Title: Structural Insight into the Mechanisms of Wnt Signaling Antagonism by Dkk
Abstract: Dickkopf (Dkk) proteins are antagonists of the canonical Wnt signaling pathway and are crucial for embryonic cell fate and bone formation. Wnt antagonism of Dkk requires the binding of the C-terminal cysteine-rich domain of Dkk to the Wnt coreceptor, LRP5/6. However, the structural basis of the interaction between Dkk and low density lipoprotein receptor-related protein (LRP) 5/6 is unknown. In this study, we examined the structure of the Dkk functional domain and elucidated its interactions with LRP5/6. Using NMR spectroscopy, we determined the solution structure of the C-terminal cysteine-rich domain of mouse Dkk2 (Dkk2C). Then, guided by mutagenesis studies, we docked Dkk2C to the YWTD β-propeller domains of LRP5/6 and showed that the ligand binding site of the third LRP5/6 β-propeller domain matches Dkk2C best, suggesting that this domain binds to Dkk2C with higher affinity. Such differential binding affinity is likely to play an essential role in Dkk function in the canonical Wnt pathway. Dickkopf (Dkk) proteins are antagonists of the canonical Wnt signaling pathway and are crucial for embryonic cell fate and bone formation. Wnt antagonism of Dkk requires the binding of the C-terminal cysteine-rich domain of Dkk to the Wnt coreceptor, LRP5/6. However, the structural basis of the interaction between Dkk and low density lipoprotein receptor-related protein (LRP) 5/6 is unknown. In this study, we examined the structure of the Dkk functional domain and elucidated its interactions with LRP5/6. Using NMR spectroscopy, we determined the solution structure of the C-terminal cysteine-rich domain of mouse Dkk2 (Dkk2C). Then, guided by mutagenesis studies, we docked Dkk2C to the YWTD β-propeller domains of LRP5/6 and showed that the ligand binding site of the third LRP5/6 β-propeller domain matches Dkk2C best, suggesting that this domain binds to Dkk2C with higher affinity. Such differential binding affinity is likely to play an essential role in Dkk function in the canonical Wnt pathway. Dickkopf (Dkk) proteins are antagonists of the canonical Wnt signaling pathway and are crucial for embryonic cell fate and bone formation, and abnormal Dkk function has been implicated in cancers, bone diseases, and Alzheimer disease (1Niehrs C. Oncogene. 2006; 25: 7469-7481Crossref PubMed Scopus (776) Google Scholar). Dkk is composed of two characteristic cysteine-rich domains, the N-terminal and C-terminal cysteine-rich domain, respectively, each containing 10 conserved cysteines, separated by a variable-length spacer region (2Krupnik V.E. Sharp J.D. Jiang C. Robison K. Chickering T.W. Amaravadi L. Brown D.E. Guyot D. Mays G. Leiby K. Chang B. Duong T. Goodearl A.D. Gearing D.P. Sokol S.Y. McCarthy S.A. Gene. 1999; 238: 301-313Crossref PubMed Scopus (418) Google Scholar). Wnt antagonism by Dkk requires the binding of the C-terminal cysteine-rich domain of Dkk to the Wnt coreceptor, low density lipoprotein receptor-related protein (LRP) 3The abbreviations used are:LRPlow density lipoprotein receptor-related proteinDkk2Cthe C-terminal cysteine-rich domain of mouse Dkk2LRP5-PD1the first β-propeller domain of mouse LRP5LRP5-PD2the second β-propeller domain of mouse LRP5LRP5-PD3the third β-propeller domain of mouse LRP5HAhemagglutininNOEnuclear Overhauser effectAPalkaline phosphataseHEKhuman embryonic kidney. 3The abbreviations used are:LRPlow density lipoprotein receptor-related proteinDkk2Cthe C-terminal cysteine-rich domain of mouse Dkk2LRP5-PD1the first β-propeller domain of mouse LRP5LRP5-PD2the second β-propeller domain of mouse LRP5LRP5-PD3the third β-propeller domain of mouse LRP5HAhemagglutininNOEnuclear Overhauser effectAPalkaline phosphataseHEKhuman embryonic kidney. 