Genes Dev. 27, 2305–2319 (2013)

Norrin is a cystine knot growth factor that interacts with the Frizzled 4 (Fz4) receptor and the Wnt co-receptor LRP5/6 to activate β-catenin signaling, thereby regulating diverse developmental processes such as angiogenesis and eye and ear development. However, the exact role of LRP5/6 in promoting Norrin-mediated signaling has remained unclear owing to the inability to detect its direct biochemical interactions. Ke et al. used a maltose binding-protein–Norrin fusion protein (Norrin-MBP), which improved solubility and aided purification, to isolate pure proteins in sufficient yield to obtain a crystal structure at 2.4-Å resolution. In addition to the cystine knot in each monomer, the structure showed a Norrin dimer stabilized by three intermolecular disulfide bonds and a hydrophobic surface. Disruption of these bonds or the hydrophobic interface resulted in a loss of Norrin-mediated signaling. The use of the fusion protein also enabled the authors to detect the direct binding of Norrin-MBP to the extracellular domains of LRP5/6, whereas competition assays revealed that Fz4 and LRP56 bind distinct sites on Norrin. Furthermore, the authors identified LRP5/6 binding sites on Norrin that, when mutated, did not affect Fz binding but decreased Norrin signaling activity. Finally, the presence of Norrin promoted the immunoprecipitation of Fz4 with LRP5, suggesting that Norrin requires direct interactions with both Fz4 and LRP5/6 to activate downstream mediators.