title: Crystal structure of LGR4-Rspo1 complex: insights into the divergent mechanisms of ligand recognition by leucine-rich repeat G-protein-coupled receptors (LGRs).
abstract: Leucine-rich repeat G-protein-coupled receptors (LGRs) are a unique class of G-protein-coupled receptors characterized by a large extracellular domain to recognize ligands and regulate many important developmental processes. Among the three groups of LGRs, group B members (LGR4-6) recognize R-spondin family proteins (Rspo1-4) to stimulate Wnt signaling. In this study, we successfully utilized the "hybrid leucine-rich repeat technique," which fused LGR4 with the hagfish VLR protein, to obtain two recombinant human LGR4 proteins, LGR415 and LGR49. We determined the crystal structures of ligand-free LGR415 and the LGR49-Rspo1 complex. LGR4 exhibits a twisted horseshoe-like structure. Rspo1 adopts a flat and β-fold architecture and is bound in the concave surface of LGR4 in the complex through electrostatic and hydrophobic interactions. All the Rspo1-binding residues are conserved in LGR4-6, suggesting that LGR4-6 bind R-spondins through an identical surface. Structural analysis of our LGR4-Rspo1 complex with the previously determined LGR4 and LGR5 structures revealed that the concave surface of LGR4 is the sole binding site for R-spondins, suggesting a one-site binding model of LGR4-6 in ligand recognition. The molecular mechanism of LGR4-6 is distinct from the two-step mechanism of group A receptors LGR1-3 and the multiple-interface binding model of group C receptors LGR7-8, suggesting LGRs utilize the divergent mechanisms for ligand recognition. Our structures, together with previous reports, provide a comprehensive understanding of the ligand recognition by LGRs.
authors.affiliation: From the Life Sciences Institute and Innovation Center for Cell Biology, Zhejiang University, Hangzhou, Zhejiang 310058 and.
authors.forename: Jin-Gen
authors.initials: JG
authors.lastname: Xu
authors.affiliation: From the Life Sciences Institute and Innovation Center for Cell Biology, Zhejiang University, Hangzhou, Zhejiang 310058 and.
authors.forename: Chunfeng
authors.initials: C
authors.lastname: Huang
authors.affiliation: the Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai 200241, China.
authors.forename: Zhengfeng
authors.initials: Z
authors.lastname: Yang
authors.affiliation: From the Life Sciences Institute and Innovation Center for Cell Biology, Zhejiang University, Hangzhou, Zhejiang 310058 and.
authors.forename: Mengmeng
authors.initials: M
authors.lastname: Jin
authors.affiliation: From the Life Sciences Institute and Innovation Center for Cell Biology, Zhejiang University, Hangzhou, Zhejiang 310058 and.
authors.forename: Panhan
authors.initials: P
authors.lastname: Fu
authors.affiliation: From the Life Sciences Institute and Innovation Center for Cell Biology, Zhejiang University, Hangzhou, Zhejiang 310058 and.
authors.forename: Ni
authors.initials: N
authors.lastname: Zhang
authors.affiliation: the Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai 200241, China.
authors.forename: Jian
authors.initials: J
authors.lastname: Luo
authors.affiliation: the Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai 200241, China.
authors.forename: Dali
authors.initials: D
authors.lastname: Li
authors.affiliation: the Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai 200241, China.
authors.forename: Mingyao
authors.initials: M
authors.lastname: Liu
authors.affiliation: From the Life Sciences Institute and Innovation Center for Cell Biology, Zhejiang University, Hangzhou, Zhejiang 310058 and zhouyanlsi@zju.edu.cn.
authors.forename: Yan
authors.initials: Y
authors.lastname: Zhou
authors.affiliation: From the Life Sciences Institute and Innovation Center for Cell Biology, Zhejiang University, Hangzhou, Zhejiang 310058 and zhuyongqun@zju.edu.cn.
authors.forename: Yongqun
authors.initials: Y
authors.lastname: Zhu
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D018360:Crystallography, X-Ray; D015195:Drug Design; D018507:Gene Expression Regulation, Developmental; D006801:Humans; D007930:Leucine / Q000737:chemistry; D008024:Ligands; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010957:Plasmids; D011485:Protein Binding; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013234:Stem Cells / Q000166:cytology; D019699:Thrombospondins / Q000737:chemistry*; D051153:Wnt Proteins / Q000378:metabolism; D000098907:R-Spondins
chemical_list: D007930:Leucine; D008024:Ligands; D043562:Receptors, G-Protein-Coupled; D011994:Recombinant Proteins; D019699:Thrombospondins; D051153:Wnt Proteins; C411562:LGR4 protein, human; C503243:RSPO1 protein, human; D000098907:R-Spondins
keywords: Hormone; Protein Structure; Receptor; Wnt Signaling; X-ray Crystallography