Structure of the Wnt-Frizzled-LRP6 initiation complex reveals the basis for coreceptor discrimination

Abstract

Wnt morphogens are critical for embryonic development and tissue regeneration. Canonical Wnts form ternary receptor complexes composed of tissue-specific Frizzled (Fzd) receptors together with the shared LRP5/6 coreceptors to initiate β-catenin signaling. The cryo-EM structure of a ternary initiation complex of an affinity-matured XWnt8-Frizzled8-LRP6 complex elucidates the basis of coreceptor discrimination by canonical Wnts by means of their N termini and linker domains that engage the LRP6 E1E2 domain funnels. Chimeric Wnts bearing modular linker "grafts" were able to transfer LRP6 domain specificity between different Wnts and enable non-canonical Wnt5a to signal through the canonical pathway. Synthetic peptides comprising the linker domain serve as Wnt-specific antagonists. The structure of the ternary complex provides a topological blueprint for the orientation and proximity of Frizzled and LRP6 within the Wnt cell surface signalosome.

Keywords: Wnt signaling; cryo-EM; crystallography; protein engineering; structural biology.

Fig. 1.

Engineering and characterization of haXWnt8. (A) Schematic for the cell-surface Wnt display and signaling of wild-type HEK293F and HEK293 expressing hFzd5_CRD-1TM (Top). Wnt expression detected by protein C (PrC) epitope tag on Wnt (Bottom). (B) Ribbon models of XWnt8–mFzd8_CRD (PDB 4F0A) and hWnt3–mFzd8_CRD (PDB 6AHY) (Top). The NC-linker is missing in the XWnt8 crystal structure, likely reflecting its flexibility. Cell surface expression and LRP6_E1E2 tetramer binding on hFzd5_CRD-1TM cell line in the absence or presence of wild-type XWnt8 or XWnt8_ΔNC (Bottom). (C) Improvements in LRP6_E1E2 tetramer binding to the XWnt8 libraries during selection compared to the wild-type XWnt8 single clone. Data were analyzed with FCS Express 7. (D) LRP6_E1E2 monomer binding to the haXWnt8 and wild-type XWnt8 (Left) and hahWnt8 and wild-type hWnt8 (Right). The squares and dots indicate the means of experimental values, and the error bars represent SD. Data were analyzed with Prism 9. The NC-linker sequences are presented on top of the binding curves. (E) Top-flash signaling assays with recombinant wild-type XWnt8, haXWnt8, and next-generation surrogate (NGS) Wnt (10) (Left) in the absence or presence of R-spondin 2. With twofold serial dilution, the agonist concentrations were 0.39 nM to 50 nM for XWnt8s and 0.016 nM to 2 nM for NGS Wnt. The bar graphs show the means of experimental values, and the error bars represent SD. Data were analyzed with Prism 9. Dose–response experiments were performed in triplicate, and representative data were shown from two independent experiments.

Fig. 2.

Cryo-EM analysis of haXWnt8–mFzd8_CRD–hLRP6_E1E2. (A) Representative 2D class averages of haXWnt8–mFzd8_CRD–hLRP6_E1E2. (B) 3D reconstruction of haXWnt8–mFzd8_CRD–hLRP6_E1E2 colored in purple (haXWnt8), cyan (mFzd8_CRD), and green (hLRP6_E1E2). (C) Close-up of the Site A and Site B interfaces viewed from the red and blue arrows indicated in the panel C Left. The cryo-EM map (transparent gray) is superimposed on the ribbon model of the complex (LRP6, green; haXWnt8, purple). The NC-linker shows continuous cryo-EM density, while N-terminal region does not. Map contour levels are set to 0.32 on ChimeraX. The sequence of the engineered haXWnt8 NC-linker is displayed at the Bottom of the Right panel and on the structure, with the wild-type sequence shown at the Bottom. (D) LRP6_E1E2 tetramer binding to hFzd5_CRD-1TM cells with or without expression of wild-type XWnt8, XWnt8_ΔN, or XWnt8_ΔNC, showing that both the N-terminal loop and NC-linker contribute to the LRP6 binding. The tetramer concentration was 50 nM and fourfold lower than the experiment shown in Fig. 1B.

Fig. 3.

