Structural and Functional Insights of Norrin Mimics Wnt for Signalling

Wnt signalling plays critical roles in cell-fate determination, tissue homeostasis and embryonic development. Not surprisingly, deregulated Wnt signalling leads to cancer, osteoporosis and degenerative illnesses. Norrin (Norrie Disease Protein, NDP; a covalent homodimer) is a secreted growth factor cystine-knot family member with angiogenic and neuroprotective properties. Norrin shows no relationship to the Wnt morphogens (secreted cysteine-rich palmitoleoylated glycoproteins). Interestingly, Norrin activates the canonical Wnt/β-catenin pathway via the interaction with Wnt receptors, Frizzled4 (Fz4), low-density lipoprotein receptor related protein 5/6 (Lrp5/6), and heparan sulphate proteoglycans (HSPGs; a component of the extracellular matrix). Mutations in the NDP gene and receptor genes lead to inherited retinal diseases including Norrie Disease, Familial Exudative Vitreoretinopathy and Coats’ disease. However, the molecular level analysis of Norrin mediated Wnt pathway remains obscure. Here, we reported crystallographic and small-angle X-ray scattering structures of Norrin in complex with Fz4 cysteine-rich domain (Fz4CRD), of this complex bound with glycosaminoglycan (GAG) analogues of HSPGs, and of unliganded Norrin and Fz4CRD. By comparative analysis with the Xenopus Wnt8-mouse Fz8CRD complex, we showed how Norrin structurally mimics Wnt for binding to the CRD of Fz receptor. In combination with biophysical and cell-based studies, we further mapped Fz4, Lrp5/6, GAG binding sites on Norrin and Norrin binding site on receptors. These results not only provide critical insights into how Norrin activates the Wnt signalling pathway, but also explain numerous disease-associated mutations on Norrin and its receptors. These structural and functional data open new avenues for the development of therapeutics to combat abnormal Norrin/Wnt signalling.