Wnt signaling plays fundamental roles in organogenesis, tissue regeneration and cancer, but high-resolution structural information of mammalian Wnt proteins is lacking. We solved a 2.8-Å resolution crystal structure of human Wnt3 in complex with mouse Frizzled 8 Cys-rich domain (CRD). Wnt3 grabs the receptor in a manner very similar to that found in Xenopus Wnt8 complexed with the same receptor. Unlike Xenopus Wnt8-bound CRD, however, Wnt3-bound CRD formed a symmetrical dimer in the crystal by exchanging the tip of the unsaturated acyl chain attached to each Wnt3, confirming the ability of Wnt and Frizzled CRD to form a 2:2 complex. The hypervariable ‘linker’ region of Wnt3 formed a β-hairpin protrusion opposite from the Frizzled binding interface, consistent with its proposed role in the coreceptor recognition. Direct binding between this segment and the Wnt coreceptor LRP6 was confirmed, enabling us to build a structural model of the Wnt–Frizzled–LRP6 ternary complex.
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Coordinates and structure factors for hWnt3–Fz8 CRD complex have been deposited in the Protein Data Bank (PDB) with the accession number 6AHY. All other data are available from the authors upon request.
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We would like to thank K. Tamura-Kawakami for the construction of expression vectors, and the staff in BL44XU, SPring-8 for their help with X-ray data collection. This work was supported in part by MEXT KAKENHI grant number JP17H01420 from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and by the Platform Project for Supporting Drug Discovery and Life Science Research (Basis for Innovative Drug Discovery and Life Science Research) funded by Japan Agency for Medical Research and Development under grant number JP18am0101075.
Integrated supplementary information
a, Structural comparison among the three Wnt proteins. Structures of Xenopus Wnt8/mFz8 CRD complex (PDB ID: 4F0A) and Drosophila WntD N-terminal domain (PDB ID: 4KRR) are shown in ribbon presentation after superposition with the hWnt3–mFz8 CRD structure at NTD, using the same color code as in Fig. 1a. b, Structural superposition of three Wnt proteins. Three Wnt structures are superposed at the core of the NTD comprised by five α-helices (magenta) and shown in Cα tracing models. c, Mobility of Wnt ligands upon the binding to Fz CRD. Three independent hWnt3 structures (chain B, skyblue; chain D, green; chain F, orange) in complex with mFz8 CRD are superposed at the Fz8 CRD (red) and shown as Cα tracings and viewed from two orientations.
a, The 2:2 symmetric dimer found in the hWnt3–mFz8 CRD crystal. The upper panel shows three independent hWnt3/mFz8 CRD units (chains AB, CD, and EF) shown with their respective packing mates (chains A’B’, C’D’, and E’F’). The bottom panel shows the crystal structure of apo-form of Fz8 CRD (chains A and B, PDB ID: 1IJY) drawn with their packing mate (chains A’ and B’), PAM-bound Fz5 CRD (chain B, PDB ID: 5URY) drawn with its packing mate (chain B’), as well as a pseudosymmetric dimer of Fz7 CRD bound by a C24 fatty acid (chains A and B, PDB ID: 5URV). The 2-fold symmetry axis is denoted by dotted lines. Lipid molecules are drawn as sphere models. b, Lipophilic cavity at the Fz-Fz interface. The volume of the cavity formed at the Fz-Fz symmetric dimer interface in the 7 dimeric structures in (a) are calculated by using the ChannelFinder program in the 3V server (http://3vee.molmovdb.org/) with the outer and inner probe radius set to 4.0 and 1.4 Å, respectively. The volume map files (.mrc format) produced by the server are opened in UCSF Chimera 1.7.0 and drawn with Fz CRD structures as in (a). The volume of the PAM chain moiety taken from chain B was calculated by the VolumeCalculation program in the same server, resulting in a value of 298 Å3.
a,b, Mouse Wnt3a in complex with mFz8 CRD prepared exactly as hWnt3–mFz8 CRD complex used in the crystallization experiments was loaded to a Superdex 200 gel filtration column at either 200 µM (a, 11 mg/ml) or 9 µM (b, 0.5 mg/ml) and eluted with HBS. The estimated molecular sizes for each peak were calculated from the elution positions of standard marker proteins. c,d, Purity and the monodispersity of the LRP6(3–4) (c) and EGFRec (d) preparations used in the SPR experiments were confirmed by gel filtration on a Superdex 200 column (upper panels) and the SDS-PAGE analysis (lower panels).
Construct design of variously tagged Fz8 (3 versions) and Wnt (12 versions) proteins are schematically shown, with each amino acid sequence of the N-terminal regions starting at the initiation Met indicated at the bottom.
a, SDS-PAGE analysis of the purified afamin-Wnt3a complex sample before and after the biotinylation. Note that the Wnt3a protein used here retains the N-terminal extra 29-residue tag sequence, appearing as larger size than the mature protein (37 kDa). The number of biotin molecules incorporated in one Wnt3a molecule ( = number of Lys modification) was estimated to be 7.35 mol/mol. b, TCF reporter assay to evaluate the signaling activity of the afamin–Wnt3a complex. Unmodified and biotinylated samples were added to the TCF reporter cells at indicated concentrations and the signaling activities were evaluated and expressed as in Fig. 3. c, d, Lack of dissociation of Wnt3a from the surface-captured Fz8 CRD-Fc during the SPR analysis. Shown are the same sensorgrams used to draw Fig. 4d (for c) and e (for d) but are before the Y-axis alignment at the time of injection (time = 190 s). The baseline value at the beginning of each binding cycle (see an expanded view in the inset) remained nearly constant with the total decline of less than 7 RU over one set of experiments, indicating the minimum dissociation of Wnt3a during this period.
Supplementary Figs. 1–5, Supplementary Notes 1–3 and Supplementary Dataset 1
Macroscopic view of the crystal packing. One set of molecules contained in the asymmetric unit are shown with the color code the same as that for Supplementary Fig. 2a, with three more sets that constitute the symmetric packing mate shown in gray.
Close-up view of the chain A/B pair at the Fz–Fz dimer interface. Fo − Fc electron density map at the tip of Ser 212 is drawn as a magenta mesh at a contour level of σ = 2.5.
Close-up view of the chain C/D pair at the Fz–Fz dimer interface. Fo – Fc electron density map at the tip of Ser 212 is drawn as a magenta mesh at a contour level of σ = 2.5.
Close-up view of the chain E/F pair at the Fz–Fz dimer interface. Fo − Fc electron density map at the tip of Ser 212 is drawn as a magenta mesh at a contour level of σ = 2.5.