Supplemental Figure 1
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1 Supplemental Figure 1 Supplemental Fig. 1. (A) The binding avidity between Fz4-FcH6 and MBP-Norrin dimers measured by biolayer interferometry. 5 µg/ml Fz4-Fc protein was loaded onto anti human Fc capture sensors, incubated with kinetic buffer, and then incubated with MBP- Norrin protein (80 nm and 32 nm) in kinetic buffer for the association phase, followed with kinetic buffer for the dissociation phase. A K d value of 4.6±1.1 nm was calculated using the associated kinetic analysis program (version 6.4). (B) Stereo view of the Norrin monomeric structure. Shown are the main chain atoms in stick models with carbon atoms in green, oxygen atoms in red, and nitrogen atoms in blue. The 2F o -F c map contoured at 1.0 σ is also shown.
2 Supplemental Figure 2
3 Supplemental Fig. 2. Crystal structure of MBP-Norrin. (A) MBP-Norrin crystal packing. Four MBP-Norrin dimers are shown in yellow, magenta, blue and grey, respectively. The unit cell box is shown in green. The majority of the interactions for crystal packing are due to MBP MBP interactions (site 1) and MBP Norrin interactions (sites 2 and 3). The figures were generated using the Pymol program. (B) Structure of the MBP-Norrin dimer. On top are two MBP molecules (green and yellow) and on the bottom are two Norrin monomers (cyan and orange). One MBP-Norrin molecule (green+cyan) is related to another molecule (yellow+orange) by a crystallographic twofold symmetry.
4 Supplemental Figure 3
5 Supplemental Fig. 3. Structural comparison of Norrin with TGF-β3. (A) The monomeric structures of Norrin and TGF-β3 (PDB code: 1TGJ) are shown in cartoon representation with a rainbow color scheme from N-terminus (blue) to C-terminus (red). The disulfide bonds including the conserved cystine knot structure (a cluster of three intramolecular disulfide bonds) are shown as stick models. The significant structural differences between them are indicated in four boxes (Boxes 1-4). (B) Comparison of the dimeric structures of Norrin with TGF-β3. Two monomers in cartoon representation are colored in green and yellow. The intermolecular disulfide bonds are shown as stick models. (C) A display of the missense Norrie disease mutations onto the Norrin dimeric structure. A Norrin dimer is shown as a Cα backbone ribbon diagram with two monomer colored in green and magenta, respectively. The residues whose mutations cause Norrie disease or related diseases are shown as spheres on the green monomer. The green spheres are cysteine residues; yellow spheres are residues involved in forming the dimer interface; blue spheres are residues involved in Fz4 binding; red spheres are residues involved in Lrp5/6 binding; grey spheres are residues involved in hydrophobic packing. The disulfide bonds are shown as stick models.
6 Supplemental Figure 4
7 Supplemental Fig. 4. The dimer structure is important for Norrin function. (A) Norrin-mediated activation of the downstream TCF luciferase reporter activity is dependent on Fz4 expression. 293STF cells were transfected with Lrp5, Lrp6, Fz4 alone or Fz4+Lrp5, Fz4+Lrp6 in the presence or absence of Norrin wild type (WT) expression vector along with TKRlu (an internal control) on day 1. Cell culture media were changed on day 2. Cells were harvested and luciferase activities were measured on day 3 using the Dual luciferase kit (Promega). TCF mediated firefly luciferase activity was normalized to renilla luciferase activity. The value for Norrin+Fz4+Lrp5 transfected cells is set to 100% and the values for the other groups are adjusted accordingly. Error bars indicate SD (n=3). (B, C) Effects of Norrin cysteine mutations and hydrophobic residue mutations at the dimer interface on Norrin function. (B) 293STF cells were transfected with Fz4 or Fz4+Lrp5 in the presence or absence of Norrin WT or mutants along with TKRlu and luciferase activities were measured similarly as described in A. Error bars indicate SD (n=3). (C) 293STF cells were transfected with Fz4+ Lrp6 in the presence or absence of Norrin WT or mutants along with TKRlu and luciferase activities were measured similarly as described in A. Error bars indicate SD (n=3).