5 or 6 (LRP5/6) (3Semenov M.V. Tamai K. Brott B.K. Kuhl M. Sokol S. He X. Curr. Biol. 2001; 11: 951-961Abstract Full Text Full Text PDF PubMed Scopus (592) Google Scholar, 4Li L. Mao J.H. Sun L. Liu W.Z. Wu D.Q. J. Biol. Chem. 2002; 277: 5977-5981Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar, 5Brott B.K. Sokol S.Y. Mol. Cell. Biol. 2002; 22: 6100-6110Crossref PubMed Scopus (190) Google Scholar, 6Mao B. Niehrs C. Gene. 2003; 302: 179-183Crossref PubMed Scopus (264) Google Scholar). The Dkk-LRP5/6 complex antagonizes canonical Wnt signaling by inhibiting LRP5/6 interaction with Wnt (4Li L. Mao J.H. Sun L. Liu W.Z. Wu D.Q. J. Biol. Chem. 2002; 277: 5977-5981Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar, 7Bafico A. Liu G. Yaniv A. Gazit A. Aaronson S.A. Nat. Cell Biol. 2001; 3: 683-686Crossref PubMed Scopus (662) Google Scholar, 8Li Y. Lu W. He X. Schwartz A.L. Bu G. Oncogene. 2004; 23: 9129-9135Crossref PubMed Scopus (73) Google Scholar) and by forming a ternary complex with the transmembrane protein Kremen (9Mao B. Wu W. Davidson G. Marhold J. Li M. Mechler B.M. Delius H. Hoppe D. Stannek P. Walter C. Glinka A. Niehrs C. Nature. 2002; 417: 664-667Crossref PubMed Scopus (859) Google Scholar, 10Zhang Y. Wang Y. Li X. Zhang J. Mao J. Li Z. Zheng J. Li L. Harris S. Wu D. Mol. Cell. Biol. 2004; 24: 4677-4684Crossref PubMed Scopus (142) Google Scholar) that promotes internalization of LRP5/6 (9Mao B. Wu W. Davidson G. Marhold J. Li M. Mechler B.M. Delius H. Hoppe D. Stannek P. Walter C. Glinka A. Niehrs C. Nature. 2002; 417: 664-667Crossref PubMed Scopus (859) Google Scholar). Despite the importance of the interaction between Dkk and LRP5/6, its structural basis is unknown. low density lipoprotein receptor-related protein the C-terminal cysteine-rich domain of mouse Dkk2 the first β-propeller domain of mouse LRP5 the second β-propeller domain of mouse LRP5 the third β-propeller domain of mouse LRP5 hemagglutinin nuclear Overhauser effect alkaline phosphatase human embryonic kidney. low density lipoprotein receptor-related protein the C-terminal cysteine-rich domain of mouse Dkk2 the first β-propeller domain of mouse LRP5 the second β-propeller domain of mouse LRP5 the third β-propeller domain of mouse LRP5 hemagglutinin nuclear Overhauser effect alkaline phosphatase human embryonic kidney. The Dkk family has at least four members (2Krupnik V.E. Sharp J.D. Jiang C. Robison K. Chickering T.W. Amaravadi L. Brown D.E. Guyot D. Mays G. Leiby K. Chang B. Duong T. Goodearl A.D. Gearing D.P. Sokol S.Y. McCarthy S.A. Gene. 1999; 238: 301-313Crossref PubMed Scopus (418) Google Scholar), and Dkk1 and Dkk2 share 50% identity in their N-terminal domains and 70% identity in their C-terminal cysteine-rich domains. We previously found that the C-terminal domain of human DKK1 and 2, which contains the second cysteine-rich region, is sufficient for antagonism of Wnt activity in mammalian cells (4Li L. Mao J.H. Sun L. Liu W.Z. Wu D.Q. J. Biol. Chem. 2002; 277: 5977-5981Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). The same was also found to be true in Xenopus; the C-terminal domain of Dkk1 and 2 is both necessary and sufficient to inhibit Wnt-stimulated induction of secondary axis development and transcriptional activation of the Siamois promoter, cooperate with a dominant-negative BMP4 receptor to induce head structure development, and physically associate with LRP5/6 (5Brott B.K. Sokol S.Y. Mol. Cell. Biol. 2002; 22: 6100-6110Crossref PubMed Scopus (190) Google Scholar, 6Mao B. Niehrs C. Gene. 2003; 302: 179-183Crossref PubMed Scopus (264) Google Scholar). In this