Xenbase Image ID: 145722

Figure 3. Crystal and solution structures of unliganded Fz4CRD.(A) Schematic domain organization (SP, signal peptide; TM. transmembrane domain; CD, cytoplasmic domain). Crystallization constructs are rainbow coloured. Disulphide bonds are drawn and blue hexagons denote N-linked glycosylation sites. Cartoon representation of Fz4CRD in rainbow colouring. N-linked N-acetyl-glucosamines (GlcNAc) and disulphide bonds are shown as blue sticks. (B) SEC-MALS experiments. The red line represents the molecular weight (left ordinate axis) and black lines show the differential refractive index (right ordinate axis) as well as SDS-PAGE (Inset). The numbers denote the corresponding molecular weights of each peak. (C and D) SAXS analyses of deglycosylated and glycosylated Fz4CRD solution structures. The experimental scattering data (black circles) and calculated scattering patterns (coloured lines) are shown and the Fz4CRD solution structure model is presented. The upper right inset shows the experimental (black circles) and calculated (coloured lines) Guinier region. The dashed lines delimit the range of fitting for Rg analysis (Rg·S ≤ 1.3). The bottom right inset shows the experimental (black circles) and calculated (coloured lines) pair distance distribution P(r) curve.DOI: http://dx.doi.org/10.7554/eLife.06554.010Figure 3—figure supplement 1. Multiple sequence alignment and structural analysis of cysteine-rich like domains.(A) Secondary structure assignment colouring corresponds to Figure 3A. The conserved cysteine residues (highlighted in cyan) form five disulphide bridges, drawn in black lines and labelled as I–V. Notably, Smo has a different cysteine pair arrangement in disulphide bridge IV. Cysteine residues of Fz4 are numbered with blue filled boxes below the sequence alignment. The red boxes denote the residues of Fz4 that contact with Norrin, whereas residues of Fz8 are boxed in blue to indicate binding to Wnt8 index finger (site 2) and in yellow for interaction with Wnt8 PAM (site 1). Coloured lines indicate Fz4CRD loops that interact with Norrin. The N-glycosylation sites are highlighted in green and those of Fz4 are marked by blue hexagons. The purple dots mark residues associated with human retinal diseases (missense mutations; http://www.uniprot.org/uniprot/Q9ULV1). Red asterisks denote the residues of Fz4 involving in GAG binding. The black arrows indicate residues potentially determining ligand-binding specificity (Figure 9D-F). Sequences are from the following UniProt entries: hFz1, Q9UP38; hFz2, Q14332; hFz3, Q9NPG1; hfz4, Q9ULV1; hfz5, Q13467; hFz6, O60353; hFz7, O75084; hfz8, Q9H461; hfz9, O00144; hfz10, Q9ULW2; sfrp1 Q8N474; sfrp2, Q96HF1; sfrp3 Q92765; sfrp4, Q6FHJ7; sfrp5, Q5T4F7; hSmo, Q99835; rMuSk, Q62838; hROR1, Q01973; hROR2, Q01974. (B) Superimposition of all molecules of unliganded Fz4CRD (crystal form I and II) reveals no major conformational changes. The main difference between Fz4CRD structures is the point at which the C-terminal region becomes disordered (black, green, and cyan arrows indicate residue 162, 165, and 172, respectively). The N-linked glycans are shown as stick models. The encircled N and C indicate the N- and C- termini. (C–H) Structural comparison of Fz4CRD with CRDs of Fz-like proteins. Ribbon diagram of superposition of Fz4CRD crystal form I (magenta) with (C) complexed mouse Fz8CRD (cyan; PDB ID: 4F0A), (D) unliganded mouse Fz8CRD (cyan; PDB ID: 1IJY), (E) mouse sFRP3CRD (green; PDB ID: 1IJX), (F) rabbit MuSkCRD (yellow; PDB ID: 3HKL), (G) zebrafish SmoCRD (red; PDB ID: 4C79), and (H) drosophila SmoCRD (red; PDB ID: 2MAH). (sfrp = secreted frizzled related protein; Smo = Smoothened; MuSk = muscle-specific kinase; ROR = tyrosine-protein kinase transmembrane receptor).DOI: http://dx.doi.org/10.7554/eLife.06554.011 Image published in: Chang TH et al. (2015) © 2015, Chang et al. This image is reproduced with permission of the journal and the copyright holder. This is an open-access article distributed under the terms of the Creative Commons Attribution license Larger Image Printer Friendly View

Return to previous page