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- EMDB-15683: Human leptin in complex with the human LEP-R ectodomain fused to ... -

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Entry
Database: EMDB / ID: EMD-15683
TitleHuman leptin in complex with the human LEP-R ectodomain fused to a C-terminal trimeric isoleucine GCN4 zipper (open 3:3 model).
Map dataSharpened cryo-EM map following non-uniform refinement in cryoSPARC
Sample
  • Complex: Human leptin in complex with the human LEP-R ectodomain C-terminally fused to a trimeric GCN4 isoleucine zipper tag
    • Protein or peptide: Leptin
    • Protein or peptide: Leptin receptor
Keywordsleptin / LEP-R / obesity / metabolism / energy balance / CYTOKINE
Function / homology
Function and homology information


regulation of transport / multicellular organism development / 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 / negative regulation of appetite by leptin-mediated signaling pathway / negative regulation of glucagon secretion / regulation of endothelial cell proliferation ...regulation of transport / multicellular organism development / 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 / negative regulation of appetite by leptin-mediated signaling pathway / negative regulation of glucagon secretion / regulation of endothelial cell proliferation / regulation of natural killer cell proliferation / leptin receptor binding / regulation of natural killer cell mediated cytotoxicity / bone growth / positive regulation of luteinizing hormone secretion / regulation of natural killer cell activation / glycerol biosynthetic process / positive regulation of monoatomic ion transport / elastin metabolic process / leptin-mediated signaling pathway / positive regulation of follicle-stimulating hormone secretion / regulation of steroid biosynthetic process / regulation of intestinal cholesterol absorption / regulation of bone remodeling / regulation of brown fat cell differentiation / positive regulation of peroxisome proliferator activated receptor signaling pathway / positive regulation of hepatic stellate cell activation / adult feeding behavior / regulation of nitric-oxide synthase activity / response to leptin / bone mineralization involved in bone maturation / sexual reproduction / regulation of feeding behavior / activation of protein kinase C activity / negative regulation of cartilage development / 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 / negative regulation of D-glucose import / cellular response to leptin stimulus / prostaglandin secretion / cardiac muscle hypertrophy / hormone metabolic process / Signaling by Leptin / aorta development / intestinal absorption / insulin secretion / cytokine receptor activity / positive regulation of p38MAPK cascade / negative regulation of vasoconstriction / eating behavior / peptide hormone receptor binding / regulation of gluconeogenesis / glycogen metabolic process / fatty acid beta-oxidation / regulation of cytokine production involved in inflammatory response / central nervous system neuron development / cytokine binding / response to dietary excess / negative regulation of lipid storage / T cell differentiation / transport across blood-brain barrier / positive regulation of TOR signaling / Synthesis, secretion, and deacylation of Ghrelin / response to vitamin E / glial cell proliferation / negative regulation of gluconeogenesis / adipose tissue development / regulation of angiogenesis / phagocytosis / positive regulation of insulin receptor signaling pathway / positive regulation of tyrosine phosphorylation of STAT protein / positive regulation of T cell proliferation / energy homeostasis / cellular response to retinoic acid / positive regulation of interleukin-12 production / regulation of insulin secretion / cholesterol metabolic process / negative regulation of autophagy / response to activity / gluconeogenesis / positive regulation of interleukin-8 production / female pregnancy / determination of adult lifespan / positive regulation of receptor signaling pathway via JAK-STAT / response to insulin / placenta development / lipid metabolic process / hormone activity / cytokine-mediated signaling pathway / regulation of blood pressure / Transcriptional regulation of white adipocyte differentiation / Synthesis, secretion, and inactivation of Glucagon-like Peptide-1 (GLP-1) / positive regulation of protein import into nucleus / positive regulation of interleukin-6 production / circadian rhythm / cellular response to insulin stimulus
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. / : ...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 speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.84 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 16, 2024-
Current statusOct 16, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_15683.png

Downloads & links

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Map

FileDownload / File: emd_15683.map.gz / Format: CCP4 / Size: 83.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened cryo-EM map following non-uniform refinement in cryoSPARC
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
1.52 Å/pix.
x 280 pix.
= 425.6 Å		1.52 Å/pix.
x 280 pix.
= 425.6 Å		1.52 Å/pix.
x 280 pix.
= 425.6 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.52 Å
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Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.408
Minimum - Maximum-1.5484812 - 3.4875438
Average (Standard dev.)0.00045017494 (±0.056957077)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions280280280
Spacing280280280
CellA=B=C: 425.6 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_15683_msk_1.map
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Additional map: Non-sharpened cryo-EM map following non-uniform refinement in cryoSPARC...

Fileemd_15683_additional_1.map
AnnotationNon-sharpened cryo-EM map following non-uniform refinement in cryoSPARC
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Half map: Half map B

Fileemd_15683_half_map_1.map
AnnotationHalf map B
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Half map: Half map A

Fileemd_15683_half_map_2.map
AnnotationHalf map A
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Sample components

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Entire : Human leptin in complex with the human LEP-R ectodomain C-termina...

