepPDB code3SOB3SOQ3SOVData collectionSSRL71/ADSC Q315007HF/Saturn 944+CMCF108ID/MAR300Space groupC2C2C2Unit cella = 167.6 Å, b = 83.3 Å, c = 62.8 Å, α = 90° β = 112° γ = 90°a = 103.3 Å, b = 47.0 Å, c = 68.8 Å, α = 90° β = 97.2° γ = 90°a = 103.3 Å, b = 46.9 Å, c = 68.3 Å, α = 90° β = 97.4° γ = 90°Resolution30–1.9 Å30–1.9 Å30–1.27 ÅTotal number of reflections217,270888,814340,026Completeness91.9 (57.7)aValues in parentheses are of the highest resolution shell.98.0 (82.9)aValues in parentheses are of the highest resolution shell.98.1 (96.5)aValues in parentheses are of the highest resolution shell.Redundancy3.8 (3.7)3.5 (2.4)3.8 (3.8)I/σ21.3(3.8)16.8 (2.3)27.0 (2.3)RsymbRsym = Σ|Ihi - Ih|/ΣIhi, where Ihi is the scaled intensity of the ith symmetry-related observation of reflection h and Ih is the mean value.0.060 (0.333)0.073 (0.454)0.046 (0.585)RefinementResolution range30–1.9 Å30–1.9 Å30–1.27 ÅRcrystcRcryst = Σh|Foh - Fch| /ΣhFoh, where Foh and Fch are the observed and calculated structure factor amplitudes for reflection h./RfreedValue of Rfree is calculated for 5% randomly chosen reflections not included in the refinement.0.174/0.2120.170/0.2210.153/0.180Nonhydrogen atoms618727803050Water molecules486266339Average B, overall37.927.523.1Protein39.926.221.3Water44.736.933.5Rmsd bond lengths0.008 Å0.004 Å0.016 ÅRmsd angles1.172°0.925°1.785°a Values in parentheses are of the highest resolution shell.b Rsym = Σ|Ihi - Ih|/ΣIhi, where Ihi is the scaled intensity of the ith symmetry-related observation of reflection h and Ih is the mean value.c Rcryst = Σh|Foh - Fch| /ΣhFoh, where Foh and Fch are the observed and calculated structure factor amplitudes for reflection h.d Value of Rfree is calculated for 5% randomly chosen reflections not included in the refinement. Open table in a new tab The antibody binds to a region at the top center of the β-propeller (Figure 1A ), an area that is frequently involved in protein-protein interactions (Springer, 1998Springer T.A. An extracellular beta-propeller module predicted in lipoprotein and scavenger receptors, tyrosine kinases, epidermal growth factor precursor, and extracellular matrix components.J. Mol. Biol. 1998; 283: 837-862Crossref PubMed Scopus (167) Google Scholar, Rudenko et al., 2002Rudenko G. Henry L. Henderson K. Ichtchenko K. Brown M.S. Goldstein J.L. Deisenhofer J. Structure of the LDL receptor extracellular domain at endosomal pH.Science. 2002; 298: 2353-2358Crossref PubMed Scopus (376) Google Scholar, Takagi et al., 2003Takagi J. Yang Y. Liu J.H. Wang J.H. Springer T.A. Complex between nidogen and laminin fragments reveals a paradigmatic beta-propeller interface.Nature. 2003; 424: 969-974Crossref PubMed Scopus (116) Google Scholar). A prominent acidic patch occupies roughly a third of the total surface area on this side of the β-propeller but barely overlaps with the YW210.09 epitope (see Figure S1A available online). The paratope is composed of residues from five of the six antibody CDRs (the exception is L2) (Figure S1B). Contacts formed by heavy chain CDRs represent 80% of the buried surface area, with the long (17 residue) CDR H3 (Figure 1A) itself accounting for over 50%. In particular, the short, contiguous sequence NAVK (100–100c; Kabat numbering) exhibits an unusually significant degree of interaction with complementary surfaces on the propeller (Figure 1B). Asn100 of the antibody makes a pair of hydrogen bonds with Asn185 of LRP6, forming a “handshake” interaction, while the Val100b side-chain docks into a hydrophobic cavity in the center channel of the β-propeller. Between these, LRP6 Arg141 is largely buried at the interface where it interacts with the main-chain carbonyl of Asn100 and appears to integrate two hydrogen-bond networks. The first of these is the Asn-Asn “handshake,” while the second involves two bridging waters (Wat1 and Wat2) that hydrogen bond to antibody main-chain amides of Val100b and Lys100c and to LRP6 Ser96, Asp98, and Arg141 side chains. Finally, the ε-amino group of Lys100c occupies a shallow cleft on the acidic patch of LRP6, where it interacts with the LRP6 main-chain carbonyls of Val70 and Leu95 and the side-chain carboxylates of Glu73 and Glu115. This interaction between the CDR H3 “NAVK” sequence and LRP6 E1 β-propeller is highly similar to the interaction reported between laminin and another YWTD β-propeller, nidogen (Takagi et al., 2003Takagi J. Yang Y. Liu J.H. Wang J.H. Springer T.A. Complex between nidogen and laminin fragments reveals a paradigmatic beta-propeller interface.Nature. 2003; 424: 969-974Crossref PubMed Scopus (116) Google Scholar) (Figure 2). In both cases, the core binding motif is “NAV,” and these residues interact with the two propellers in the same way. In particular, the interactions with Fab Asn100 have exact counterparts in the laminin-nidogen complex (Figure 2A), and in both cases the Val side chain enters a central hydrophobic cavity. Nidogen and LRP6 each interact with a charged residue outside of the core binding motif: in the case of LRP6, this is the Lys of “NAVK” (i.e., Lys100c from the Fab, Figure 2B), while in nidogen, it is the Asp of “DPNAV” from laminin (Figure 2C). In contrast to LDLr, the central channels of nidogen and LRP6 E1 are closed off from solvent by a Phe residue held in place by a nearby Trp side chain, or “hydrophobic shutter” (Takagi et al., 2003Takagi J. Yang Y. Liu J.H. Wang J.H. Springer T.A. Complex between nidogen and laminin fragments reveals a paradigmatic beta-propeller interface.Nature. 