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.06554.020