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- EMDB-15681: Human leptin in complex with the human LEP-R ectodomain fused to ... -
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Open data
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Basic information
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Title | Human leptin in complex with the human LEP-R ectodomain fused to a C-terminal trimeric isoleucine GCN4 zipper (closed 3:3 model) | |||||||||
![]() | Sharpened cryo-EM map followin non-uniform refinement. | |||||||||
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Function / homology | ![]() regulation of transport / 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 ...regulation of transport / 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 / positive regulation of luteinizing hormone secretion / regulation of natural killer cell mediated cytotoxicity / bone growth / regulation of natural killer cell activation / positive regulation of monoatomic ion transport / glycerol biosynthetic process / 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 / adult feeding behavior / positive regulation of hepatic stellate cell activation / 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 / multicellular organism development / ovulation from ovarian follicle / negative regulation of appetite / positive regulation of developmental growth / leukocyte tethering or rolling / energy reserve metabolic process / prostaglandin secretion / negative regulation of glucose import / bile acid metabolic process / cellular response to leptin stimulus / hormone metabolic process / cardiac muscle hypertrophy / Signaling by Leptin / intestinal absorption / insulin secretion / aorta development / positive regulation of p38MAPK cascade / cytokine receptor activity / peptide hormone receptor binding / negative regulation of vasoconstriction / eating behavior / regulation of gluconeogenesis / regulation of nitric-oxide synthase activity / glycogen metabolic process / cytokine binding / central nervous system neuron development / fatty acid beta-oxidation / regulation of cytokine production involved in inflammatory response / regulation of insulin secretion / response to dietary excess / negative regulation of lipid storage / transport across blood-brain barrier / T cell differentiation / positive regulation of TOR signaling / response to vitamin E / Synthesis, secretion, and deacylation of Ghrelin / glial cell proliferation / regulation of angiogenesis / adipose tissue development / negative regulation of gluconeogenesis / phagocytosis / energy homeostasis / positive regulation of insulin receptor signaling pathway / cellular response to retinoic acid / positive regulation of T cell proliferation / positive regulation of tyrosine phosphorylation of STAT protein / positive regulation of interleukin-12 production / cholesterol metabolic process / negative regulation of autophagy / response to activity / positive regulation of interleukin-8 production / gluconeogenesis / determination of adult lifespan / female pregnancy / positive regulation of receptor signaling pathway via JAK-STAT / placenta development / response to insulin / lipid metabolic process / hormone activity / Synthesis, secretion, and inactivation of Glucagon-like Peptide-1 (GLP-1) / regulation of blood pressure / cytokine-mediated signaling pathway / Transcriptional regulation of white adipocyte differentiation / positive regulation of protein import into nucleus / circadian rhythm / cellular response to insulin stimulus / transmembrane signaling receptor activity Similarity search - Function | |||||||||
Biological species | ![]() | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 6.45 Å | |||||||||
![]() | Verstraete K / Savvides SN / Verschueren KG / Tsirigotaki A | |||||||||
Funding support | ![]()
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![]() | ![]() 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 |
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Structure visualization
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 79.1 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 25.4 KB 25.4 KB | Display Display | ![]() |
FSC (resolution estimation) | ![]() | 9.3 KB | Display | ![]() |
Images | ![]() | 88.7 KB | ||
Masks | ![]() | 83.7 MB | ![]() | |
Others | ![]() ![]() ![]() | 41.4 MB 77.7 MB 77.7 MB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Validation report
Summary document | ![]() | 893.2 KB | Display | ![]() |
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Full document | ![]() | 892.8 KB | Display | |
Data in XML | ![]() | 17.4 KB | Display | |
Data in CIF | ![]() | 22.3 KB | Display | |
Arichive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 8avfMC ![]() 7z3pC ![]() 7z3qC ![]() 7z3rC ![]() 8av2C ![]() 8avbC ![]() 8avcC ![]() 8avdC ![]() 8aveC ![]() 8avoC ![]() 8b7qC M: atomic model generated by this map C: citing same article ( |
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Similar structure data | Similarity search - Function & homology ![]() |
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Links
EMDB pages | ![]() ![]() |
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Related items in Molecule of the Month |
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Map
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Annotation | Sharpened cryo-EM map followin non-uniform refinement. | ||||||||||||||||||||
Voxel size | X=Y=Z: 1.52 Å | ||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Mask #1
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Density Histograms |
-Additional map: Non-sharpened cryo-EM map followin non-uniform refinement
File | emd_15681_additional_1.map | ||||||||||||
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Annotation | Non-sharpened cryo-EM map followin non-uniform refinement | ||||||||||||
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Density Histograms |
-Half map: Half map A
File | emd_15681_half_map_1.map | ||||||||||||
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Annotation | Half map A | ||||||||||||
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Density Histograms |
-Half map: Half map B
File | emd_15681_half_map_2.map | ||||||||||||
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Annotation | Half map B | ||||||||||||
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Density Histograms |
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Sample components
-Entire : Human leptin in complex with the human LEP-R ectodomain C-termina...
Entire | Name: Human leptin in complex with the human LEP-R ectodomain C-terminally fused to a trimeric GCN4 isoleucine zipper tag |
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Components |
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-Supramolecule #1: Human leptin in complex with the human LEP-R ectodomain C-termina...
Supramolecule | Name: Human leptin in complex with the human LEP-R ectodomain C-terminally fused to a trimeric GCN4 isoleucine zipper tag type: complex / ID: 1 / Chimera: Yes / 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. |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 350 KDa |
-Macromolecule #1: Leptin
Macromolecule | Name: 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 |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 18.605061 KDa |
Recombinant expression | Organism: ![]() |
Sequence | String: AHHHHHHPGG PGSENLYFQG GSTGGVPIQK VQDDTKTLIK TIVTRINDIS HTQSVSSKQK VTGLDFIPGL HPILTLSKMD QTLAVYQQI LTSMPSRNVI QISNDLENLR DLLHVLAFSK SCHLPWASGL ETLDSLGGVL EASGYSTEVV ALSRLQGSLQ D MLWQLDLS PGC |
-Macromolecule #2: Leptin receptor
Macromolecule | Name: 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 |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 98.822062 KDa |
Recombinant expression | Organism: ![]() |
Sequence | String: 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 |
-Experimental details
-Structure determination
Method | cryo EM |
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![]() | single particle reconstruction |
Aggregation state | particle |
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Sample preparation
Concentration | 0.3 mg/mL | |||||||||
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Buffer | pH: 7.4 Component:
Details: 20 mM HEPES, 150 mM NaCl, pH 7.4 | |||||||||
Grid | Model: 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 | |||||||||
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 295 K / Instrument: FEI VITROBOT MARK IV | |||||||||
Details | This sample was monodisperse. |
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Electron microscopy
Microscope | JEOL CRYO ARM 300 |
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Image recording | Film or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 13755 / Average electron dose: 62.4 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: ![]() |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: OTHER / Nominal defocus max: 3.0 µm / Nominal defocus min: 0.8 µm |
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Image processing
-Atomic model buiding 1
Initial model | (PDB ID: , ) |
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Details | Atomic 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. |
Refinement | Space: REAL / Protocol: FLEXIBLE FIT |
Output model | ![]() PDB-8avf: |