2003; 424: 969-974Crossref PubMed Scopus (116) Google Scholar) (Figure S2). This feature is thought to distinguish YWTD propeller domains that can bind to low molecular-weight ligands, and, based on sequence, it would appear that multiple propellers from the Wnt coreceptors LRP5 and LRP6 might resemble nidogen in this respect (Takagi et al., 2003Takagi J. Yang Y. Liu J.H. Wang J.H. Springer T.A. Complex between nidogen and laminin fragments reveals a paradigmatic beta-propeller interface.Nature. 2003; 424: 969-974Crossref PubMed Scopus (116) Google Scholar) (Figure S2C). However, it was not anticipated that LRP5/6 and nidogen would recognize the same core binding motif. To discover optimal binding sequences for LRP6 E1, we used peptide phage display. Peptide sequence motifs are remarkably consistent with the binding motif observed in the antibody complex structure. For peptides selected from both linear (Figure 3) and cyclic peptides libraries (Figure S3), an invariant Asn is present (position 0). At position + 2, there is a branched hydrophobic residue, with Ile rather than Val being present in the overwhelming majority of cases. A basic residue (most often Lys) is generally present at position +3, but this preference varies somewhat for different cyclic sequence subgroups (Figure S3). Affinities (IC50 values) for representative linear peptides were in the low micromolar range, while affinities of cyclic peptides ranged from low micromolar to submicromolar (not shown). The strong selection for this short motif confirms that the E1 propeller recognizes a specific linear sequence. A sequence present in human DKK1 (NAIKN; amino acids 40–44) is very similar to the motif found in the CDR H3 loop of YW210.09 and to the preferred LRP6 E1 binding motif identified by phage display. The motif is present in multiple DKK proteins (Figure 4A ) and is conserved among species (Figure S4A). This N-terminal region of DKK1 precedes the two predicted cysteine-rich domains (CRDs) and, unlike the CRDs, has not been identified previously as functionally important (Brott and Sokol, 2002Brott B.K. Sokol S.Y. Regulation of Wnt/LRP signaling by distinct domains of Dickkopf proteins.Mol. Cell. Biol. 2002; 22: 6100-6110Crossref PubMed Scopus (190) Google Scholar). A similar motif appears in two other proteins regulating Wnt signaling via interaction with LRP5/6, namely SOST (Semënov et al., 2005Semënov M. Tamai K. He X. SOST is a ligand for LRP5/LRP6 and a Wnt signaling inhibitor.J. Biol. Chem. 2005; 280: 26770-26775Crossref PubMed Scopus (605) Google Scholar) and WISE (Itasaki et al., 2003Itasaki N. Jones C.M. Mercurio S. Rowe A. Domingos P.M. Smith J.C. Krumlauf R. Wise, a context-dependent activator and inhibitor of Wnt signalling.Development. 2003; 130: 4295-4305Crossref PubMed Scopus (271) Google Scholar) (Figure 4A; Figure S4A). SOST and WISE are highly similar and belong to the superfamily of cystine-knot proteins (McDonald and Hendrickson, 1993McDonald N.Q. Hendrickson W.A. A structural superfamily of growth factors containing a cystine knot motif.Cell. 1993; 73: 421-424Abstract Full Text PDF PubMed Scopus (475) Google Scholar); the motif occurs in the extended loop 2, also called the “heel” (Lintern et al., 2009Lintern K.B. Guidato S. Rowe A. Saldanha J.W. Itasaki N. Characterization of wise protein and its molecular mechanism to interact with both Wnt and BMP signals.J. Biol. Chem. 2009; 284: 23159-23168Crossref PubMed Scopus (99) Google Scholar, Veverka et al., 2009Veverka V. Henry A.J. Slocombe P.M. Ventom A. Mulloy B. Muskett F.W. Muzylak M. Greenslade K. Moore A. Zhang L. et al.Characterization of the structural features and interactions of sclerostin: molecular insight into a key regulator of Wnt-mediated bone formation.J. Biol. Chem. 2009; 284: 10890-10900Crossref PubMed Scopus (191) Google Scholar). In the case of SOST, the “heel” is the binding epitope for a neutralizing antibody (Lintern et al., 2009Lintern K.B. Guidato S. Rowe A. Saldanha J.W. Itasaki N. Characterization of wise protein and its molecular mechanism to interact with both Wnt and BMP signals.J. Biol. Chem. 2009; 284: 23159-23168Crossref PubMed Scopus (99) Google Scholar). Based on the aligned sequences, the core motif would appear to be “NXI,” where “X” is a small residue (Ala, Ser) or Trp. The presence of this motif suggests that the inhibito" @default.
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• W2022121540 title "Wnt Antagonists Bind through a Short Peptide to the First β-Propeller Domain of LRP5/6" @default.
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