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- PDB-8b7q: Cryo-EM structure for the mouse LEPR-CRH2:Leptin:LEPR-Ig complex ... -

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Basic information

Entry
Database: PDB / ID: 8b7q
TitleCryo-EM structure for the mouse LEPR-CRH2:Leptin:LEPR-Ig complex following symmetry expansion in combination with local refinement
Components
  • Leptin
  • Leptin receptor
KeywordsCYTOKINE / leptin / LEP-R / obesity / metabolism / energy balance
Function / homology
Function and homology information


Synthesis, secretion, and deacylation of Ghrelin / regulation of lipoprotein lipid oxidation / cellular response to L-ascorbic acid / positive regulation of fat cell apoptotic process / negative regulation of glutamine transport / leptin receptor activity / regulation of transport / negative regulation of appetite by leptin-mediated signaling pathway / Synthesis, secretion, and inactivation of Glucagon-like Peptide-1 (GLP-1) / negative regulation of glucagon secretion ...Synthesis, secretion, and deacylation of Ghrelin / regulation of lipoprotein lipid oxidation / cellular response to L-ascorbic acid / positive regulation of fat cell apoptotic process / negative regulation of glutamine transport / leptin receptor activity / regulation of transport / negative regulation of appetite by leptin-mediated signaling pathway / Synthesis, secretion, and inactivation of Glucagon-like Peptide-1 (GLP-1) / negative regulation of glucagon secretion / regulation of endothelial cell proliferation / regulation of natural killer cell mediated cytotoxicity / regulation of natural killer cell proliferation / leptin receptor binding / bone growth / positive regulation of luteinizing hormone secretion / regulation of natural killer cell activation / glycerol biosynthetic process / elastin metabolic process / leptin-mediated signaling pathway / positive regulation of hepatic stellate cell activation / positive regulation of follicle-stimulating hormone secretion / regulation of steroid biosynthetic process / positive regulation of monoatomic ion transport / regulation of intestinal cholesterol absorption / regulation of bone remodeling / regulation of brown fat cell differentiation / positive regulation of peroxisome proliferator activated receptor signaling pathway / regulation of nitric-oxide synthase activity / adult feeding behavior / response to leptin / activation of protein kinase C activity / bone mineralization involved in bone maturation / regulation of lipid biosynthetic process / sexual reproduction / negative regulation of cartilage development / regulation of feeding behavior / ovulation from ovarian follicle / negative regulation of appetite / positive regulation of developmental growth / leukocyte tethering or rolling / energy reserve metabolic process / bile acid metabolic process / cellular response to leptin stimulus / negative regulation of D-glucose import / prostaglandin secretion / regulation of protein localization to nucleus / cardiac muscle hypertrophy / hormone metabolic process / positive regulation of p38MAPK cascade / intestinal absorption / regulation of fat cell differentiation / insulin secretion / regulation of metabolic process / aorta development / negative regulation of vasoconstriction / regulation of gluconeogenesis / peptide hormone receptor binding / glycogen metabolic process / eating behavior / fatty acid beta-oxidation / regulation of cytokine production involved in inflammatory response / central nervous system neuron development / response to dietary excess / negative regulation of lipid storage / T cell differentiation / response to vitamin E / cell surface receptor signaling pathway via JAK-STAT / positive regulation of TOR signaling / regulation of angiogenesis / adipose tissue development / negative regulation of gluconeogenesis / phagocytosis / glial cell proliferation / positive regulation of insulin receptor signaling pathway / positive regulation of T cell proliferation / energy homeostasis / cellular response to retinoic acid / positive regulation of interleukin-12 production / regulation of insulin secretion / negative regulation of autophagy / cholesterol metabolic process / response to activity / gluconeogenesis / positive regulation of interleukin-8 production / female pregnancy / positive regulation of receptor signaling pathway via JAK-STAT / determination of adult lifespan / response to insulin / placenta development / hormone activity / regulation of blood pressure / lipid metabolic process / positive regulation of interleukin-6 production / positive regulation of protein import into nucleus / cellular response to insulin stimulus / glucose metabolic process / circadian rhythm / positive regulation of reactive oxygen species metabolic process / positive regulation of tumor necrosis factor production
