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Figure 8. The potential Lrp5/6 binding site on Norrin.(A) Cartoon representation of Norrin (grey) in complex with Fz4CRD (cyan). Residues in the potential Lrp5/6 binding site are shown as spheres (atom colouring: magenta, carbon; blue, nitrogen; red, oxygen). The boxes highlight residues associated with disease mutations. (B) Cartoon model of Norrin showing three distinct binding sites.DOI: http://dx.doi.org/10.7554/eLife.06554.024Figure 8—figure supplement 1. Verification of Norrin potential Lrp5/6 binding site.(A) Luciferase reporter assay. The luciferase activities were normalized to a maximum activity value (100%) for Norrin wild-type and error bars represent standard deviations (n = 3). (B) SPR binding assay of Norrin mutant R121W. Biotinylated Norrin mutant R121W was coated onto a CM5 sensor chip and a series of concentrations of Fz4CRD as analyte were injected over the chip. Graphs show sensorgrams (top panels) and fitted plot of equilibrium binding response (bottom panels). (C) Heparin binding assay of Norrin mutant R121W. Protein elution profiles (left panel) were monitored by absorbance at 280 nm (blue curves) for a NaCl gradient (0.25–2 M; black dashed lines). An input sample, flow-through (green line) and peak fractions (yellow and red lines) are analysed on SDS-PAGE (right panel). Norrin mutant R121W was eluted at 1.5 M NaCl concentration. Notably, we found almost 50% of the Norrin mutant R121W precipitated as the NaCl concentration was reduced before injection for heparin affinity chromatography (see ‘Materials and methods’ for detailed information).DOI: http://dx.doi.org/10.7554/eLife.06554.025 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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