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- EMDB-15678: Mouse leptin:LEP-R complex cryoEM structure (3:3 model) -

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Basic information

Entry
Database: EMDB / ID: EMD-15678
TitleMouse leptin:LEP-R complex cryoEM structure (3:3 model)
Map dataSharpenend cryo-EM map following non-uniform refinement
Sample
  • Complex: Stucture of mouse leptin in complex with a trimerized form of the mouse Lep-R extracellular region
    • Protein or peptide: Leptin
    • Protein or peptide: Leptin receptor
  • Ligand: NICKEL (II) ION
Keywordsleptin / LEP-R / obesity / metabolism / energy balance / CYTOKINE
Function / homology
Function and homology information


negative regulation of locomotor rhythm / negative regulation of metabolic process / positive regulation of protein modification process / regulation of intestinal cholesterol absorption / Synthesis, secretion, and deacylation of Ghrelin / leptin receptor activity / regulation of lipoprotein lipid oxidation / cellular response to L-ascorbic acid / positive regulation of fat cell apoptotic process / negative regulation of glutamine transport ...negative regulation of locomotor rhythm / negative regulation of metabolic process / positive regulation of protein modification process / regulation of intestinal cholesterol absorption / Synthesis, secretion, and deacylation of Ghrelin / leptin receptor activity / regulation of lipoprotein lipid oxidation / cellular response to L-ascorbic acid / positive regulation of fat cell apoptotic process / negative regulation of glutamine transport / activation of protein kinase C activity / negative regulation of appetite by leptin-mediated signaling pathway / negative regulation of eating behavior / positive regulation of luteinizing hormone secretion / Synthesis, secretion, and inactivation of Glucagon-like Peptide-1 (GLP-1) / negative regulation of glucagon secretion / regulation of steroid biosynthetic process / negative regulation of cartilage development / elastin metabolic process / leptin receptor binding / sexual reproduction / regulation of natural killer cell mediated cytotoxicity / regulation of bone remodeling / regulation of endothelial cell proliferation / regulation of natural killer cell proliferation / positive regulation of follicle-stimulating hormone secretion / glycerol biosynthetic process / leptin-mediated signaling pathway / ovulation from ovarian follicle / regulation of transport / regulation of brown fat cell differentiation / bone growth / positive regulation of monoatomic ion transport / protein-hormone receptor activity / energy reserve metabolic process / positive regulation of hepatic stellate cell activation / positive regulation of peroxisome proliferator activated receptor signaling pathway / regulation of nitric-oxide synthase activity / regulation of catalytic activity / regulation of feeding behavior / adult feeding behavior / bone mineralization involved in bone maturation / regulation of lipid biosynthetic process / regulation of natural killer cell activation / response to leptin / fatty acid catabolic process / negative regulation of hydrolase activity / negative regulation of D-glucose import across plasma membrane / hormone metabolic process / bile acid metabolic process / negative regulation of appetite / positive regulation of developmental growth / prostaglandin secretion / leukocyte tethering or rolling / aorta development / cellular response to leptin stimulus / cardiac muscle hypertrophy / intestinal absorption / regulation of protein localization to nucleus / positive regulation of p38MAPK cascade / regulation of fat cell differentiation / cell surface receptor signaling pathway via STAT / regulation of gluconeogenesis / cytokine receptor activity / insulin secretion / response to dietary excess / glycogen metabolic process / negative regulation of vasoconstriction / response to vitamin E / eating behavior / fatty acid beta-oxidation / peptide hormone receptor binding / regulation of cytokine production involved in inflammatory response / adipose tissue development / central nervous system neuron development / cytokine binding / regulation of insulin secretion / peptide hormone binding / T cell differentiation / negative regulation of lipid storage / regulation of angiogenesis / negative regulation of gluconeogenesis / positive regulation of insulin receptor signaling pathway / positive regulation of TOR signaling / cholesterol metabolic process / glial cell proliferation / energy homeostasis / positive regulation of interleukin-12 production / cellular response to retinoic acid / phagocytosis / cell surface receptor signaling pathway via JAK-STAT / determination of adult lifespan / positive regulation of T cell proliferation / placenta development / negative regulation of autophagy / response to activity / positive regulation of insulin secretion involved in cellular response to glucose stimulus / positive regulation of interleukin-8 production / lipid metabolic process / positive regulation of receptor signaling pathway via JAK-STAT
Similarity search - Function
Leptin / Leptin receptor, immunoglobulin-like domain / Leptin / 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 receptor, immunoglobulin-like domain / Leptin / 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)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.6 Å
AuthorsVerstraete K / Savvides SN / Verschueren KG / Tsirigotaki A
Funding support Belgium, 1 items
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
DepositionAug 26, 2022-
Header (metadata) releaseApr 5, 2023-
Map releaseApr 5, 2023-
UpdateOct 23, 2024-
Current statusOct 23, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_15678.png

