Structural basis of Wnt recognition by Frizzled

Abstract

Wnts are lipid-modified morphogens that play critical roles in development principally through engagement of Frizzled receptors. The 3.25 angstrom structure of Xenopus Wnt8 (XWnt8) in complex with mouse Frizzled-8 (Fz8) cysteine-rich domain (CRD) reveals an unusual two-domain Wnt structure, not obviously related to known protein folds, resembling a "hand" with "thumb" and "index" fingers extended to grasp the Fz8-CRD at two distinct binding sites. One site is dominated by a palmitoleic acid lipid group projecting from serine 187 at the tip of Wnt's thumb into a deep groove in the Fz8-CRD. In the second binding site, the conserved tip of Wnt's "index finger" forms hydrophobic amino acid contacts with a depression on the opposite side of the Fz8-CRD. The conservation of amino acids in both interfaces appears to facilitate ligand-receptor cross-reactivity, which has important implications for understanding Wnt's functional pleiotropy and for developing Wnt-based drugs for cancer and regenerative medicine.

Figure 1. Formation of the XWnt8 complex with mouse Fz8-CRD for structure determination

(A) Strategy for purification of the XWnt8/Fz8-CRD complex. The mouse Fz8-CRD was co-expressed as an Fc-fusion protein with XWnt8 in Drosophila S2 cells and the complex was captured with Protein-A. The XWnt8/Fz-CRD complex was eluted from the resin using 3C protease, which cleaved the Fz8-CRD from the Fc. (B) The complex was then purified by gel filtration chromatography. The doublet band for XWnt8 represents glycosylation heterogeneity. (C) Initial density-modified electron density map calculated with experimental phases derived from Selenomethionine sites (shown in green spheres). N-Glycan evident in experimentally-phased map is labeled. The initial backbone trace built into this map is shown within the electron density, along with neighboring symmetry mates. See also Table S1, and Fig. S1 for electron density of the refined structure.

Figure 2. Overall structure of XWnt8 in complex with Fz8-CRD

Ribbon models of XWnt8 (violet) and Fz8-CRD (blue) as viewed ‘face on’ (A) and ‘side-on’ (B). N-linked glycans are drawn as green sticks, disulfide bonds are drawn as orange sticks. (C) Surface representation of XWnt8 after removal of the Fz8-CRD from the complex structure. The extended palmitoleic acid (PAM) group is shown in red extending from the Wnt thumb. See also Fig. S5 for mapping of potential Lrp5/6 binding site. (D) Secondary structure diagram of the XWnt8 fold. Disulfide connectivity is indicated by orange lines, visible N-glycan addition sites by green cartoon, PAM addition site by red cartoon. See also Fig. S2 for images of the bound versus unbound structure of the Fz8-CRD, and a molecular surface of the entire complex.

Figure 3. Acylation of the XWnt8 thumb loop mediates site 1 binding to Fz8-CRD

(A) Shape and chemical complementarity of the lipid-CRD interaction is evident when the electron density of the lipid modification (red lipid in grey mesh, sigmaA-weighted 2Fo-Fc map contoured at 0.8σ) at Ser-187 of XWnt8 (violet) is shown together with the molecular surface of the site 1 groove in the Fz8-CRD (blue). (B) Amino acid interactions mediating recognition of the XWnt8 thumb by the Fz8-CRD at site 1 (Table S1). Several hydrogen bonds are drawn as dashed lines. (C) Site 1 recognition occurs largely through chemically or strictly conserved Wnt and Fz amino acids (see also Figs S3 and S4). Residues of the Fz8-CRD that contact XWnt8 or the lipid are indicated with blue labels. Alternative residues at each position in other Fz-CRD are indicated by residues within parentheses. The relative font sizes of the different amino acids within the parentheses reflects an approximate percentage of the ten Fz that use that amino acid at the respective position. “mc” label indicates that the residue contacts Wnt through main chain interaction rather than side chain.

Figure 4. A conserved Wnt/Fz recognition mode in the site 2 interface

(A) Electron density of the XWnt8 finger loop (grey mesh, sigmaA-weighted 2Fo-Fc map contoured at 0.83) bound in a concave depression on the Fz8-CRD surface (blue). (B) XWnt8 index finger loop (violet) and Fz8-CRD (blue) amino acids mediating recognition at site 2 (Table S1). Disulfide bonds are drawn as yellow sticks. (C) Conservation analysis of site 2 interactions reveals that the majority of side-chain specific interactions are either strictly or chemically conserved in Fz (see also Figs S3 and S4). Alternative residues at each position in other Fz-CRD are indicated by residues within parentheses. The relative font sizes of the different amino acids within the parentheses reflects an approximate percentage of the ten Fz that use that amino acid at the respective position. “mc” label indicates that the residue contacts Wnt through main chain interaction rather than side chain. At the C-terminal end of the Fz8-CRD construct, positions 151 and 152 are linker-derived residues (Ala), but are Asn and Tyr in the wild type Fz8-CRD sequence.

Figure 5. “Mini-Wnt” discriminates between different Fz-CRD and engages site 2 independently of site 1

(A) The C-terminal 90-amino acids of XWnt8 were displayed on the surface of yeast and shown to bind robustly to Fz5-, and Fz8-CRD, and weakly to F4-CRD by FACS analysis using fluorescent Fz-CRD-Streptavidin-PE tetramers. The cartoon of mini-Wnt displayed on yeast depicts the C-terminal 90-amino acids seen in the structure. The population of mini-Wnt yeast stained by the Fz-CRD tetramers by FACS was ~50%, ~30%, and ~4% for Fz8, Fz5, and Fz4-CRD, respectively. (B) Surface plasmon resonance analysis of mini-XWnt8 binding to Fz5- and F8-CRD immobilized on a BIAcore T100 sensor chip. Dose-response titrations are plotted of the equilibrium binding experiments, the insets of each panel show the titration data and that each concentration reached steady state.