Structural basis of Norrin recognition by Lgr4/5/6

Lgr4/5/6 have been identified as stem cell markers and canonical Wnt signalling enhancers via complexing with R-spondin and Rnf43/Znrf3. Lgr4/5/6 have been reported to interact with Norrin, a cystine-knot growth factor that activates Wnt signalling via binding to Frizzled4 cysteine-rich domain (Fz4CRD). Norrin-Fz4 signalling axis regulates central nervous system vascularization. However, the molecular basis of Norrin and Lgr4/5/6 recognition remains lacking. Here, we present the structure of Norrin in complex with an ectodomain variant of Lgr4 (Norrin-Lgr4ECD), revealing a 2:2 stoichiometry. Our analyses show that residues of Lgr4/5/6ECD for Norrin binding are highly conserved. Structural comparisons of Norrin-Lgr4ECD with Rspo1- Lgr4ECD and Norrin-Fz4CRD complexes reveal that Norrin binding site on Lgr4 overlaps with Rspo1 binding site on Lgr4 and Fz4 binding site on Norrin overlaps with Lgr4 binding site on Norrin. The present work opens new avenues to elucidate the function of Norrin-Lgr4/5/6 signalling axis.