EntireName: Human leptin in complex with the human LEP-R ectodomain C-terminally fused to a trimeric GCN4 isoleucine zipper tag
Components
  • Complex: Human leptin in complex with the human LEP-R ectodomain C-terminally fused to a trimeric GCN4 isoleucine zipper tag
    • Protein or peptide: Leptin
    • Protein or peptide: Leptin receptor

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Supramolecule #1: Human leptin in complex with the human LEP-R ectodomain C-termina...

SupramoleculeName: Human leptin in complex with the human LEP-R ectodomain C-terminally fused to a trimeric GCN4 isoleucine zipper tag
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Human leptin carrying an N-terminal His-tag and the human LEP-R ectodomain C-terminally fused to a trimeric GCN4 isoleucine zipper tag (without His-tag) were co-expressed in HEK293 FreeStyle ...Details: Human leptin carrying an N-terminal His-tag and the human LEP-R ectodomain C-terminally fused to a trimeric GCN4 isoleucine zipper tag (without His-tag) were co-expressed in HEK293 FreeStyle cells in the presence of kifunensine. The resulting complex was purified from the conditioned medium by IMAC and SEC.
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 350 KDa

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Macromolecule #1: Leptin

MacromoleculeName: Leptin / type: protein_or_peptide / ID: 1
Details: N-terminally His-tagged human leptin was co-expressed with the human LEP-R ectodomain fused a trimeric GCN4 isoleucine zipper tag in HEK293 FreeStyle cells.
Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 18.605061 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString:
AHHHHHHPGG PGSENLYFQG GSTGGVPIQK VQDDTKTLIK TIVTRINDIS HTQSVSSKQK VTGLDFIPGL HPILTLSKMD QTLAVYQQI LTSMPSRNVI QISNDLENLR DLLHVLAFSK SCHLPWASGL ETLDSLGGVL EASGYSTEVV ALSRLQGSLQ D MLWQLDLS PGC

UniProtKB: Leptin

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Macromolecule #2: Leptin receptor

MacromoleculeName: Leptin receptor / type: protein_or_peptide / ID: 2
Details: Human leptin carrying an N-terminal His-tag and the human LEP-R ectodomain C-terminally fused to a trimeric GCN4 isoleucine zipper tag (without His-tag) were co-expressed in HEK293 FreeStyle ...Details: Human leptin carrying an N-terminal His-tag and the human LEP-R ectodomain C-terminally fused to a trimeric GCN4 isoleucine zipper tag (without His-tag) were co-expressed in HEK293 FreeStyle cells in the presence of kifunensine. The resulting complex was purified from the conditioned medium by IMAC and SEC.
Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 98.822062 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: FNLSYPITPW RFKLSCMPPN STYDYFLLPA GLSKNTSNSN GHYETAVEPK FNSSGTHFSN LSKTTFHCCF RSEQDRNCSL CADNIEGKT FVSTVNSLVF QQIDANWNIQ CWLKGDLKLF ICYVESLFKN LFRNYNYKVH LLYVLPEVLE DSPLVPQKGS F QMVHCNCS ...String:
FNLSYPITPW RFKLSCMPPN STYDYFLLPA GLSKNTSNSN GHYETAVEPK FNSSGTHFSN LSKTTFHCCF RSEQDRNCSL CADNIEGKT FVSTVNSLVF QQIDANWNIQ CWLKGDLKLF ICYVESLFKN LFRNYNYKVH LLYVLPEVLE DSPLVPQKGS F QMVHCNCS VHECCECLVP VPTAKLNDTL LMCLKITSGG VIFQSPLMSV QPINMVKPDP PLGLHMEITD DGNLKISWSS PP LVPFPLQ YQVKYSENST TVIREADKIV SATSLLVDSI LPGSSYEVQV RGKRLDGPGI WSDWSTPRVF TTQDVIYFPP KIL TSVGSN VSFHCIYKKE NKIVPSKEIV WWMNLAEKIP QSQYDVVSDH VSKVTFFNLN ETKPRGKFTY DAVYCCNEHE CHHR YAELY VIDVNINISC ETDGYLTKMT CRWSTSTIQS LAESTLQLRY HRSSLYCSDI PSIHPISEPK DCYLQSDGFY ECIFQ PIFL LSGYTMWIRI NHSLGSLDSP PTCVLPDSVV KPLPPSSVKA EITINIGLLK ISWEKPVFPE NNLQFQIRYG LSGKEV QWK MYEVYDAKSK SVSLPVPDLC AVYAVQVRCK RLDGLGYWSN WSNPAYTVVM DIKVPMRGPE FWRIINGDTM KKEKNVT LL WKPLMKNDSL CSVQRYVINH HTSCNGTWSE DVGNHTKFTF LWTEQAHTVT VLAINSIGAS VANFNLTFSW PMSKVNIV Q SLSAYPLNSS CVIVSWILSP SDYKLMYFII EWKNLNEDGE IKWLRISSSV KKYYIHDHFI PIEKYQFSLY PIFMEGVGK PKIINSFTQD DIEKHQSDST GGSGGSGGSG GSGGSRMKQI EDKIEEILSK IYHIENEIAR IKKLIGER