8 Supplemental Figure 5
9 Supplemental Fig. 5. Control experiments to examine Fz4 cell surface expression, Fz4 protein expression and Fz4-YFP fluorescence. (A) Fz4 surface expression for HEK293 cells transfected with Fz4 receptor with or without different tags. HEK293 cells were transfected with Fz4 receptor with or without a v5, YFP, YFP-N, YFP-C or Rlu tag at the C-terminus for 2 d. Cells were harvested by Cell Dissociation Solution (Sigma), and stained with a biotinylated Fz4 antibody (R&D systems) on ice for 30 min. Then cells were incubated with Streptavidin-Allophycocyanin (APC) (BD Biosciences) on ice for 30 min. Cells were washed with FACS buffer (1xPBS, 2%FBS, 1% Azide) between different steps and analyzed by flow cytometry using MoFlo Astrios (Beckman Coulter, Inc.). (B) Coexpression with Norrin or treatment with MBP-Norrin protein did not change overall protein expression for Fz4 receptor. HEK293 cells were transfected with Fz4±Norrin on day 1. HEK293 cells transfected with Fz4 were treated with or without 5 µg/ml MBP-Norrin on day 2 and harvested on day 3 for Western blot analysis. The blot was first probed with an anti-v5 antibody (Thermo Scientific), and then stripped and reprobed for β-actin as a loading control. (C) The cell surface YFP fluorescence for HEK293 cells transfected with Fz4YFP±Norrin DNAs. The surface YFP fluorescence is used as an indication for Fz4 surface expression. (D) The cell surface YFP fluorescence for cells transfected with Fz4-YFP, Fz4-YFP-N, Fz4-YFP-C or Fz4-YFP-N+Fz4-YFP-C. COS-1 cells were transfected with either intact YFP construct or the complementary YFP-N and YFP-C constructs.
10 Supplemental Figure 6
11 Supplemental Fig. 6. Both BP1 and BP2 domains of Lrp6 can bind to Norrin. (A) MBP-Norrin competes the interaction between biotinylated DKK1 peptide and His8- tagged BP1-2 protein. The AlphaScreen binding data were plotted with increasing concentrations of MBP-Norrin protein. The first two columns are negative controls. The binding signal of biotinylated DKK1 peptide and His8-tagged Lrp6 BP1-2 protein in the absence of MBP-Norrin protein was set to 100%. Error bars indicate SD (n=3). (B, C) Lrp6 BP2-Fc fusion protein binds to Norrin. (B) Binding of Lrp6 BP2-Fc protein to MBP-Norrin was determined by biolayer interferometry. Protein A sensors were loaded with 5 µg/ml Fz4-Fc, Lrp6 BP2-Fc, or human IgG and then incubated with buffer. The loaded sensors were incubated sequentially with 20 µg/ml MBP-Norrin in the association step and with buffer in the dissociation step. The interaction between Fz4-Fc and MBP- Norrin was used as a positive control. (C) The binding of MBP-Norrin to Lrp6 BP2-Fc measured by AlphaScreen assay. 50 nm biotinylated MBP-Norrin was mixed with 50 nm His6 tagged Lrp6-BP2-Fc protein at room temperature for 4 h. The reaction mixtures also contained 10 µg/ml of the streptavidin-coated donor beads and Ni-chelate-coated acceptor beads. The data represent the average of triplicate samples.
12 Supplemental Figure 7
13 Supplemental Fig. 7. Norrin binds to Fz4 and Lrp6 extracellular domains to promote their interaction. (A) Norrin forms a ternary complex with Fz4-CRD and Lrp6 BP1-2 domains measured by biolayer interferometry. Protein A sensors were incubated sequentially with 5 µg/ml Fz4-FcH6 (step1, loading), kinetic buffer (step2, baseline), 10 µg/ml MBP-Norrin (step3, association 1). The bound sensors were further incubated sequentially in kinetic buffer (step4, baseline), different Lrp6 ECD proteins (40 µg/ml Lrp6BP1-2, 40 µg/ml Lrp6BP3-4 and 80 µg/ml Lrp6BP1-4) (step5, association 2), and kinetic buffer (step6, dissociation). All the curves are aligned to the beginning point of Step 5 using the OctetRed analysis program (version 6.4). (B) Fz4-FcH6 protein does not directly bind Lrp6 β-propeller domains. Protein A sensors were loaded with 5 µg/ml Fz4-FcH6 protein and then incubated with kinetic buffer. The loaded sensors were then incubated sequentially with the indicated proteins (20 µg/ml MBP-Norrin, 70 µg/ml Lrp6 BP1-2, 70 µg/ml BP3-4, or 140 µg/ml BP1-4) in the association step and with kinetic buffer in the dissociation step. MBP-Norrin protein was used as a positive control.