Downloads & links

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Map

FileDownload / File: emd_15678.map.gz / Format: CCP4 / Size: 42.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpenend cryo-EM map following non-uniform refinement
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.66 Å/pix.
x 224 pix.
= 371.392 Å		1.66 Å/pix.
x 224 pix.
= 371.392 Å		1.66 Å/pix.
x 224 pix.
= 371.392 Å

Surface

Projections

Slices (1/3)

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.658 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.178
Minimum - Maximum-0.38276014 - 1.9273546
Average (Standard dev.)0.00032553636 (±0.025291251)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions224224224
Spacing224224224
CellA=B=C: 371.392 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_15678_msk_1.map
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Additional map: Non-sharpened cryo-EM map following non-uniform refinement

Fileemd_15678_additional_1.map
AnnotationNon-sharpened cryo-EM map following non-uniform refinement
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Half map: Half map A

Fileemd_15678_half_map_1.map
AnnotationHalf map A
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Half map: Half map B

Fileemd_15678_half_map_2.map
AnnotationHalf map B
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Sample components

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Entire : Stucture of mouse leptin in complex with a trimerized form of the...

EntireName: Stucture of mouse leptin in complex with a trimerized form of the mouse Lep-R extracellular region
Components
  • Complex: Stucture of mouse leptin in complex with a trimerized form of the mouse Lep-R extracellular region
    • Protein or peptide: Leptin
    • Protein or peptide: Leptin receptor
  • Ligand: NICKEL (II) ION

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Supramolecule #1: Stucture of mouse leptin in complex with a trimerized form of the...

SupramoleculeName: Stucture of mouse leptin in complex with a trimerized form of the mouse Lep-R extracellular region
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Details: The mLEP-R ectodomain was C-terminally fused to a trimeric GCN4 isoleucine zipper tag and secreted from HEK93 FreeStyle cells and complex with refolded mouse leptin produced in E.coli.
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 444 KDa

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Macromolecule #1: Leptin

MacromoleculeName: Leptin / type: protein_or_peptide / ID: 1
Details: N-terminally His-tagged mouse leptin was refolded from inclusion bodies produced in E.coli.
Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 18.873283 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
MGSSHHHHHH PGGPGSENLY FQGGSTGGVP IQKVQDDTKT LIKTIVTRIN DISHTQSVSA KQRVTGLDFI PGLHPILSLS KMDQTLAVY QQVLTSLPSQ NVLQIANDLE NLRDLLHLLA FSKSCSLPQT SGLQKPESLD GVLEASLYST EVVALSRLQG S LQDILQQL DVSPEC

UniProtKB: Leptin

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Macromolecule #2: Leptin receptor

