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- PDB-8avb: Cryo-EM structure for mouse leptin in complex with the mouse LEP-... -
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Basic information
Entry | Database: PDB / ID: 8avb | ||||||
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Title | Cryo-EM structure for mouse leptin in complex with the mouse LEP-R ectodomain (1:2 mLEP:mLEPR model). | ||||||
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![]() | CYTOKINE / leptin / LEP-R / obesity / metabolism / energy balance | ||||||
Function / homology | ![]() negative regulation of metabolic process / negative regulation of locomotor rhythm / Synthesis, secretion, and deacylation of Ghrelin / negative regulation of eating behavior / 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 metabolic process / negative regulation of locomotor rhythm / Synthesis, secretion, and deacylation of Ghrelin / negative regulation of eating behavior / 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 / Synthesis, secretion, and inactivation of Glucagon-like Peptide-1 (GLP-1) / protein-hormone receptor activity / 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 / regulation of nitric-oxide synthase activity / bone mineralization involved in bone maturation / sexual reproduction / regulation of feeding behavior / regulation of lipid biosynthetic process / activation of protein kinase C activity / negative regulation of cartilage development / fatty acid catabolic process / ovulation from ovarian follicle / negative regulation of appetite / positive regulation of developmental growth / leukocyte tethering or rolling / energy reserve metabolic process / regulation of metabolic process / negative regulation of glucose import / prostaglandin secretion / bile acid metabolic process / tyrosine phosphorylation of STAT protein / cellular response to leptin stimulus / hormone metabolic process / regulation of protein localization to nucleus / cardiac muscle hypertrophy / aorta development / intestinal absorption / insulin secretion / regulation of fat cell differentiation / positive regulation of p38MAPK cascade / peptide hormone receptor binding / cytokine receptor activity / negative regulation of vasoconstriction / eating behavior / regulation of gluconeogenesis / glycogen metabolic process / cytokine binding / fatty acid beta-oxidation / central nervous system neuron development / regulation of cytokine production involved in inflammatory response / positive regulation of insulin secretion involved in cellular response to glucose stimulus / peptide hormone binding / regulation of insulin secretion / response to dietary excess / negative regulation of lipid storage / T cell differentiation / positive regulation of TOR signaling / response to vitamin E / glial cell proliferation / regulation of angiogenesis / adipose tissue development / negative regulation of gluconeogenesis / phagocytosis / energy homeostasis / cellular response to retinoic acid / positive regulation of insulin receptor signaling pathway / 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 / regulation of protein phosphorylation / response to insulin / placenta development Similarity search - Function | ||||||
Biological species | ![]() ![]() | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.43 Å | ||||||
![]() | Verstraete, K. / Savvides, S.N. / Verschueren, K.G. / 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
Structure viewer | Molecule: ![]() ![]() |
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PDBx/mmCIF format | ![]() | 314.5 KB | Display | ![]() |
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-Validation report
Summary document | ![]() | 1.1 MB | Display | ![]() |
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Full document | ![]() | 1.2 MB | Display | |
Data in XML | ![]() | 51.4 KB | Display | |
Data in CIF | ![]() | 75.2 KB | Display | |
Arichive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 15677MC ![]() 7z3pC ![]() 7z3qC ![]() 7z3rC ![]() 8av2C ![]() 8avcC ![]() 8avdC ![]() 8aveC ![]() 8avfC ![]() 8avoC ![]() 8b7qC C: citing same article ( M: map data used to model this data |
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Similar structure data | Similarity search - Function & homology ![]() |
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Links
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Assembly
Deposited unit | ![]()
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Components
#1: Protein | Mass: 16434.676 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Details: Mouse leptin was expressed with an N-terminal His-tag. Before complex formation with the mouse LEP-R ecotodomain, the His-tag was removed with TEV protease Source: (gene. exp.) ![]() ![]() ![]() ![]() |
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#2: Protein | Mass: 94081.344 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Details: The N-terminally His-tagged LEP-R ecotomain was secreted from HEK293 FreeStyle cells. The N-terminal His-tag was not removed before complex formation with refolded mouse leptin. Source: (gene. exp.) ![]() ![]() ![]() |
