title: Structures of Wnt-antagonist ZNRF3 and its complex with R-spondin 1 and implications for signaling.
abstract: Zinc RING finger 3 (ZNRF3) and its homolog RING finger 43 (RNF43) antagonize Wnt signaling in adult stem cells by ubiquitinating Frizzled receptors (FZD), which leads to endocytosis of the Wnt receptor. Conversely, binding of ZNRF3/RNF43 to LGR4-6 - R-spondin blocks Frizzled ubiquitination and enhances Wnt signaling. Here, we present crystal structures of the ZNRF3 ectodomain and its complex with R-spondin 1 (RSPO1). ZNRF3 binds RSPO1 and LGR5-RSPO1 with micromolar affinity via RSPO1 furin-like 1 (Fu1) domain. Anonychia-related mutations in RSPO4 support the importance of the observed interface. The ZNRF3-RSPO1 structure resembles that of LGR5-RSPO1-RNF43, though Fu2 of RSPO1 is variably oriented. The ZNRF3-binding site overlaps with trans-interactions observed in 2:2 LGR5-RSPO1 complexes, thus binding of ZNRF3/RNF43 would disrupt such an arrangement. Sequence conservation suggests a single ligand-binding site on ZNRF3, consistent with the proposed competing binding role of ZNRF3/RNF43 in Wnt signaling.
authors.affiliation: Crystal and Structural Chemistry, Bijvoet Center for Biomolecular Research, Department of Chemistry, Faculty of Science, Utrecht University, Utrecht, The Netherlands.
authors.forename: Weng Chuan
authors.initials: WC
authors.lastname: Peng
authors.affiliation: Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences and University Medical Centre Utrecht, Utrecht, The Netherlands.
authors.forename: Wim
authors.initials: W
authors.lastname: de Lau
authors.affiliation: Crystal and Structural Chemistry, Bijvoet Center for Biomolecular Research, Department of Chemistry, Faculty of Science, Utrecht University, Utrecht, The Netherlands.
authors.forename: Pramod K
authors.initials: PK
authors.lastname: Madoori
authors.affiliation: Crystal and Structural Chemistry, Bijvoet Center for Biomolecular Research, Department of Chemistry, Faculty of Science, Utrecht University, Utrecht, The Netherlands.
authors.forename: Federico
authors.initials: F
authors.lastname: Forneris
authors.affiliation: Crystal and Structural Chemistry, Bijvoet Center for Biomolecular Research, Department of Chemistry, Faculty of Science, Utrecht University, Utrecht, The Netherlands.
authors.forename: Joke C M
authors.initials: JC
authors.lastname: Granneman
authors.affiliation: Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences and University Medical Centre Utrecht, Utrecht, The Netherlands.
authors.forename: Hans
authors.initials: H
authors.lastname: Clevers
authors.affiliation: Crystal and Structural Chemistry, Bijvoet Center for Biomolecular Research, Department of Chemistry, Faculty of Science, Utrecht University, Utrecht, The Netherlands.
authors.forename: Piet
authors.initials: P
authors.lastname: Gros
mesh_terms: D053687:Adult Stem Cells / Q000378:metabolism; D000818:Animals; D018360:Crystallography, X-Ray; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D046912:Multiprotein Complexes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015513:Oncogene Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020836:Protein Structure, Quaternary; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019699:Thrombospondins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D060449:Wnt Signaling Pathway; D000098907:R-Spondins
chemical_list: D004268:DNA-Binding Proteins; D046912:Multiprotein Complexes; D015513:Oncogene Proteins; D043562:Receptors, G-Protein-Coupled; D019699:Thrombospondins; D044767:Ubiquitin-Protein Ligases; C116884:LGR5 protein, human; C493597:Lgr5 protein, mouse; C503243:RSPO1 protein, human; D000098907:R-Spondins; C518903:RSPO1 protein, mouse; C496945:RNF43 protein, human; C575493:RNF43 protein, mouse; C572950:ZNRF3 protein, human; C572951:Znrf3 protein, mouse