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- PDB-5gje: Three-dimensional reconstruction of human LRP6 ectodomain complex... -

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Basic information

Entry
Database: PDB / ID: 5gje
TitleThree-dimensional reconstruction of human LRP6 ectodomain complexed with Dkk1
Components
  • (Low-density lipoprotein receptor-related protein ...) x 2
  • Dickkopf-related protein 1
KeywordsSIGNALING PROTEIN / Wnt signaling / Wnt co-receptor / LRP6 / glycoprotein / antagonist / Dkk1 / conformational change
Function / homology
Function and homology information


negative regulation of mesodermal cell fate specification / regulation of endodermal cell fate specification / positive regulation of Wnt signaling pathway, calcium modulating pathway / negative regulation of Wnt-Frizzled-LRP5/6 complex assembly / positive regulation of midbrain dopaminergic neuron differentiation / negative regulation of presynapse assembly / Signaling by LRP5 mutants / Wnt signaling pathway involved in somitogenesis / regulation of dopaminergic neuron differentiation / motor learning ...negative regulation of mesodermal cell fate specification / regulation of endodermal cell fate specification / positive regulation of Wnt signaling pathway, calcium modulating pathway / negative regulation of Wnt-Frizzled-LRP5/6 complex assembly / positive regulation of midbrain dopaminergic neuron differentiation / negative regulation of presynapse assembly / Signaling by LRP5 mutants / Wnt signaling pathway involved in somitogenesis / regulation of dopaminergic neuron differentiation / motor learning / Wnt-Frizzled-LRP5/6 complex / negative regulation of cardiac muscle cell differentiation / Negative regulation of TCF-dependent signaling by WNT ligand antagonists / endoderm formation / synapse pruning / Signaling by RNF43 mutants / neural crest formation / receptor-mediated endocytosis involved in cholesterol transport / endocardial cushion development / regulation of receptor internalization / heart induction / receptor antagonist activity / kinase inhibitor activity / Wnt receptor activity / low-density lipoprotein particle receptor activity / toxin transmembrane transporter activity / co-receptor binding / negative regulation of smooth muscle cell apoptotic process / Wnt-protein binding / positive regulation of Wnt signaling pathway, planar cell polarity pathway / cellular response to cholesterol / midbrain dopaminergic neuron differentiation / negative regulation of protein serine/threonine kinase activity / dopaminergic neuron differentiation / heart valve development / frizzled binding / negative regulation of ossification / neural crest cell differentiation / Wnt signalosome / embryonic limb morphogenesis / face morphogenesis / Disassembly of the destruction complex and recruitment of AXIN to the membrane / low-density lipoprotein particle receptor binding / limb development / negative regulation of Wnt signaling pathway / negative regulation of SMAD protein signal transduction / mesoderm formation / negative regulation of BMP signaling pathway / hair follicle development / canonical Wnt signaling pathway / coreceptor activity / positive regulation of cell cycle / negative regulation of peptidyl-serine phosphorylation / regulation of neuron apoptotic process / response to retinoic acid / forebrain development / regulation of synaptic transmission, glutamatergic / Regulation of FZD by ubiquitination / negative regulation of protein binding / TCF dependent signaling in response to WNT / protein localization to plasma membrane / positive regulation of JNK cascade / negative regulation of canonical Wnt signaling pathway / growth factor activity / cell morphogenesis / cell-cell adhesion / response to peptide hormone / Wnt signaling pathway / positive regulation of DNA-binding transcription factor activity / negative regulation of neuron projection development / positive regulation of cytosolic calcium ion concentration / chemical synaptic transmission / cytoplasmic vesicle / early endosome membrane / learning or memory / membrane raft / signaling receptor binding / neuronal cell body / synapse / positive regulation of gene expression / negative regulation of apoptotic process / positive regulation of DNA-templated transcription / negative regulation of transcription by RNA polymerase II / cell surface / endoplasmic reticulum / protein homodimerization activity / positive regulation of transcription by RNA polymerase II / extracellular space / extracellular region / identical protein binding / plasma membrane
Similarity search - Function
