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- PDB-9kb8: Cryo-EM structure of LGR4-RSPO2-ZNRF3 complex (1:1:2) -

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Basic information

Entry
Database: PDB / ID: 9kb8
TitleCryo-EM structure of LGR4-RSPO2-ZNRF3 complex (1:1:2)
Components
  • E3 ubiquitin-protein ligase ZNRF3
  • Leucine-rich repeat-containing G-protein coupled receptor 4
  • R-spondin-2
KeywordsMEMBRANE PROTEIN / Wnt signal
Function / homology
Function and homology information


trachea cartilage morphogenesis / negative regulation of odontogenesis of dentin-containing tooth / lung growth / regulation of Wnt signaling pathway, planar cell polarity pathway / Wnt receptor catabolic process / metanephric glomerulus development / metanephric nephron tubule morphogenesis / negative regulation of non-canonical Wnt signaling pathway / epithelial cell proliferation involved in renal tubule morphogenesis / protein-hormone receptor activity ...trachea cartilage morphogenesis / negative regulation of odontogenesis of dentin-containing tooth / lung growth / regulation of Wnt signaling pathway, planar cell polarity pathway / Wnt receptor catabolic process / metanephric glomerulus development / metanephric nephron tubule morphogenesis / negative regulation of non-canonical Wnt signaling pathway / epithelial cell proliferation involved in renal tubule morphogenesis / protein-hormone receptor activity / intestinal stem cell homeostasis / BMP receptor binding / negative regulation of toll-like receptor signaling pathway / positive regulation of branching involved in ureteric bud morphogenesis / male genitalia development / bone remodeling / dopaminergic neuron differentiation / frizzled binding / embryonic forelimb morphogenesis / embryonic hindlimb morphogenesis / digestive tract development / limb development / negative regulation of cold-induced thermogenesis / negative regulation of cytokine production / epithelial tube branching involved in lung morphogenesis / bone mineralization / hair follicle development / positive regulation of Wnt signaling pathway / canonical Wnt signaling pathway / Regulation of FZD by ubiquitination / stem cell proliferation / circadian regulation of gene expression / negative regulation of canonical Wnt signaling pathway / G protein-coupled receptor activity / RING-type E3 ubiquitin transferase / Wnt signaling pathway / ubiquitin-protein transferase activity / osteoblast differentiation / ubiquitin protein ligase activity / transmembrane signaling receptor activity / positive regulation of canonical Wnt signaling pathway / heparin binding / ubiquitin-dependent protein catabolic process / spermatogenesis / protein ubiquitination / signaling receptor binding / innate immune response / cell surface / extracellular space / extracellular region / zinc ion binding / plasma membrane
Similarity search - Function
E3 ubiquitin-protein ligase ZNRF3, Zinc finger, RING-type / ZNRF-3, ectodomain / ZNRF-3 Ectodomain / : / R-spondin, Fu-CRD domain / : / Furin-like repeat, cysteine-rich / Glycoprotein hormone receptor family / Ring finger domain / Leucine rich repeat N-terminal domain ...E3 ubiquitin-protein ligase ZNRF3, Zinc finger, RING-type / ZNRF-3, ectodomain / ZNRF-3 Ectodomain / : / R-spondin, Fu-CRD domain / : / Furin-like repeat, cysteine-rich / Glycoprotein hormone receptor family / Ring finger domain / Leucine rich repeat N-terminal domain / Leucine-rich repeat N-terminal domain / Leucine rich repeat N-terminal domain / Thrombospondin type-1 (TSP1) repeat superfamily / Thrombospondin type-1 (TSP1) repeat profile. / Thrombospondin type 1 repeats / Thrombospondin type-1 (TSP1) repeat / Leucine Rich Repeat / Leucine-rich repeats, bacterial type / Leucine-rich repeat, SDS22-like subfamily / Furin-like repeat / Furin-like repeats / Leucine rich repeat / Leucine-rich repeat, typical subtype / Leucine-rich repeats, typical (most populated) subfamily / Leucine-rich repeat profile. / Ring finger / Growth factor receptor cysteine-rich domain superfamily / Leucine-rich repeat / Leucine-rich repeat domain superfamily / Zinc finger RING-type profile. / Zinc finger, RING-type / G protein-coupled receptor, rhodopsin-like / GPCR, rhodopsin-like, 7TM / G-protein coupled receptors family 1 profile. / 7 transmembrane receptor (rhodopsin family) / Zinc finger, RING/FYVE/PHD-type
Similarity search - Domain/homology
R-spondin-2 / Leucine-rich repeat-containing G-protein coupled receptor 4 / E3 ubiquitin-protein ligase ZNRF3
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.25 Å
AuthorsPeng, Y. / Fujimura, A. / Asami, J. / Zhang, Z. / Shimizu, T. / Ohto, U.
Funding support Japan, 1items
OrganizationGrant numberCountry
Ministry of Education, Culture, Sports, Science and Technology (Japan)22K15046 and 24K09349 (Z.Z.); 22H05184 and 23H00366 (T.S.); and 22H02556 (U.O.) Japan
CitationJournal: Nat Commun / Year: 2025
Title: Structural insights into Wnt/β-catenin signaling regulation by LGR4, R-spondin, and ZNRF3.
Authors: Yuxuan Peng / Akiko Fujimura / Jinta Asami / Zhikuan Zhang / Toshiyuki Shimizu / Umeharu Ohto /
Abstract: Leucine-rich repeat-containing G protein-coupled receptor 4 (LGR4) plays a critical role in regulating the wingless-related integration site (Wnt) signaling pathway and is essential for organ ...Leucine-rich repeat-containing G protein-coupled receptor 4 (LGR4) plays a critical role in regulating the wingless-related integration site (Wnt) signaling pathway and is essential for organ development and carcinogenesis. LGR4, along with its ligand R-spondin (RSPO), potentiates Wnt/β-catenin signaling by recruiting its signaling suppressor, E3 ligase Zinc and Ring Finger 3 (ZNRF3), and inducing its membrane clearance. However, detailed mechanisms underlying this process remain unknown. In this study, we present the cryo-electron microscopy structures of human LGR4, the LGR4-RSPO2 and LGR4-RSPO2-ZNRF3 complexes. Upon RSPO2 binding, LGR4 undergoes no significant conformational changes in its transmembrane and extracellular domain structures or their relative orientations. LGR4, RSPO2, and ZNRF3 assemble into a 2:2:2 complex with the ZNRF3 dimer enclosed at the center. This ternary arrangement and forced dimerization of ZNRF3 likely underpin how LGR4 and RSPO2 potentiate Wnt/β-catenin signaling by sequestering ZNRF3 from Wnt receptors and facilitating its auto-inactivation. This study provides a structural basis for understanding the regulatory mechanism of Wnt/β-catenin signaling through the LGR4-RSPO2-ZNRF3 pathway and may offer opportunities for future drug development targeting this axis.
History
DepositionOct 30, 2024Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Oct 15, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
B: E3 ubiquitin-protein ligase ZNRF3
A: Leucine-rich repeat-containing G-protein coupled receptor 4
C: R-spondin-2
D: E3 ubiquitin-protein ligase ZNRF3
hetero molecules