14 Supplemental Figure 8
15 Supplemental Fig. 8. The sequence alignment of Norrin proteins from different species and with other growth factors. (A) Norrin sequences from different species were aligned by using the ClustalW program. All the DNA sequences were obtained from the NCBI protein database. The identical residues are highlighted in yellow whereas the conserved residues are highlighted in light yellow. The secondary structures based on human Norrin structure are noted above the sequence alignment. The black stars denote the cysteines forming intramolecular disulfide bonds whereas the red stars denote the cysteines forming intermolecular disulfide bonds. The filled triangles denote the residues for Fz4 binding whereas the filled circles denote residues for Lrp5/6 binding. (B) Sequence alignment of human Norrin, von Willebrand factor (vwf), mucin-2, TGF-β and pig submaxillary mucin proteins. The initial alignment was made by using ClustalW program and improved by small manual adjustments. All the DNA sequences were obtained from the NCBI protein database. The highly conserved residues are highlighted in yellow whereas the less conserved residues are highlighted in light yellow. The black stars denote these cysteines forming the cystine knot structure, whereas the red stars denote the cysteines forming intermolecular disulfide bonds. The green stars denote the two cysteines forming the TGF-β specific intramolecular disulfide bond, whereas the blue stars denote the two cysteines forming the Norrin specific intramolecular disulfide bond, which are also conserved in vwf and mucin proteins. The intramolecular disulfide bond pattern for Norrin is shown with black lines. The alignment figures were prepared by using the Aline program.
16 Supplemental Table 1. IC 50 values obtained by using purified MBP-Norrin wild type and mutant proteins to compete the Fz4-Fc-H6 and biotin-mbp-norrin interaction or H8- Lrp6 BP1-2 and biotin DKK1 peptide interaction. IC 50 (nm) Competition with Fz4-CRD interaction Lrp6 BP1-2 interaction Wild type R41E NC 288 K54E/R109E 18 NC C55A/C110A NC stands for no competition.
17 Supplemental Table 2. Functional categorization of Norrin missense mutations, related to Figure 2 and Figure 5. Mutations were categorized into six groups based on our structural and functional studies of Norrin (Cysteine mutations, Dimer interface mutations, Hydrophobic packing and protein stability mutations, Fz4 binding site mutations, Lrp5/6 binding site mutations, and other mutations). The full names of the disease acronyms are shown below the table. The information for Norrin mutations is obtained from the website ( Cysteine mutations Fz4 binding site Lp5/6 binding site Mutation Disease Mutation Disease C39R ND Y44C ND C55R, C55F ND L62P ND C65Y,C65W ND A63D,A63S ND C69S ND E66K PFV C95R,C95S ND G67R,G67E ND (severe) C96Y ND,EVR R74C ND,FEVR C96W CD S75P,S75C ND C110R,C110S ND F89L ND Dimer C110G FEVR R97P ND Interface C126S ND P98L ND C128R ND L108P ROP R41K EVR A118D ND R41T ND Y120C EVR R41S PFV R121G ND,PRDX H43R, H43Q ND R121W ND,FEVR,ROP V45M, V45E ND R121Q ND,FEVR L61F ND, VI R121L FEVR L61I FEVR I123N ND L61P ND Hydrophobic V60E ND L124F FEVR packing and protein R90C, ND stability R90P K54N ND, L103V FEVR FEVR R115L FEVR L108P ROP K104Q ND Y120C EVR (mild) K104N ND I123N ND S92P ND S101F ND, PHPV A105T ND ND = Norrie Disease (F)EVR = (Familial) Exudative Vitreoretinopathy PFV = Persistent Fetal Vasculature ROP = Retinopathy Of Prematurity CD = Coats Disease PRDX = Primary Retinal Dysplasia VI = Venous Insufficiency PHPV = Primary Hyperplastic Persistent Vitreous
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Supplementary Figure 1.
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