: / : / : / : / Dickkopf-related protein 1/2/4, C-terminal subdomain 2 / Dickkopf-related protein 1/2/4, C-terminal subdomain 1 / Dickkopf, N-terminal cysteine-rich / Dickkopf-like protein / Dickkopf N-terminal cysteine-rich region / Low density lipoprotein receptor-related protein 5/6 ...: / : / : / : / Dickkopf-related protein 1/2/4, C-terminal subdomain 2 / Dickkopf-related protein 1/2/4, C-terminal subdomain 1 / Dickkopf, N-terminal cysteine-rich / Dickkopf-like protein / Dickkopf N-terminal cysteine-rich region / Low density lipoprotein receptor-related protein 5/6 / Low-density lipoprotein receptor repeat class B / LDL-receptor class B (LDLRB) repeat profile. / LDLR class B repeat / Low-density lipoprotein-receptor YWTD domain / Low-density lipoprotein receptor domain class A / Low-density lipoprotein (LDL) receptor class A, conserved site / LDL-receptor class A (LDLRA) domain signature. / LDL-receptor class A (LDLRA) domain profile. / Low-density lipoprotein receptor domain class A / Low-density lipoprotein (LDL) receptor class A repeat / LDL receptor-like superfamily / Six-bladed beta-propeller, TolB-like / Coagulation Factor Xa inhibitory site / Epidermal growth factor-like domain. / EGF-like domain signature 2. / EGF-like domain
Similarity search - Domain/homology
PHOSPHATE ION / Low-density lipoprotein receptor-related protein 6 / Dickkopf-related protein 1
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / negative staining / Resolution: 21 Å
AuthorsMatoba, K. / Mihara, E. / Tamura-Kawakami, K. / Hirai, H. / Thompson, S. / Iwasaki, K. / Takagi, J.
CitationJournal: Cell Rep / Year: 2017
Title: Conformational Freedom of the LRP6 Ectodomain Is Regulated by N-glycosylation and the Binding of the Wnt Antagonist Dkk1.
Authors: Kyoko Matoba / Emiko Mihara / Keiko Tamura-Kawakami / Naoyuki Miyazaki / Shintaro Maeda / Hidenori Hirai / Samuel Thompson / Kenji Iwasaki / Junichi Takagi /
Abstract: LDL-receptor-related protein 6 (LRP6) is a single-pass membrane glycoprotein with a large modular ectodomain and forms a higher order signaling platform upon binding Wnt ligands on the cell surface. ...LDL-receptor-related protein 6 (LRP6) is a single-pass membrane glycoprotein with a large modular ectodomain and forms a higher order signaling platform upon binding Wnt ligands on the cell surface. Although multiple crystal structures are available for fragments of the LRP6 ectodomain, we lack a consensus view on the overall molecular architecture of the full-length LRP6 and its dynamic aspects. Here, we used negative-stain electron microscopy to probe conformational states of the entire ectodomain of LRP6 in solution and found that the four-module ectodomain undergoes a large bending motion hinged at the junction between the second and the third modules. Importantly, the extent of inter-domain motion is modulated by evolutionarily conserved N-glycan chains proximal to the joint. We also found that the LRP6 ectodomain becomes highly compact upon complexation with the Wnt antagonist Dkk1, suggesting a potential role for the ectodomain conformational change in the regulation of receptor oligomerization and signaling.
History
DepositionJun 29, 2016Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jan 18, 2017Provider: repository / Type: Initial release
Revision 2.0Jul 29, 2020Group: Advisory / Atomic model ...Advisory / Atomic model / Data collection / Derived calculations / Structure summary
Category: atom_site / chem_comp ...atom_site / chem_comp / em_entity_assembly / em_software / entity / pdbx_branch_scheme / pdbx_chem_comp_identifier / pdbx_entity_branch / pdbx_entity_branch_descriptor / pdbx_entity_branch_link / pdbx_entity_branch_list / pdbx_entity_nonpoly / pdbx_nonpoly_scheme / pdbx_struct_assembly_gen / pdbx_validate_close_contact / struct_asym / struct_conn / struct_site / struct_site_gen
Item: _atom_site.B_iso_or_equiv / _atom_site.Cartn_x ..._atom_site.B_iso_or_equiv / _atom_site.Cartn_x / _atom_site.Cartn_y / _atom_site.Cartn_z / _atom_site.auth_asym_id / _atom_site.auth_atom_id / _atom_site.auth_comp_id / _atom_site.auth_seq_id / _atom_site.label_asym_id / _atom_site.label_atom_id / _atom_site.label_comp_id / _atom_site.label_entity_id / _atom_site.type_symbol / _chem_comp.name / _chem_comp.type / _em_entity_assembly.entity_id_list / _em_software.name / _pdbx_struct_assembly_gen.asym_id_list / _pdbx_validate_close_contact.auth_asym_id_1 / _pdbx_validate_close_contact.auth_seq_id_1 / _struct_conn.pdbx_role / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id
Description: Carbohydrate remediation / Provider: repository / Type: Remediation

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Structure visualization

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Assembly

Deposited unit
A: Low-density lipoprotein receptor-related protein 6
B: Low-density lipoprotein receptor-related protein 6
C: Dickkopf-related protein 1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)151,66614
Polymers148,4533
Non-polymers3,21411
Water23413
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area6340 Å2
ΔGint31 kcal/mol
Surface area56270 Å2

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Components

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Low-density lipoprotein receptor-related protein ... , 2 types, 2 molecules AB