report, we defined the structure of the Dkk functional domain, the C-terminal cysteine-rich domain of mouse Dkk2 (amino acids Met172-Ile259) (Dkk2C), and elucidated its interactions with the extracellular β-propeller domains of LRP5/6. Our structural studies suggest that, comparing with other β-propeller domains, the third β-propeller domain of LRP5/6 binds to Dkk2C with greatest affinity. Such differential binding affinity is likely to play an essential role in Dkk function in the canonical Wnt pathway. Furthermore, this finding not only increases our understanding of the regulation of canonical Wnt signaling by Dkk but also may expand the range of options for innovative targeted therapies. Dkk2C-mediated Inhibition of Wnt Activity—The recombinant protein Dkk2C (amino acids Met172-Ile259 of mouse Dkk2) was expressed and purified from an Escherichia coli system as described previously (11Wong H.C. Mao J. Nguyen J.T. Srinivas S. Zhang W. Liu B. Li L. Wu D. Zheng J. Nat. Struct. Biol. 2000; 7: 1178-1184Crossref PubMed Scopus (126) Google Scholar). The recombinant protein contained an N-terminal S tag and a thrombin cleavage site between the S tag and Dkk2C. The purified recombinant Dkk2C contained only one single band in SDS-PAGE. NIH3T3 cells were seeded in 24-well plates at 4 × 105 cells/well and transfected with a LEF-1 luciferase reporter plasmid, an enhanced green fluorescent protein plasmid, and LacZ plasmid (total 0.5 μg of DNA/well) by using Lipofectamine and Plus (Invitrogen), as suggested by the manufacturer. 24 h after transfection, cells were treated with Wnt3a conditioned medium and different dosages of purified Dkk2C for 6 h. Cells were treated with Wnt3a and vehicle as the control for 6 h. Then cells were lysed, and luciferase activity in the cell lysate was measured as described previously (12Li L. Yuan H. Xie W. Mao J. Caruso A.M. McMahon A. Sussman D.J. Wu D. J. Biol. Chem. 1999; 274: 129-134Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar). Luminescence intensity, which represents Wnt activity, was normalized against the fluorescence intensity of enhanced green fluorescent protein. S-protein Pulldown Experiments and Western Blotting Analysis—For preparation of the first β-propeller domain of mouse LRP5 with HA tag (LRP5-PD1-HA)-containing conditioned medium, HEK cells were seeded in 6-well plates at 4 × 105 cells/well and transfected with 1 μg of DNA/well. The conditioned medium was collected 30 h after transfection by centrifugation. S-protein-agarose was obtained from Novagen, and the pulldown experiments were conducted in accordance with a standard protocol provided by the manufacturer. Briefly, for each pulldown experiment, 300 μl of S-tagged Dkk2C (5 μm) was incubated with 200 μl of S-protein-agarose at room temperature for 2 h with gentle agitation to allow the binding of S-tagged Dkk2C to S-protein-agarose, followed by washing four times to remove the unbound Dkk2C by centrifugation. Then, the S-tagged Dkk2C-charged agarose was incubated with the LRP5-PD1-HA conditioned medium in indicated concentrations at 4 °C for 4 h with gentle agitation. After the agarose was washed three times with buffer, it underwent SDS-PAGE and then Western blotting analysis. Western blotting analysis was performed following a standard protocol by using mouse monoclonal IgG3 against an HA tag (from Millipore) as the primary antibody and goat anti-mouse horseradish peroxidase-conjugated IgG (from Cell Signaling Technology) as the secondary antibody. Finally, the membrane was incubated in SuperSignal West Femto maximum sensitivity substrate (Pierce) at room temperature for 5 min, and the results were developed on the film (Eastman Kodak Co.). Structural Determination of the Solution Structure of Dkk2C—The method used to determine the solution structure of Dkk2C is similar to those described previously (11Wong H.C. Mao J. Nguyen J.T. Srinivas S. Zhang W. Liu B. Li L. Wu D. Zheng J. Nat. Struct. Biol. 2000; 7: 1178-1184Crossref PubMed Scopus (126) Google Scholar, 13Wong H.C. Bourdelas A. Krauss A. Lee H.