Sclerostin inhibits Wnt signaling through tandem interaction with two LRP6 ectodomains

Jinuk Kim,Jeesoo Kim,Yejin Jeong,Taeyong Park,Eun Jin Kim,Jong-Seo Kim,Wonhee Han,Injin Bang,Hee-Jung Choi,Hyun Sik Lee,Kyeonghwan Roh,Chanhee Kang,Chaok Seok,Jin-Kwan Han

doi:10.1038/s41467-020-19155-4

Abstract

Low-density lipoprotein receptor-related protein 6 (LRP6) is a coreceptor of the β-catenin-dependent Wnt signaling pathway. The LRP6 ectodomain binds Wnt proteins, as well as Wnt inhibitors such as sclerostin (SOST), which negatively regulates Wnt signaling in osteocytes. Although LRP6 ectodomain 1 (E1) is known to interact with SOST, several unresolved questions remain, such as the reason why SOST binds to LRP6 E1E2 with higher affinity than to the E1 domain alone. Here, we present the crystal structure of the LRP6 E1E2–SOST complex with two interaction sites in tandem. The unexpected additional binding site was identified between the C-terminus of SOST and the LRP6 E2 domain. This interaction was confirmed by in vitro binding and cell-based signaling assays. Its functional significance was further demonstrated in vivo using Xenopus laevis embryos. Our results provide insights into the inhibitory mechanism of SOST on Wnt signaling.

Highlights

Low-density lipoprotein receptor-related protein 6 (LRP6) is a coreceptor of the β-catenindependent Wnt signaling pathway
The crystal structure of the LRP6 E1E2–SOSTtr[177] complex was solved at 3.8 Å resolution by the molecular replacement (MR) method using the known LRP6 E1E2 structure (PDB ID 3S94) and the core cystine-knot nuclear magnetic resonance (NMR) structure of SOST (PDB ID 2K8P) with the loop 2 region removed as search models
The mFo-DFc difference electron density map calculated after rigid body fitting of the MR solution revealed clear density for SOST NXI motif at the top surface of LRP6 ectodomain 1 (E1) (Supplementary Fig. 1)

Summary

Introduction

Low-density lipoprotein receptor-related protein 6 (LRP6) is a coreceptor of the β-catenindependent Wnt signaling pathway. The unexpected additional binding site was identified between the C-terminus of SOST and the LRP6 E2 domain. This interaction was confirmed by in vitro binding and cell-based signaling assays. 1234567890():,; Low-density lipoprotein (LDL) receptor-related protein 5/6 (LRP5/6) is an essential co-receptor of the canonical Wnt signaling pathway. Structural information regarding the Wnt–LRP6 complex is yet to be determined, biochemical studies have shown that Wnt3a and Wnt9b interact with LRP6 E3E4 and E1E2, respectively, with KD values in the range of 10–100 nM2. In addition to Wnts, various Wnt signaling inhibitors, including Dickkopf-1 (DKK1), sclerostin (SOST), and sclerostin domain-containing protein 1 (SOSTDC1, called as WISE), are known to bind to LRP6 ECD3–5.

Methods

Results

Conclusion