Architecture of the Norrin signaling complex and structure-guided surrogate design

Norrin binds Frizzled 4 (FZD₄), tetraspanin 12 (TSPAN12) and low-density lipoprotein receptor-related protein 5 (LRP5) to regulate angiogenesis and the integrity of the blood-brain barrier and inner blood-retina barrier (BRB). Aberrant Norrin signal causes retinopathies, BRB defects and deafness. Therapeutic mimetics of Norrin are currently in clinical trials for eye diseases. To characterize the mechanism of Norrin signal biogenesis, we determined the structure of a Norrin-FZD₄-TSPAN12-LRP5 signaling complex using a de novo designed FZD₄ binder as a 'protein glue'. Our structure shows that two curved Norrin homodimers bridge two FZD₄-TSPAN12 heterodimers. LRP5_LA3 simultaneously interacts with TSPAN12 and two Norrin homodimers, positioning the cytosolic FZD₄ and LRP5 tails for signal activation. On the basis of our findings, we designed FZD_4CRD-LRP5_LA3 bispecific binders that act as Norrin surrogates. Our signaling assays showed that oligomerization of the Norrin surrogate enhances signaling potency. Thus, this work reveals how the Norrin signal is generated and may assist in the development of Norrin/Wnt mimetics.