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- EMDB-15677: Cryo-EM structure for mouse leptin in complex with the mouse LEP-... -

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Basic information

Entry
Database: EMDB / ID: EMD-15677
TitleCryo-EM structure for mouse leptin in complex with the mouse LEP-R ectodomain (1:2 mLEP:mLEPR model)
Map dataSharpened cryo-EM map following non-uniform refinement
Sample
  • Complex: Complex between mouse leptin and the mouse LEP-R ectodomain.
    • Protein or peptide: Leptin
    • Protein or peptide: Leptin receptor
Keywordsleptin / LEP-R / obesity / metabolism / energy balance / CYTOKINE
Function / homology
Function and homology information


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 / prostaglandin secretion / negative regulation of glucose import / 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 / cytokine receptor activity / peptide hormone receptor binding / 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 / negative regulation of autophagy / cholesterol metabolic process / response to activity / positive regulation of interleukin-8 production / gluconeogenesis / female pregnancy / determination of adult lifespan / positive regulation of receptor signaling pathway via JAK-STAT / regulation of protein phosphorylation / response to insulin / placenta development
Similarity search - Function
Leptin / Leptin / Leptin receptor, immunoglobulin-like domain / Obesity receptor immunoglobulin like domain / Immunoglobulin C2-set-like, ligand-binding / Ig-like C2-type domain / Short hematopoietin receptor, family 1, conserved site / 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 / Immunoglobulin C2-set-like, ligand-binding / Ig-like C2-type domain / Short hematopoietin receptor, family 1, conserved site / Long hematopoietin receptor, Gp130 family 2, conserved site / Long hematopoietin receptor, gp130 family signature. / Four-helical cytokine-like, core / 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: 4.43 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-
UpdateJul 26, 2023-
Current statusJul 26, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_15677.png

Downloads & links

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Map

FileDownload / File: emd_15677.map.gz / Format: CCP4 / Size: 40.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened cryo-EM map following non-uniform refinement
Voxel sizeX=Y=Z: 1.51 Å
Density
Contour LevelBy AUTHOR: 0.39
Minimum - Maximum-0.746262 - 2.2452404
Average (Standard dev.)-0.0006971231 (±0.0479572)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions220220220
Spacing220220220
CellA=B=C: 332.2 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_15677_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
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Histogram

Histogram (log scale)

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

Fileemd_15677_additional_1.map
AnnotationNon-sharpened cryo-EM map following non-uniform refinement
Projections & Slices
AxesZYX

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Half map: Half map A

Fileemd_15677_half_map_1.map
AnnotationHalf map A
Projections & Slices
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Half map: Half map B

Fileemd_15677_half_map_2.map
AnnotationHalf map B
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Histogram (log scale)

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Sample components

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Entire : Complex between mouse leptin and the mouse LEP-R ectodomain.

EntireName: Complex between mouse leptin and the mouse LEP-R ectodomain.
Components
  • Complex: Complex between mouse leptin and the mouse LEP-R ectodomain.
    • Protein or peptide: Leptin
    • Protein or peptide: Leptin receptor

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Supramolecule #1: Complex between mouse leptin and the mouse LEP-R ectodomain.

SupramoleculeName: Complex between mouse leptin and the mouse LEP-R ectodomain.
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
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.
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 230 KDa

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Macromolecule #1: Leptin

MacromoleculeName: Leptin / type: protein_or_peptide / ID: 1
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
Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 16.434676 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
GGSTGGVPIQ KVQDDTKTLI KTIVTRINDI SHTQSVSAKQ RVTGLDFIPG LHPILSLSKM DQTLAVYQQV LTSLPSQNVL QIANDLENL RDLLHLLAFS KSCSLPQTSG LQKPESLDGV LEASLYSTEV VALSRLQGSL QDILQQLDVS PEC

UniProtKB: Leptin

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Macromolecule #2: Leptin receptor

MacromoleculeName: Leptin receptor / type: protein_or_peptide / ID: 2
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.
Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 94.081344 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: AHHHHHHPGG PGSDELDLNL AYPISPWKFK LFCGPPNTTD DSFLSPAGAP NNASALKGAS EAIVEAKFNS SGIYVPELSK TVFHCCFGN EQGQNCSALT DNTEGKTLAS VVKASVFRQL GVNWDIECWM KGDLTLFICH MEPLPKNPFK NYDSKVHLLY D LPEVIDDS ...String:
AHHHHHHPGG PGSDELDLNL AYPISPWKFK LFCGPPNTTD DSFLSPAGAP NNASALKGAS EAIVEAKFNS SGIYVPELSK TVFHCCFGN EQGQNCSALT DNTEGKTLAS VVKASVFRQL GVNWDIECWM KGDLTLFICH MEPLPKNPFK NYDSKVHLLY D LPEVIDDS PLPPLKDSFQ TVQCNCSLRG CECHVPVPRA KLNYALLMYL EITSAGVSFQ SPLMSLQPML VVKPDPPLGL HM EVTDDGN LKISWDSQTM APFPLQYQVK YLENSTIVRE AAEIVSATSL LVDSVLPGSS YEVQVRSKRL DGSGVWSDWS SPQ VFTTQD VVYFPPKILT SVGSNASFHC IYKNENQIIS SKQIVWWRNL AEKIPEIQYS IVSDRVSKVT FSNLKATRPR GKFT YDAVY CCNEQACHHR YAELYVIDVN INISCETDGY LTKMTCRWSP STIQSLVGST VQLRYHRRSL YCPDSPSIHP TSEPK NCVL QRDGFYECVF QPIFLLSGYT MWIRINHSLG SLDSPPTCVL PDSVVKPLPP SNVKAEITVN TGLLKVSWEK PVFPEN NLQ FQIRYGLSGK EIQWKTHEVF DAKSKSASLL VSDLCAVYVV QVRCRRLDGL GYWSNWSSPA YTLVMDVKVP MRGPEFW RK MDGDVTKKER NVTLLWKPLT KNDSLCSVRR YVVKHRTAHN GTWSEDVGNR TNLTFLWTEP AHTVTVLAVN SLGASLVN F NLTFSWPMSK VSAVESLSAY PLSSSCVILS WTLSPDDYSL LYLVIEWKIL NEDDGMKWLR IPSNVKKFYI HDNFIPIEK YQFSLYPVFM EGVGKPKIIN GFTKDAIDKQ QNDAG

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

Concentration0.2 mg/mL
BufferpH: 7.4
Component:
ConcentrationFormulaName
150.0 mMNaClsodium chloride
20.0 mMC8H18N2O4SHepes

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
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 295 K / Instrument: FEI VITROBOT MARK IV
DetailsThe 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.

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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: 7100 / Average electron dose: 62.0 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: 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.
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

7z3r


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.
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.43 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. v3.3.2) / Software - details: Non-uniform refinement / Number images used: 28296
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. v3.3.2) / Software - details: Ab initio
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. v3.3.2) / Software - details: Non-uniform refinement
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

7z3r

DetailsAn 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.
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-8avb:
Cryo-EM structure for mouse leptin in complex with the mouse LEP-R ectodomain (1:2 mLEP:mLEPR model).

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