Similarity search - Function
Leptin / Leptin / Leptin receptor, immunoglobulin-like domain / Obesity receptor immunoglobulin like domain / Short hematopoietin receptor, family 1, conserved site / Immunoglobulin C2-set-like, ligand-binding / Ig-like C2-type domain / Long hematopoietin receptor, Gp130 family 2, conserved site / Long hematopoietin receptor, gp130 family signature. / Four-helical cytokine-like, core ...Leptin / Leptin / Leptin receptor, immunoglobulin-like domain / Obesity receptor immunoglobulin like domain / Short hematopoietin receptor, family 1, conserved site / Immunoglobulin C2-set-like, ligand-binding / Ig-like C2-type domain / Long hematopoietin receptor, Gp130 family 2, conserved site / Long hematopoietin receptor, gp130 family signature. / Four-helical cytokine-like, core / Fibronectin type III domain / Fibronectin type 3 domain / Fibronectin type-III domain profile. / Fibronectin type III / Fibronectin type III superfamily / Ig-like domain profile. / Immunoglobulin-like domain / Immunoglobulin-like fold
Similarity search - Domain/homology
Leptin / Leptin receptor
Similarity search - Component
Biological speciesMus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.02 Å
AuthorsVerstraete, K. / Savvides, S.N. / Verschueren, K.G. / Tsirigotaki, A.
Funding support Belgium, 1items
OrganizationGrant numberCountry
Research Foundation - Flanders (FWO)G0G0619N Belgium
CitationJournal: Nat Struct Mol Biol / Year: 2023
Title: Mechanism of receptor assembly via the pleiotropic adipokine Leptin.
Authors: Alexandra Tsirigotaki / Ann Dansercoer / Koen H G Verschueren / Iva Marković / Christoph Pollmann / Maximillian Hafer / Jan Felix / Catherine Birck / Wouter Van Putte / Dominiek Catteeuw / ...Authors: Alexandra Tsirigotaki / Ann Dansercoer / Koen H G Verschueren / Iva Marković / Christoph Pollmann / Maximillian Hafer / Jan Felix / Catherine Birck / Wouter Van Putte / Dominiek Catteeuw / Jan Tavernier / J Fernando Bazan / Jacob Piehler / Savvas N Savvides / Kenneth Verstraete /
Abstract: The adipokine Leptin activates its receptor LEP-R in the hypothalamus to regulate body weight and exerts additional pleiotropic functions in immunity, fertility and cancer. However, the structure and ...The adipokine Leptin activates its receptor LEP-R in the hypothalamus to regulate body weight and exerts additional pleiotropic functions in immunity, fertility and cancer. However, the structure and mechanism of Leptin-mediated LEP-R assemblies has remained unclear. Intriguingly, the signaling-competent isoform of LEP-R is only lowly abundant amid several inactive short LEP-R isoforms contributing to a mechanistic conundrum. Here we show by X-ray crystallography and cryo-EM that, in contrast to long-standing paradigms, Leptin induces type I cytokine receptor assemblies featuring 3:3 stoichiometry and demonstrate such Leptin-induced trimerization of LEP-R on living cells via single-molecule microscopy. In mediating these assemblies, Leptin undergoes drastic restructuring that activates its site III for binding to the Ig domain of an adjacent LEP-R. These interactions are abolished by mutations linked to obesity. Collectively, our study provides the structural and mechanistic framework for how evolutionarily conserved Leptin:LEP-R assemblies with 3:3 stoichiometry can engage distinct LEP-R isoforms to achieve signaling.
History
DepositionSep 30, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Apr 5, 2023Provider: repository / Type: Initial release
Revision 1.1Apr 26, 2023Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 1.2Oct 23, 2024Group: Data collection / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Leptin
B: Leptin receptor
F: Leptin receptor
hetero molecules