Crystal structure of the YW210-fused haXWnt8 NC-linker in complex with hLRP6_E1E2. (A) Overall view of the crystal structure of hLRP6_E1E2 (green) bound to YW210 (yellow and wheat) and the haXWnt8 NC-linker (pink). A blue box indicates site B. (B) mFo-DFc electron density (gray) from a simulated annealing omit map contoured at 3σ around the NC-linker peptide. (C) Close-up view of the boxed region in panel A, with the solvent-accessible surface of LRP6 rendered transparently and key interacting side chains labeled; W550 omitted for clarity. (D) Alignment of NC-linkers from hWnt8 (orange), XWnt8 (blue), and haXWnt8 (pink), with interactions to LRP6 residues (green) indicated for hydrogen bonds (red dashed line) and van der Waals interactions (blue dashed lines). The engineered disulfide bond in the crystallized NC-linker is indicated by a yellow line. Residues unchanged between XWnt8 and haXWnt8 are boxed.

Fig. 4.

Exchange of the NC-linker between Wnt proteins modifies the signaling characteristics of their parental Wnt. (A) Representative TOPbrite dual-luciferase reporter assay showing normalized Wnt signaling induced by various Wnt1 NC-linker alanine mutants from five biological replicates. Sequences are provided in SI Appendix, Fig. S5. Bar and error bar represent the mean and SD of five technical replicates. (B) Representative TOPbrite dual-luciferase reporter assay measuring Wnt signaling with overexpression of Wnts and Wnt chimeras. Signaling was normalized to the negative control (empty vector). Bar and error bar represent the mean and SD of three technical replicates from one of the three biological replicates. (C) Representative Western blot showing β-catenin and LRP6 levels with overexpression of Wnts and Wnt chimeras. (D) Quantitative PCR analysis of Wnt target gene Axin2 with overexpression of Wnts and Wnt chimeras. The expression was normalized to the negative control in each group. Bars represent the means and SEM of four biological replicates. (E) Schematic representation of LRP6 domains and domain-specific bound antibodies, YW210 and YW211. (F) Representative TOPbrite dual-luciferase reporter assay showing the signaling characteristics of Wnt1, Wnt3a, the Wnt1-mimicking chimeras, Wnt3a_1 and Wnt5a_1, and the haXWnt8-mimicking chimera, Wnt3a_haXWnt8. The signaling was normalized to the negative control (without any treatment) in each group. Bar and error bar represent the means and SD of six technical replicates. ****P < 0.0001, one-way ANOVA with Tukey’s multiple comparison test from one of the three biological replicates. Data were analyzed with Prism 9.

Fig. 5.

Synthetic Wnt linker peptides show LRP6 domain specific binding. (A) A ribbon-and-surface model of haXWnt8–mFzd8_CRD–hLRP6_E1E2 colored in purple (haXWnt8), cyan (mFzd8_CRD), and green (hLRP6_E1E2) (Left). The sequences of W1.cys, W2b.cys, W3a.cys, and W7a.cys are portrayed in the schematic form showing the constrained peptides by a disulfide bond (Right). (B) Representative ELISA binding assay of Wnt.cys peptides to LRP6_E1E2 (Left) and LRP6_E3E4 (Right). The signals from the binding of various Wnt.cys peptides to the assay plate were subtracted from their binding signals to LRP6. Data are shown as the means with SD of three technical replicates from one of the three biological replicates. (C) Representative ELISA competition assay of LRP6 E1E2-binding Wnt.cys peptides with Fab YW210. (D) Dose-dependent inhibition of Wnt1-mediated signaling by W1.cys, W2b.cys, and W7a.cys. Data are shown as the means with SD of three technical replicates from one of the three biological replicates. (E) Representative ELISA competition assay of LRP6 E3E4-binding Wnt3a.cys with Fab YW211 and H07 VHH. Only the peptides showing binding to each functional domain of LRP6 were tested in the competition ELISA. Data are shown as the means with SD of three technical replicates from one of the three biological replicates. Data were analyzed with Prism 9.

Fig. 6.

Modeling initiation and inhibition of Wnt signaling. (A) Binding modes of E1E2-binding Wnt8 and E3E4-binding Wnt3 to LRP6. Crystal structures of LRP6_E1E2 and LRP6_E3E4 are placed linearly, and mFzd8_CRD–XWnt8 and mFzd8_CRD–hWnt3 are anchored to the propeller funnels in the orientation suggested by the competition assays. LRP6, mFzd8, XWnt8, and hWnt3 are colored in green, cyan, purple, and pink, respectively. (B) The structural models of hLRP6_E1E4 binding to haXWnt8–mFzd8_CRD (Left), sclerostin (Center), or DKK1 (Right). (C) Schematic for the transmembrane LRP6–Fzd5 signaling complex with endogenous Wnts or Surrogate Wnt (10).