-J. Shao Y.-M. Wu D. Mlodzik M. Shi D.L. Zheng J. Mol. Cell. 2003; 12: 1251-1260Abstract Full Text Full Text PDF PubMed Scopus (392) Google Scholar). Briefly, the 13C/15N double-labeled protein was produced in an E. coli system, and the N-terminal S tag was removed by thrombin. Typical NMR samples consisted of 1 mm 15N/13C-Dkk2C in 5 mm D4-acetic acid (pH 5.0) buffer with 10% (v/v) D2O. All NMR experiments were performed with Bruker 600- and 800-MHz NMR spectrometers at 25 °C. NMR spectra were processed and displayed by the NMRPipe (14Delaglio F. Grzesiek S. Vuister G.W. Zhu G. Pfeifer J. Bax A. J. Biomol. NMR. 1995; 6: 277-293Crossref PubMed Scopus (11533) Google Scholar) software package. The program XEASY (15Eccles C. Guntert P. Billeter M. Wuthrich K. J. Biomol. NMR. 1991; 1: 111-130Crossref PubMed Scopus (269) Google Scholar) was used for data analysis and structure assignment. Backbone assignment was based mainly on HNCA, HN(CO)CA, HNCACB, and CBCA(CO)NH experiments. Side chain proton resonance was assigned by using 15N HSQC-TOCSY and HCCH-TOCSY. Aromatic side chain proton resonance was assigned with CB(CGCD)HD and CB(CGC-DCE)HE experiments. NOE distance constraints were obtained from NOE peaks in two-dimensional 1H-1H NOE spectroscopy, three-dimensional 15N HSQC-NOE spectroscopy, and three-dimensional 13C HSQC-NOE spectroscopy experiments. Intensities of NOE peaks were calibrated and converted to distance constraints by the program CALIBA (16Guntert P. Braun W. Wuthrich K. J. Mol. Biol. 1991; 217: 517-530Crossref PubMed Scopus (914) Google Scholar). CYANA2.1 (17Guntert P. Mumenthaler C. Wuthrich K. J. Mol. Biol. 1997; 273: 283-298Crossref PubMed Scopus (2553) Google Scholar) software was used for structure calculations, which were based on 1,879 proton-proton distance constraints and 112 dihedral angle restraints. Through the space proximity, the five disulfide bridges within the structure could be clearly identified in the earlier structural calculations. Based on such information, 30 disulfide distance constraints (three upper limits and three lower limits for each disulfide bridge) were added in the final structural calculation. The superimposition of backbone atoms of 20 conformers with smallest target function values among 200 calculated structures yielded a root mean square deviation of 0.36 ± 0.11 Å relative to the average structure, and the average target function value of ensemble structures was 1.57 ± 0.15 Å2 with no distance violations >0.2 Å or dihedral angle violations >5°. The statistical characteristics of these 20 best conformers are described in Table 1.TABLE 1Statistical characteristics of the 20 conformers of the solution structure of Dkk2CParameterNo. of NOE distance restraintsIntraresidue448InterresidueSequential561Medium range243Long range627Total1879No. of disulfides restraints30No. of Talos dihedral angle restraintsπ56ϰ56r.m.s. deviations from the mean (Å)aThe average root mean square deviation (r.m.s.d.) between the 20 structures with the lowest target functions and the mean coordinates of the protein.Overall structure,bResidues Gly177-Ile259. backbone0.36 ± 0.11Gly177-Gln217 and Glu226-Ile259, backbone0.18 ± 0.04Overall structure,bResidues Gly177-Ile259. heavy atoms0.82 ± 0.10Gly177-Gln217 and Glu226-Ile259, heavy atoms0.68 ± 0.10ResiduesbResidues Gly177-Ile259. in