Theoretical massNumber of molelcules
Total (without water)214,0534
Polymers213,8323
Non-polymers2211
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: light scattering
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area3220 Å2
ΔGint-14 kcal/mol
Surface area23140 Å2

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Components

#1: Protein Leptin / Obesity factor


Mass: 18873.283 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Mouse leptin was produced with an N-terminal His-tag and refolded from inclusion bodies produced in E. coli
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Lep, Ob / Plasmid: pET15b / Details (production host): pET15b-His-TEV-mLeptin / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P41160
#2: Protein Leptin receptor / LEP-R / B219 / OB receptor / OB-R


Mass: 97479.391 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: The mLEP-R ectodomain was C-terminally fused to a trimeric GCN4 isoleucine zipper tag and secreted from HEK93 FreeStyle cells and complexed with refolded mouse leptin produced in E.coli.
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Lepr, Db, Obr / Plasmid: pTwist / Cell line (production host): HEK293 / Production host: Homo sapiens (human) / Variant (production host): FreeStyle / References: UniProt: P48356
#3: 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.: 1 / Source method: obtained synthetically / Formula: C8H15NO6 / Feature type: SUBJECT OF INVESTIGATION
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 interestY
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: Mouse leptin in complex with a trimerized form of the mouse Lep-R extracellular region
Type: COMPLEX
Details: The mLEP-R ectodomain was C-terminally fused to a trimeric GCN4 isoleucine zipper tag and secreted from HEK93 FreeStyle cells and complexed with refolded mouse leptin produced in E.coli.
Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightValue: 0.444 MDa / Experimental value: YES
Source (natural)Organism: Mus musculus (house mouse)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7.4 / Details: 20 mM HEPES, 150 mM NaCl, pH 7.4
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPESC8H18N2O4S1
2150 mMsodium chlorideNaCl1
SpecimenConc.: 0.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: This sample was monodisperse.
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: C-flat-1.2/1.3
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 99 % / Chamber temperature: 295 K

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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: OTHER / Nominal magnification: 105000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1200 nm / Cs: 2.7 mm
Image recordingElectron dose: 45 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 13230

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Processing

Software
NameVersionClassificationNB
phenix.real_space_refine1.19.2_4158refinement
PHENIX1.19.2_4158refinement
EM software
IDNameVersionCategoryDetails
5cryoSPARCv3.3.1CTF correction
8UCSF Chimera1.17model fitting
9PHENIX1.19.2-4158model refinement
10cryoSPARCv3.3.1initial Euler assignment
11cryoSPARCv3.3.1classificationAb initio 3D classification
12cryoSPARCversion 3.3.23D reconstructionSymmetry expansion in combination with local refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 141157
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.02 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 163914
Details: To address the breakage of symmetry in the ring-like core region of the complex and possibly revolve the mLEP-RCRH2:mLeptin:mLEP-RCRH1-IgCRH2 subcomplex at higher resolution, the pseudo-C3 ...Details: To address the breakage of symmetry in the ring-like core region of the complex and possibly revolve the mLEP-RCRH2:mLeptin:mLEP-RCRH1-IgCRH2 subcomplex at higher resolution, the pseudo-C3 symmetric volume following consenus refinement was aligned to the pseudo-C3 symmetry axis via the Volume Alignment Tool job in cryoSPARC. The associated particle set was re-extracted without binning and symmetry expanded around the C3 axis resulting in 163,914 particles. Using the molmap function in Chimera, a volume blurred to 25 Angstrom around one mLEP-RCRH2:mLeptin:mLEP-RCRH1-IgCRH2 subcomplex was generated, and transformed into a mask with the Volume Tools job in cryoSPARC. Local refinement was performed by limiting the rotation and shift search extent around the original consensus refinement. The center of mass of the mask was used as a fulcrum point. This approach resulted in a cryo-EM map with an FSC0.143 resolution of 4.02 Angstrom in which the crystallographic model for the mLEP-RCRH2:mLeptin:mLEP-R_IgCRH2' complex was fitted using Chimera and real-space refined in Phenix using reference restraints to the starting model.
Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Details: The crystallographic model for the mLEP-RCRH2:mLeptin:mLEP-R_IgCRH2' complex (pdb 7z3r) was fitted in the cryo-EM map using Chimera and real-space refined in Phenix using reference ...Details: The crystallographic model for the mLEP-RCRH2:mLeptin:mLEP-R_IgCRH2' complex (pdb 7z3r) was fitted in the cryo-EM map using Chimera and real-space refined in Phenix using reference restraints to the starting model.
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 101.59 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00243588
ELECTRON MICROSCOPYf_angle_d0.61114885
ELECTRON MICROSCOPYf_chiral_restr0.0425572
ELECTRON MICROSCOPYf_plane_restr0.0041615
ELECTRON MICROSCOPYf_dihedral_angle_d9.8271325

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