MacromoleculeName: Leptin receptor / type: protein_or_peptide / ID: 2 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 97.479391 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: LNLAYPISPW KFKLFCGPPN TTDDSFLSPA GAPNNASALK GASEAIVEAK FNSSGIYVPE LSKTVFHCCF GNEQGQNCSA LTDNTEGKT LASVVKASVF RQLGVNWDIE CWMKGDLTLF ICHMEPLPKN PFKNYDSKVH LLYDLPEVID DSPLPPLKDS F QTVQCNCS ...String:
LNLAYPISPW KFKLFCGPPN TTDDSFLSPA GAPNNASALK GASEAIVEAK FNSSGIYVPE LSKTVFHCCF GNEQGQNCSA LTDNTEGKT LASVVKASVF RQLGVNWDIE CWMKGDLTLF ICHMEPLPKN PFKNYDSKVH LLYDLPEVID DSPLPPLKDS F QTVQCNCS LRGCECHVPV PRAKLNYALL MYLEITSAGV SFQSPLMSLQ PMLVVKPDPP LGLHMEVTDD GNLKISWDSQ TM APFPLQY QVKYLENSTI VREAAEIVSA TSLLVDSVLP GSSYEVQVRS KRLDGSGVWS DWSSPQVFTT QDVVYFPPKI LTS VGSNAS FHCIYKNENQ IISSKQIVWW RNLAEKIPEI QYSIVSDRVS KVTFSNLKAT RPRGKFTYDA VYCCNEQACH HRYA ELYVI DVNINISCET DGYLTKMTCR WSPSTIQSLV GSTVQLRYHR RSLYCPDSPS IHPTSEPKNC VLQRDGFYEC VFQPI FLLS GYTMWIRINH SLGSLDSPPT CVLPDSVVKP LPPSNVKAEI TVNTGLLKVS WEKPVFPENN LQFQIRYGLS GKEIQW KTH EVFDAKSKSA SLLVSDLCAV YVVQVRCRRL DGLGYWSNWS SPAYTLVMDV KVPMRGPEFW RKMDGDVTKK ERNVTLL WK PLTKNDSLCS VRRYVVKHRT AHNGTWSEDV GNRTNLTFLW TEPAHTVTVL AVNSLGASLV NFNLTFSWPM SKVSAVES L SAYPLSSSCV ILSWTLSPDD YSLLYLVIEW KILNEDDGMK WLRIPSNVKK FYIHDNFIPI EKYQFSLYPV FMEGVGKPK IINGFTKDAI DKQQNDAGST GGSGGSGGSG GSGGSRMKQI EDKIEEILSK IYHIENEIAR IKKLIGER

UniProtKB: Leptin receptor

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Macromolecule #3: NICKEL (II) ION

MacromoleculeName: NICKEL (II) ION / type: ligand / ID: 3 / Number of copies: 3 / Formula: NI
Molecular weightTheoretical: 58.693 Da
Chemical component information

ChemComp-NI:
NICKEL (II) ION

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.3 mg/mL
BufferpH: 7.4
Component:
ConcentrationFormulaName
20.0 mMC8H18N2O4SHEPES
150.0 mMNaClsodium chloride

Details: 20 mM HEPES, 150 mM NaCl, pH 7.4
GridModel: C-flat-1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 99 % / Chamber temperature: 22 K / Instrument: LEICA EM GP
DetailsThis sample was monodisperse.

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Number real images: 13230 / Average electron dose: 45.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: OTHER / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.2 µm / Nominal magnification: 105000
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 141157
Details: Movies were processed via patch-based motion correction and CTF estimation as implemented in cryoSPARC v3.3.1. Initial 2D classes were obtained by the blob picker function in cryoSPARC, ...Details: Movies were processed via patch-based motion correction and CTF estimation as implemented in cryoSPARC v3.3.1. Initial 2D classes were obtained by the blob picker function in cryoSPARC, followed by particle picking via TOPAZ as implemented in cryoSPARC. Junk particles were removed by multiple rounds of 2D classification and ab initio 3D classification resulting in a particle set of 141,157 particles.
Startup modelType of model: INSILICO MODEL
In silico model: Hybrid model: crystallographic structure (pdb 7z3r) + Alphafold model mouse LEPR
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. v3.3.1) / Software - details: Non-uniform refinement / Number images used: 37530
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. v3.3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. v3.3.1)
Final 3D classificationNumber classes: 2 / Avg.num./class: 33128 / Software - Name: cryoSPARC (ver. v3.3.1) / Software - details: Ab initio
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-8avc:
Mouse leptin:LEP-R complex cryoEM structure (3:3 model)

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