Ramachandran plot (%)cExcluding glycines and prolines and calculated using the Ramachandran macro in CYANA software.Most favorable regions84.7Additionally allowed regions13.4Generously allowed regions1.9Disallowed regions0.0a The average root mean square deviation (r.m.s.d.) between the 20 structures with the lowest target functions and the mean coordinates of the protein.b Residues Gly177-Ile259.c Excluding glycines and prolines and calculated using the Ramachandran macro in CYANA software. Open table in a new tab Mutagenesis Studies—To determine the effect of representative point mutants of Dkk1 on the inhibition of Wnt activity (10Zhang Y. Wang Y. Li X. Zhang J. Mao J. Li Z. Zheng J. Li L. Harris S. Wu D. Mol. Cell. Biol. 2004; 24: 4677-4684Crossref PubMed Scopus (142) Google Scholar), NIH3T3 cells were transfected with LEF-1 luciferase reporter plasmids and 1 day later were treated with Wnt3a conditioned medium and Dkk1 or Dkk1 mutants conditioned medium prepared from HEK cells for 6 h. Then luciferase activity was measured as described above. To measure the binding of Dkk1 and its mutants to LRP6, HEK cells were transfected with LRP6 plasmids and 1 day later were incubated on ice with wild-type or mutant Dkk1-alkaline phosphatase (AP) fusion protein conditioned medium for 2 h (10Zhang Y. Wang Y. Li X. Zhang J. Mao J. Li Z. Zheng J. Li L. Harris S. Wu D. Mol. Cell. Biol. 2004; 24: 4677-4684Crossref PubMed Scopus (142) Google Scholar). Then the cells were washed and lysed, and AP activity in cell lysate was measured by using a Tropix luminescence AP assay kit as described previously (10Zhang Y. Wang Y. Li X. Zhang J. Mao J. Li Z. Zheng J. Li L. Harris S. Wu D. Mol. Cell. Biol. 2004; 24: 4677-4684Crossref PubMed Scopus (142) Google Scholar). Elucidation of the Complex of LRP5 Bound with Dkk—The software package ICM (Molsoft) was used to build the structures of the first three β-propeller domains of LRP5 (termed as LRP5-PD1, PRP5-PD2, and LRP5-PD3) using the crystal structure of the low density lipoprotein receptor YWTD β-propeller domain (Protein Data Bank code 1IJQ (18Jeon H. Meng W.Y. Takagi J. Eck M.J. Springer T.A. Blacklow S.C. Nat. Struct. Biol. 2001; 8: 499-504Crossref PubMed Scopus (183) Google Scholar)) as the template. The initial homology model structures were refined and evaluated by using software package AMBER8 (19Case D.A. Cheatham III, T.E. Darden T. Gohlke H. Luo R. Merz Jr., K.M. Onufriev A. Simmerling C. Wang B. Woods R.J. J. Comput. Chem. 2005; 26: 1668-1688Crossref PubMed Scopus (6519) Google Scholar). In this step, a 5-ns molecular dynamic simulation with 2 femtoseconds/step was performed by placing the individual propeller domain in a TIP3P water box. The docking studies used the HADDOCK (20Dominguez C. Boelens R. Bonvin A.M. J. Am. Chem. Soc. 2003; 125: 1731-1737Crossref PubMed Scopus (2169) Google Scholar) program. First, the homology model of the third β-propeller domain of LRP5 (LRP5-PD3) and the solution structure of Dkk2C were used as starting structures. Mutation of the Tyr719, Glu721, Arg764, Trp780, Asp887, and Phe888 residues of LRP5-PD3 had an effect of >10% on Dkk-mediated inhibition of Wnt activity. These residues were defined as active residues, and the neighboring surface residues (Arg652, Ala653, Val694, Lys697, Asp718, Gln737, Gly738, Asn762, Gly781, Pro784, Arg805, Trp863, His866, and Met890) were defined as passive residues. In Dkk2C, mutation of His198 (His210 in Dkk1), Lys205 (Lys217), Arg230 (Arg242), and His254 (His267) strongly disrupted both Dkk-mediated Wnt inhibition and LRP6 binding; these residues were defined as active residues. Passive residues were neighboring surface residues