Theoretical massNumber of molelcules
Total (without water)164,9878
Polymers164,1024
Non-polymers8854
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein E3 ubiquitin-protein ligase ZNRF3 / RING finger protein 203 / RING-type E3 ubiquitin transferase ZNRF3 / Zinc/RING finger protein 3


Mass: 25888.084 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ZNRF3, KIAA1133, RNF203 / Production host: Homo sapiens (human)
References: UniProt: Q9ULT6, RING-type E3 ubiquitin transferase
#2: Protein Leucine-rich repeat-containing G-protein coupled receptor 4 / G-protein coupled receptor 48


Mass: 93372.047 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: LGR4, GPR48 / Production host: Homo sapiens (human) / References: UniProt: Q9BXB1
#3: Protein R-spondin-2 / Roof plate-specific spondin-2 / hRspo2


Mass: 18953.715 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: RSPO2, UNQ9384/PRO34209 / Production host: Homo sapiens (human) / References: UniProt: Q6UXX9
#4: Sugar
ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0
Has ligand of interestN
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: LGR4-RSPO2-ZNRF3 complex (1:1:2) / Type: COMPLEX / Entity ID: #1-#3 / Source: RECOMBINANT
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 48 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM softwareName: PHENIX / Version: 1.20.1_4487: / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.25 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 693808 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0039601
ELECTRON MICROSCOPYf_angle_d0.58513019
ELECTRON MICROSCOPYf_dihedral_angle_d3.5541354
ELECTRON MICROSCOPYf_chiral_restr0.0421501
ELECTRON MICROSCOPYf_plane_restr0.0031667

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