-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
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Sample preparation
Component | Name: Complex between mouse leptin and the mouse LEP-R ectodomain. Type: COMPLEX Details: The mouse leptin:LEP-R complex was isolated from the excess of mouse leptin via size-exclusion chromatography. The elution peak corresponding to the leptin:LEP-R was concentrated to 5 mg/mL, ...Details: The mouse leptin:LEP-R complex was isolated from the excess of mouse leptin via size-exclusion chromatography. The elution peak corresponding to the leptin:LEP-R was concentrated to 5 mg/mL, aliquoted and flash frozen into liquid nitrogen. Just before plunge freezing the sample was diluted to 0.2 mg/mL. Entity ID: all / Source: RECOMBINANT | |||||||||||||||
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Molecular weight | Value: 0.230 MDa / Experimental value: YES | |||||||||||||||
Source (natural) | Organism: ![]() ![]() | |||||||||||||||
Source (recombinant) | Organism: ![]() | |||||||||||||||
Buffer solution | pH: 7.4 / Details: 20 mM Hepes, 150 mM NaCl, pH 7.4 | |||||||||||||||
Buffer component |
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Specimen | Conc.: 0.2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES Details: The mouse leptin:LEP-R complex was isolated from the excess of mouse leptin via size-exclusion chromatography. The elution peak corresponding to the leptin:LEP-R was concentrated to 5 mg/mL, ...Details: The mouse leptin:LEP-R complex was isolated from the excess of mouse leptin via size-exclusion chromatography. The elution peak corresponding to the leptin:LEP-R was concentrated to 5 mg/mL, aliquoted and flash frozen into liquid nitrogen. Just before plunge freezing the sample was diluted to 0.2 mg/mL. | |||||||||||||||
Specimen support | Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R0.6/1 | |||||||||||||||
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 295 K |
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Electron microscopy imaging
Microscopy | Model: JEOL CRYO ARM 300 |
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Electron gun | Electron source: ![]() |
Electron lens | Mode: OTHER / Nominal defocus max: 3000 nm / Nominal defocus min: 800 nm |
Image recording | Electron dose: 62 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 7100 |
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Processing
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EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 28684 Details: Movies were motion corrected via MotionCor2 1.4.0 and had their contrast transfer functions (CTFs) determined via patch-based CTF estimation ain cryoSPARC v3.3.2. Initial high-resolution 2D ...Details: Movies were motion corrected via MotionCor2 1.4.0 and had their contrast transfer functions (CTFs) determined via patch-based CTF estimation ain cryoSPARC v3.3.2. Initial high-resolution 2D classes were obtained via the blob picker function and reference-free 2D classification in cryoSPARC, These 2D classes were then used to seed template-based and neural network-based particle picking via Topaz 0.2.4. Junk particles were removed by multiple rounds of 2D classification. High-resolution 2D classes corresponding to an apparent dimeric mLeptin:mLEP-R were manually selected. | ||||||||||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: C1 (asymmetric) | ||||||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 4.43 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 28296 / Num. of class averages: 1 / Symmetry type: POINT | ||||||||||||||||||||||||||||||||||||||||
Atomic model building | Protocol: FLEXIBLE FIT / Space: REAL Details: An atomic model for a 1:2 mLeptin:LEP-RIgCRH2FnIII complex was created based on the AlphaFold prediction for mLEP-RECD and the determined mLeptin:mLEP-RIgCRH2 and mLEP-RFnIII module crystal ...Details: An atomic model for a 1:2 mLeptin:LEP-RIgCRH2FnIII complex was created based on the AlphaFold prediction for mLEP-RECD and the determined mLeptin:mLEP-RIgCRH2 and mLEP-RFnIII module crystal structures and fitted in the cryo-EM map via Chimera followed by real space refinement in Phenix using rigid body refinement and coordinate refinement with reference restraints to the starting model and hydrogen-bonding restraints across the site II and site III mLeptin:mLEP-R interface regions. | ||||||||||||||||||||||||||||||||||||||||
Atomic model building | PDB-ID: 7Z3R | ||||||||||||||||||||||||||||||||||||||||
Refinement | Cross valid method: NONE Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2 | ||||||||||||||||||||||||||||||||||||||||
Displacement parameters | Biso mean: 509.52 Å2 | ||||||||||||||||||||||||||||||||||||||||
Refine LS restraints |
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