Glu179, Phe199, Trp200, Thr201, Leu203, Pro206, Glu212, Val213, Lys216, Gln217, Glu226, Ile227, Gln229, Val241, Thr246, Ser249, Arg252, and Leu253. The flexible interface was defined as active and passive residues ±2 sequential residues for the purpose of docking. Ambiguous interaction restraints used in the docking process were defined as an ambiguous distance between all active and passive residues shown to be involved at the interaction interface. The docking calculation was initiated with two proteins separated by 150 Å with random starting orientations. Three stages of docking solutions (rigid-body docking, semi-flexible simulated annealing, and a final refinement in water) were executed sequentially by energy minimization. Complex structures were sorted according to the intermolecular interaction energy (the sum of intermolecular van der Waals and electrostatic energies and restraint energies). In the last water refinement stage, the 100 docking structures with the lowest intermolecular interaction energies were generated and clustered on the basis of a 1.0-Å backbone root mean square deviation tolerance at the binding interface. The final docking complex structure was the structure that had the lowest intermolecular interaction energy within the cluster with the lowest average intermolecular interaction energy. Using an E. coli expression system, we expressed and purified the C-terminal cysteine-rich domain of mouse Dkk2 (Dkk2C) (residues Met172-Ile259). The purified recombinant Dkk2C contained only one single band in SDS-PAGE (Fig. 1A). The Dkk2C possessed a significant inhibitory activity on canonical Wnt signaling; it inhibited Wnt3a activities with an IC50 value around 8 nm in the Wnt reporter gene assay (Fig. 1B), indicating the protein we produced is fully functional. Furthermore, in vitro pulldown experiments showed that Dkk2C directly bound to the first propeller domain of LRP5 (LRP5-PD1), further confirming that the protein we produced is well folded and functional (Fig. 1C). Using NMR spectroscopy, we determined the solution structure of Dkk2C (Fig. 2A). The structure was well defined except for one loop region and the N-terminal region (Fig. 2B and Table 1), and the five disulfide bonds were clearly identified. Within the structure of Dkk2C are two subdomains sharing very similar topology; each has a central anti-parallel β-sheet region consisting of three β strands (β1-β3 in subdomain 1 and β4-β6 in subdomain 2) and two finger-shaped loops linking the three β strands (Fig. 2C). The second subdomain has longer and flexible "finger loops" and is thus much larger than the first one. The flexibilities of the two finger loops in the second subdomain are clear in the relaxation data. The steady-state heteronuclear 15N[1H] NOE values versus the residue number of Dkk2C are shown in Fig. 3. Because the lengths of the N-H bonds are fixed, the 15N[1H] NOE values report information about the dynamics of N-H bonds and are used to determine the motion of a particular residue (21Wong H.C. Liu G. Zhang Y.M. Rock C.O. Zheng J. J. Biol. Chem. 2002; 277: 15874-15880Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). Typically, the value for the heteronuclear 15N[1H] NOE of folded residues is ∼1-0.7, and the NOE for a flexible loop is <0.5. The dynamic study showed that the first finger loop in the second subdomain (loop β4-β5) is most flexible. The study also showed that the folded Dkk2C should start at Gly177.FIGURE 3Plot of backbone amide heteronuclear 15N[1H] NOE values versus residue number for the Dkk2C. The steady-state heteronuclear 15N[1H] NOE value is plotted versus the residue number measured with a Bruker 600-MHz spectrometer at 25 °C. The secondary structure elements and disulfide brides in the Dkk2C are indicated at the top; amino acids that contact the third β-propeller domain of LRP5 in the docked model are indicated by the red dots. Because the lengths of the N-H bonds are fixed, the 15N[1H] NOE provides information about the dynamics of N-H bonds that can be used to determine whether a particular amide is in a well folded or a flexible region of a protein. The arrow indicates the starting residue, Gly177, of the folded Dkk2C.