#1: Protein Low-density lipoprotein receptor-related protein 6 / LRP-6


Mass: 69034.883 Da / Num. of mol.: 1 / Fragment: UNP residues 20-630
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: LRP6 / Production host: Cricetulus griseus (Chinese hamster) / References: UniProt: O75581
#2: Protein Low-density lipoprotein receptor-related protein 6 / LRP-6


Mass: 69810.609 Da / Num. of mol.: 1 / Fragment: UNP residues 631-1246 / Mutation: V1062I
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: LRP6 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: O75581

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Protein , 1 types, 1 molecules C

#3: Protein Dickkopf-related protein 1 / hDkk-1 / SK


Mass: 9607.076 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: DKK1, UNQ492/PRO1008 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: O94907

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Sugars , 3 types, 8 molecules

#4: Polysaccharide
2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 424.401 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpNAcb1-4DGlcpNAcb1-Glycam Condensed SequenceGMML 1.0
WURCS=2.0/1,2,1/[a2122h-1b_1-5_2*NCC/3=O]/1-1/a4-b1WURCSPDB2Glycan 1.1.0
[]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}}}LINUCSPDB-CARE
#5: Polysaccharide 2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-[alpha-L-fucopyranose-(1-6)]2-acetamido-2-deoxy-beta- ...2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-[alpha-L-fucopyranose-(1-6)]2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 570.542 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpNAcb1-4[LFucpa1-6]DGlcpNAcb1-Glycam Condensed SequenceGMML 1.0
WURCS=2.0/2,3,2/[a2122h-1b_1-5_2*NCC/3=O][a1221m-1a_1-5]/1-1-2/a4-b1_a6-c1WURCSPDB2Glycan 1.1.0
[]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}[(6+1)][a-L-Fucp]{}}}LINUCSPDB-CARE
#7: Sugar ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-Acetylglucosamine


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
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

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Non-polymers , 3 types, 16 molecules

#6: Chemical ChemComp-PO4 / PHOSPHATE ION / Phosphate


Mass: 94.971 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: PO4
#8: Chemical ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL / Glycerol


Mass: 92.094 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C3H8O3
#9: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 13 / Source method: isolated from a natural source / Formula: H2O

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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

Component
IDNameTypeDetailsEntity IDParent-IDSource
1Human LRP6 ectodomain complexed with full length of Dkk1COMPLEXHuman Dkk1 fused with human growth hormone at the N-terminal (hGH-Dkk1) was co-expressed with LRP6ec in the HEK293S GnT1- cells.#1-#30RECOMBINANT
2LRP6 ectodomainCOMPLEX#1-#21RECOMBINANT
3Human Dickkopf-related protein1COMPLEXN-terminal human growth hormone fused full length of Dkk1#31RECOMBINANT
Molecular weightValue: 0.21 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
11Homo sapiens (human)9606
21Homo sapiens (human)9606
31Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDCellPlasmid
11Homo sapiens (human)9606HEK293S GnTI-pCDNA3.1/pEBMulti-Hyg
22Homo sapiens (human)9606HEK293S GnTI-pCDNA3.1
33Homo sapiens (human)9606HEK293S GnTI-pEBMulti-Hyg
Buffer solutionpH: 7.2
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: YES / Vitrification applied: NO / Details: This sample was monodisperse.
EM stainingType: NONE / Material: Uranyl acetate
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in.

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Electron microscopy imaging

MicroscopyModel: HITACHI H-9500SD
Electron gunElectron source: LAB6 / Accelerating voltage: 200 kV / Illumination mode: SPOT SCAN
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 80000 X / Cs: 2.8 mm
Specimen holderSpecimen holder model: SIDE ENTRY, EUCENTRIC
Image recordingAverage exposure time: 2 sec. / Electron dose: 20 e/Å2 / Film or detector model: TVIPS TEMCAM-F224 (2k x 2k)
Image scansSampling size: 24 µm / Width: 2048 / Height: 2048

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Processing

EM software
IDNameVersionCategory
1SPIDER & WEB17particle selection
2EM-Menu4image acquisition
4SPIDERCTF correction
7Chimera1.10.1model fitting
10SPIDERinitial Euler assignment
11SPIDERfinal Euler assignment
12SPIDERclassification
13SPIDER3D reconstruction
Image processingDetails: The selected images were band-pass filtered.
CTF correctionDetails: This was not done for RCT but for following single-particle reconstruction
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionDetails: 10974 tilt pairs for RCT
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 21 Å / Resolution method: FSC 0.5 CUT-OFF / Num. of particles: 5390 / Algorithm: BACK PROJECTION / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT
Atomic model building
IDPDB-IDPdb chain-ID 3D fitting-IDPdb chain residue range
14DG6

4dg6

A11-611
23S2K

3s2k

B1631-1243
33S2K

3s2k

C1182-266

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