UniProtKB: Leptin receptor

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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: Quantifoil R0.6/1 / Material: GOLD / Mesh: 300 / Support film - Material: GRAPHENE / Support film - topology: CONTINUOUS / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 1 sec. / Pretreatment - Atmosphere: AIR
Details: 4 microliter of hLeptin:hLEP-RECD-tGCN4 complex at 0.3 mg.mL-1 was applied to a glow-discharged Quantifoil R 0.6/1 300 mesh golden grid coated with graphene (PUXANO), blotted for 1 s (blot ...Details: 4 microliter of hLeptin:hLEP-RECD-tGCN4 complex at 0.3 mg.mL-1 was applied to a glow-discharged Quantifoil R 0.6/1 300 mesh golden grid coated with graphene (PUXANO), blotted for 1 s (blot force = 1) under 100% humidity at 295K and plunged into liquid ethane using an FEI Vitribot Mark IV
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 295 K / Instrument: FEI VITROBOT MARK IV
DetailsThis sample was monodisperse.

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Electron microscopy

MicroscopeJEOL CRYO ARM 300
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 13755 / Average electron dose: 62.4 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: OTHER / Nominal defocus max: 3.0 µm / Nominal defocus min: 0.8 µm

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Image processing

Particle selectionNumber selected: 206218
Details: Initial 2D-classes were obtained via template-based particle picking using 2D-classes for a mLeptin:mLEP-R-deltaFnIII-tGCN4 complex lowpass filtered to 20 Angstrom. These 2D classes were ...Details: Initial 2D-classes were obtained via template-based particle picking using 2D-classes for a mLeptin:mLEP-R-deltaFnIII-tGCN4 complex lowpass filtered to 20 Angstrom. These 2D classes were then used to seed template-based picking and neural network-based particle picking via Topaz 0.2.4. Junk particles were removed by multiple rounds of 2D classification resulting in a particle set of 206,218 particles. High-resolution 2D classes were selected for ab initio 3D classification followed by heterogeneous refinement and non-uniform refinement.
Startup modelType of model: INSILICO MODEL
In silico model: A hybrid model for the 3:3 hLeptin:hLEP-RECD complex was created based on the AlphaFold predictions for hLEP-RECD and hLeptin, and the determined crystal structures for the mouse 3:3 ...In silico model: A hybrid model for the 3:3 hLeptin:hLEP-RECD complex was created based on the AlphaFold predictions for hLEP-RECD and hLeptin, and the determined crystal structures for the mouse 3:3 Leptin:LEP-RIgCRH2 complex (pdb 7z3r) and human Leptin:LEP-RCRH2 complex (pdb 7z3q)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 6.84 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. v3.3.2) / Software - details: Non-uniform refinement / Number images used: 30353
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. v3.1.0) / Software - details: Ab initio
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final 3D classificationNumber classes: 5 / Avg.num./class: 20028
Software:
Namedetails
cryoSPARC (ver. v3.1.0)Ab initio
cryoSPARC (ver. v3.1.0)Hetero-refinement

Details: High-resolution 2D classes were selected for ab initio 3D classification followed by heterogeneous refinement and non-uniform refinement which resulted in a cryo-EM map that showed well- ...Details: High-resolution 2D classes were selected for ab initio 3D classification followed by heterogeneous refinement and non-uniform refinement which resulted in a cryo-EM map that showed well-defined density for a 2:2 hLeptin:hLEP-RECD subcomplex (100,139 particles, FSC0.143 = 4.94 Angstrom). This particle set was further subclassified via ab initio 3D classification (5 classes) and heterogeneous refinement followed by non-uniform refinement which resulted in cryo-EM maps representing open (31,968 particles, FSC0.143 = 6.56 Angstrom) and closed (31,432 particles, FSC0.143 = 6.06 Angstrom) states of the 3:3 hLeptin:hLEP-RECD complex.
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChain

7z3r

source_name: PDB, initial_model_type: experimental model

7z3q

source_name: PDB, initial_model_type: experimental model
DetailsAtomic models for the 2:2 and 3:3 hLeptin:hLEP-R complexes were created based on the Alphafold2 predictions for hLEP-R and hLeptin, and the crystal structures for the mouse 3:3 Leptin:LEP-RIgCRH2 complex (pdb 7z3r) and the human Leptin:LEP-RCRH2 complex (pdb 7z3q) and fitted in the cryo-EM maps via Chimera. Atomic models were further refined via real space refinement in Phenix using rigid body refinement and coordinate refinement with reference restraints to the starting model.
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-8avo:
Human leptin in complex with the human LEP-R ectodomain fused to a C-terminal trimeric isoleucine GCN4 zipper (open 3:3 model).

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