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Within each subdomain are two disulfide bonds to stabilize the β-core region; one connects the first and second β-sheets (Cys183-Cys195 connecting β1 and β2 in the subdomain 1 and Cys214-Cys239 connecting β4 and β5 in subdomain 2), and another (Cys189-Cys204 and Cys233-Cys256) connects the third β sheet (β3 and β6, respectively) to the first finger loop (loop β1-β2 and loop β4-β5, respectively). The fifth disulfide bond (Cys194-Cys231) links the two subdomains together. The five dihedral angles in each of five disulfide bonds in the solution structure of Dkk2C are well within the range of the established stereochemical preferences of a single disulfide bridge (22Sali A. Overington J.P. Protein Sci. 1994; 3: 1582-1596Crossref PubMed Scopus (261) Google Scholar). Among the five disulfide bonds in the Dkk2C structure, only the first one (Cys183-Cys195) has the right-handed conformation; the rest of the four disulfide bonds are all in the left-handed conformation (23Thornton J.M. J. Mol. Biol. 1981; 151: 261-287Crossref PubMed Scopus (674) Google Scholar). Analysis with DALI (24Holm L. Sander C. J. Mol. Biol. 1993; 233: 123-138Crossref PubMed Scopus (3561) Google Scholar) software showed that the Dkk2C structure shares some features with those of colipase (Protein Data Bank code 1PCN) (25van T.H. Sarda L. Verger R. Cambillau C. Nature. 1992; 359: 159-162Crossref PubMed Scopus (321) Google Scholar) and MIT1 (mamba intestinal toxin 1) (Protein Data Bank code 1IMT) (26Boisbouvier J. Albrand J.P. Blackledge M. Jaquinod M. Schweitz H. Lazdunski M. Marion D. J. Mol. Biol. 1998; 283: 205-219Crossref PubMed Scopus (63) Google Scholar), which belong to a family of proteins lacking extensive secondary structures and stabilized by abundant disulfide bridges (27van T.H. Bezzine S. Cambillau C. Verger R. Carriere F. Biochim. Biophys. Acta. 1999; 1441: 173-184Crossref PubMed Scopus (72) Google Scholar). All three can be described as an assembly of protruding fingers, held together at one end by a network of five disulfide bridges. However, only the two central β-core regions of Dkk2C, colipase, and MIT1 are similar, and the connectivity patterns of disulfide bridges among the three proteins share a highly conserved feature (23Thornton J.M. J. Mol. Biol. 1981; 151: 261-287Crossref PubMed Scopus (674) Google Scholar). Indeed, the sequence identity shared by Dkk2C with the two proteins (24% with colipase and 29% with MIT1) is concentrated in the two central β-core regions, all of the finger-loop regions of Dkk2C are unique, and each has a different length and conformation. The C-terminal cysteine-rich domains of Dkk1/2 interact with LRP5/6 (5Brott B.K. Sokol S.Y. Mol. Cell. Biol. 2002; 22: 6100-6110Crossref PubMed Scopus (190) Google Scholar, 10Zhang Y. Wang Y. Li X. Zhang J. Mao J. Li Z. Zheng J. Li L. Harris S. Wu D. Mol. Cell. Biol. 2004; 24: 4677-4684Crossref PubMed Scopus (142) Google Scholar), whose extracellular region contains four well defined YWTD repeat domains that have a typical symmetrical six-bladed β-propeller fold (28Springer T.A. J. Mol. Biol. 1998; 283: 837-862Crossref PubMed Scopus (167) Google Scholar, 29Springer T.A. Curr. Opin. Struct. Biol. 