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

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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


Synthesis, secretion, and deacylation of Ghrelin / leptin receptor activity / 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 / negative regulation of appetite by leptin-mediated signaling pathway / Synthesis, secretion, and inactivation of Glucagon-like Peptide-1 (GLP-1) / negative regulation of glucagon secretion ...Synthesis, secretion, and deacylation of Ghrelin / leptin receptor activity / 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 / negative regulation of appetite by leptin-mediated signaling pathway / Synthesis, secretion, and inactivation of Glucagon-like Peptide-1 (GLP-1) / negative regulation of glucagon secretion / regulation of endothelial cell proliferation / regulation of natural killer cell proliferation / regulation of natural killer cell mediated cytotoxicity / leptin receptor binding / regulation of bone remodeling / positive regulation of luteinizing hormone secretion / bone growth / regulation of natural killer cell activation / glycerol biosynthetic process / regulation of steroid biosynthetic process / elastin metabolic process / leptin-mediated signaling pathway / positive regulation of follicle-stimulating hormone secretion / positive regulation of monoatomic ion transport / regulation of intestinal cholesterol absorption / positive regulation of hepatic stellate cell activation / regulation of feeding behavior / regulation of brown fat cell differentiation / positive regulation of peroxisome proliferator activated receptor signaling pathway / regulation of nitric-oxide synthase activity / adult feeding behavior / activation of protein kinase C activity / bone mineralization involved in bone maturation / regulation of lipid biosynthetic process / sexual reproduction / response to leptin / negative regulation of cartilage development / ovulation from ovarian follicle / negative regulation of D-glucose import / negative regulation of appetite / positive regulation of developmental growth / energy reserve metabolic process / leukocyte tethering or rolling / cellular response to leptin stimulus / bile acid metabolic process / prostaglandin secretion / cardiac muscle hypertrophy / hormone metabolic process / positive regulation of p38MAPK cascade / regulation of protein localization to nucleus / cell surface receptor signaling pathway via STAT / regulation of fat cell differentiation / intestinal absorption / regulation of metabolic process / eating behavior / insulin secretion / aorta development / regulation of gluconeogenesis / negative regulation of vasoconstriction / response to vitamin E / glycogen metabolic process / peptide hormone receptor binding / fatty acid beta-oxidation / regulation of cytokine production involved in inflammatory response / central nervous system neuron development / response to dietary excess / peptide hormone binding / T cell differentiation / negative regulation of lipid storage / positive regulation of TOR signaling / regulation of angiogenesis / cell surface receptor signaling pathway via JAK-STAT / negative regulation of gluconeogenesis / positive regulation of insulin receptor signaling pathway / adipose tissue development / phagocytosis / glial cell proliferation / cholesterol metabolic process / energy homeostasis / cellular response to retinoic acid / positive regulation of T cell proliferation / positive regulation of interleukin-12 production / regulation of insulin secretion / negative regulation of autophagy / placenta development / response to activity / gluconeogenesis / determination of adult lifespan / positive regulation of interleukin-8 production / positive regulation of receptor signaling pathway via JAK-STAT / female pregnancy / response to insulin / circadian rhythm / hormone activity / positive regulation of interleukin-6 production / positive regulation of protein import into nucleus / lipid metabolic process / regulation of blood pressure / glucose metabolic process / cellular response to insulin stimulus
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: 4.43 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-
UpdateOct 16, 2024-
Current statusOct 16, 2024Processing 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
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.51 Å/pix.
x 220 pix.
= 332.2 Å		1.51 Å/pix.
x 220 pix.
= 332.2 Å		1.51 Å/pix.
x 220 pix.
= 332.2 Å

Surface

Projections

Slices (1/3)

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.51 Å
Density

Histogram

Histogram (log scale)
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
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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
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Half map: Half map A

Fileemd_15677_half_map_1.map
AnnotationHalf map A
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Half map: Half map B

Fileemd_15677_half_map_2.map
AnnotationHalf map B
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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


Chain - Source name: PDB / Chain - Initial model type: experimental model
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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