title: Crystal structure of R-spondin 2 in complex with the ectodomains of its receptors LGR5 and ZNRF3.
abstract: The four secreted R-spondin (Rspo1-4) proteins of vertebrates function as stem cell growth factors and potentiate canonical Wnt signalling. Rspo proteins act by cross-linking members of two cell surface receptor families, complexing the stem cell markers LGR4-6 with the Frizzled-specific E3 ubiquitin ligases ZNRF3/RNF43. The consequent internalisation of the ternary LGR-Rspo-E3 complex removes the E3 ligase activity, which otherwise targets the Wnt receptor Frizzled for degradation, and thus enhances Wnt signalling. Multiple combinations of LGR4-6, Rspo1-4 and ZNRF3/RNF43 are possible, implying the existence of generic interaction determinants, but also of specific differences in complex architecture and activity. We present here a high resolution crystal structure of an ectodomain variant of human LGR5 (hLGR5ecto) complexed with a signalling competent fragment of mouse Rspo2 (mRspo2Fu1-Fu2). The structure shows that the particularly potent Rspo2 ligand engages LGR5 in a fashion almost identical to that reported for hRSPO1. Comparison of our hLGR5ecto structure with previously published structures highlights a surprising plasticity of the LGR ectodomains, characterised by a nearly 9° or larger rotation of the N-terminal half of the horseshoe-like fold relative to the C-terminal half. We also report a low resolution hLGR5-mRspo2Fu1-Fu2-mZNRF3ecto ternary complex structure. This crystal structure confirms our previously suggested hypothesis, showing that Rspo proteins cross-link LGRs and ZNRF3 into a 2:2:2 complex, whereas a 1:1:1 complex is formed with RNF43.
authors.affiliation: Division of Structural Biology, Henry Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford OX3 7BN, United Kingdom.
authors.forename: Matthias
authors.initials: M
authors.lastname: Zebisch
authors.affiliation: Division of Structural Biology, Henry Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford OX3 7BN, United Kingdom. Electronic address: yvonne@strubi.ox.ac.uk.
authors.forename: E Yvonne
authors.initials: EY
authors.lastname: Jones
mesh_terms: D001665:Binding Sites; D018360:Crystallography, X-Ray; D008958:Models, Molecular; D054730:Protein Interaction Domains and Motifs; D017434:Protein Structure, Tertiary; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry*; D019699:Thrombospondins / Q000737:chemistry*; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry*; D000098907:R-Spondins
chemical_list: D043562:Receptors, G-Protein-Coupled; D019699:Thrombospondins; D044767:Ubiquitin-Protein Ligases; C116884:LGR5 protein, human; C518904:RSPO2 protein, mouse; D000098907:R-Spondins; C572951:Znrf3 protein, mouse
keywords: Complex crystal structure; RING finger ubiquitin ligase; Signalling; Stem cells; Wnt agonist