2002; 12: 802-813Crossref PubMed Scopus (33) Google Scholar). Although each of the first three β-propeller domains can interact with Dkk1/2, only the third is required for Dkk1/2-mediated inhibition of Wnt signaling, presumably because it binds favorably to Dkk1/2 (10Zhang Y. Wang Y. Li X. Zhang J. Mao J. Li Z. Zheng J. Li L. Harris S. Wu D. Mol. Cell. Biol. 2004; 24: 4677-4684Crossref PubMed Scopus (142) Google Scholar). Furthermore, the residues involved in Dkk-mediated Wnt inhibition in the third β-propeller domain of LRP5 (LRP5-PD3) were found by alanine substitution mapping (10Zhang Y. Wang Y. Li X. Zhang J. Mao J. Li Z. Zheng J. Li L. Harris S. Wu D. Mol. Cell. Biol. 2004; 24: 4677-4684Crossref PubMed Scopus (142) Google Scholar) to be clustered on a concave, amphitheatre-shaped surface centered on the pseudo-6-fold axis atop the β-propeller (10Zhang Y. Wang Y. Li X. Zhang J. Mao J. Li Z. Zheng J. Li L. Harris S. Wu D. Mol. Cell. Biol. 2004; 24: 4677-4684Crossref PubMed Scopus (142) Google Scholar). This amphitheatre-shaped ligand binding site is likely to be the common feature among these β-propeller domains (supplemental Fig. 1) (30Takagi J. Yang Y. Liu J.H. Wang J.H. Springer T.A. Nature. 2003; 424: 969-974Crossref PubMed Scopus (116) Google Scholar, 31Rudenko G. Henry L. Henderson K. Ichtchenko K. Brown M.S. Goldstein J.L. Deisenhofer J. Science. 2002; 298: 2353-2358Crossref PubMed Scopus (376) Google Scholar). Because of the network of hydrogen bonds within the β-propeller domains and the nature of the concave surface, the ligand binding sites of the β-propeller domains are rigid (30Takagi J. Yang Y. Liu J.H. Wang J.H. Springer T.A. Nature. 2003; 424: 969-974Crossref PubMed Scopus (116) Google Scholar). Typical ligands of these the β-propeller domains are rigid as well (30Takagi J. Yang Y. Liu J.H. Wang J.H. Springer T.A. Nature. 2003; 424: 969-974Crossref PubMed Scopus (116) Google Scholar, 31Rudenko G. Henry L. Henderson K. Ichtchenko K. Brown M.S. Goldstein J.L. Deisenhofer J. Science. 2002; 298: 2353-2358Crossref PubMed Scopus (376) Google Scholar). The rigidity of the ligands and receptors minimizes loss of entropy upon binding and promotes a high affinity (30Takagi J. Yang Y. Liu J.H. Wang J.H. Springer T.A. Nature. 2003; 424: 969-974Crossref PubMed Scopus (116) Google Scholar). The rigidity also makes it possible to model the complex by docking ligands to β-propeller domains. For example, at CAPRI, different groups successfully docked laminin to the β-propeller of nidogen successfully (32Gray J.J. Curr. Opin. Struct. Biol. 2006; 16: 183-193Crossref PubMed Scopus (162) Google Scholar, 33Mendez R. Leplae R. Lensink M.F. Wodak S.J. Proteins. 2005; 60: 150-169Crossref PubMed Scopus (300) Google Scholar). We therefore conducted a docking study to examine the interaction between Dkk2C and LRP5-PD3. To maximize the accuracy of this study, we used the program HADDOCK (20Dominguez C. Boelens R. Bonvin A.M. J. Am. Chem. Soc. 2003; 125: 1731-1737Crossref PubMed Scopus (2169) Google Scholar) to incorporate data from mutagenesis studies of the binding of Dkk1/2 to LRP5/6. The mutation E721A on the amphitheatre surface of LRP5-PD3 that binds to Dkk had the strongest effect on Dkk1-mediated inhibition of Wnt1 activity (∼70% reduction) and abolished binding of LRP5-PD3 to Dkk1 (10Zhang Y. Wang Y. Li X. Zhang J. Mao J. Li Z. Zheng J. Li L. Harris S. Wu D. Mol. Cell. Biol. 2004; 24: 4677-4684Crossref PubMed Scopus (142) Google Scholar). Because the corresponding residue in nidogen, Glu994, forms a salt bridge with a lysine residue in bound laminin (30Takagi J. Yang Y. Liu J.H. Wang J.H. Springer T.A. Nature. 2003; 424: 969-974Crossref PubMed Scopus (116)