Xenbase Image ID: 145729

Figure 6. Biophysical and functional characterisation of Fz4 binding site.Surface representation of Norrin–Fz4CRD complex in open book view. (A) Interface residues are coloured orange (Norrin) and blue (loop I), green (loop II), yellow (loop III), and cyan (Phe96) on Fz4CRD. Norrin mutation sites used in functional assays are labelled (red, residues involved in Fz4CRD binding; grey filled box, residues associated with diseases; black, residues located outside the Fz4 binding site). (B) Norrin and Fz4CRD coloured by electrostatic potential from red (acidic; −7 kbT/ec) to blue (basic; 7 kbT/ec). (C) Disease-associated mutations mapped onto the surface of Norrin and Fz4CRD (purple, missense mutations; red, missense mutations of cysteine residues). (D) Surfaces colour-coded according to sequence conservation from white (not conserved) to black (conserved). (E) SPR results for Fz4CRD binding to Norrin wild-type (WT) and Norrin V45E mutant. Inset SPR sensorgrams are of equilibrium-based binding assays with reference subtraction. (F) Luciferase reporter assays histograms with Kd values from SPR measurements (Figure 6—figure supplement 1) shown above. Residues involved in the Fz4CRD binding site are coloured red. Residues without contact with Fz4CRD are coloured black. Grey filled boxes highlight disease-associated residues (Figure 2—figure supplement 2). The luciferase activities were normalized to a maximum activity value (100%) for Norrin wild-type and error bars represent standard deviations (n = 3).DOI: http://dx.doi.org/10.7554/eLife.06554.020Figure 6—figure supplement 1. SPR equilibrium binding data.SPR equilibrium binding experiments using Fz4CRD as analyte and biotinylated human Norrin wild-type (WT) and mutants as immobilized ligands on CM5 sensor chips. SPR sensorgrams (left panels) and fitted plots of equilibrium binding response (right panels; 1:1 Langmuir binding model) against a series of concentrations of Fz4CRD are shown. The binding parameters are shown as Surface (the amounts of biotinylated Norrin coated onto sensor chip), Bmax (the maximum response), Kd (binding constant), and RU (response unit). N/A indicates not applicable for calculation of Kd. Residues associated with diseases are highlighted as filled grey boxes. (A) The interaction of Norrin wild-type with Fz4CRD has a Kd of 1.1 μM. (B) Introduction of an N-linked glycosylation site on the Fz4 binding site of Norrin results in the complete loss of Fz4CRD binding. (C) Norrin mutants identified from diseases show no binding to Fz4CRD. (D) Mutations designed to disrupt the hydrogen bond and salt bridge interactions of the Fz4 binding site completely abolish binding to Fz4CRD. (E) Introduction of an N-linked glycosylation site on the Norrin surface locating outside the Fz4 binding site does not affect the binding affinity of Norrin to Fz4CRD. (F) Norrin discriminates between different FzCRD proteins. Biotinylated human Norrin was immobilized onto a CM5 sensor chip to give 410 response units (RU) and different FzCRD proteins were used as analytes. The injected human Fz4CRD proteins have a concentration ranging from 2.3 nM to 75 μM. Other FzCRD concentrations ranged from 0.3 μM to 150 μM (mouse Fz5CRD) or 0.2 μM–100 μM (human Fz7CRD and mouse Fz8CRD). Because of the limitations of detection, the Kd values of Fz5CRD and Fz8CRD are too low to measure accurately, resulting in variable standard deviations. The binding affinity of Fz7CRD to Norrin is not sufficient to calculate a Kd.DOI: http://dx.doi.org/10.7554/eLife.06554.021 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

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