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- PDB-8avo: Human leptin in complex with the human LEP-R ectodomain fused to ... -

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Basic information

Entry
Database: PDB / ID: 8avo
TitleHuman leptin in complex with the human LEP-R ectodomain fused to a C-terminal trimeric isoleucine GCN4 zipper (open 3:3 model).
Components
  • Leptin
  • Leptin receptor
KeywordsCYTOKINE / leptin / LEP-R / obesity / metabolism / energy balance
Function / homology
Function and homology information


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
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 speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 6.84 Å
AuthorsVerstraete, K. / Savvides, S.N. / Verschueren, K.G. / Tsirigotaki, A.
Funding support Belgium, 1items
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, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Apr 5, 2023Provider: repository / Type: Initial release
Revision 1.1Apr 26, 2023Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Leptin
B: Leptin receptor
C: Leptin
D: Leptin receptor
E: Leptin
F: Leptin receptor


Theoretical massNumber of molelcules
Total (without water)352,2816
Polymers352,2816
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: light scattering, The molecular weight of the trimeric human leptin:LEP-R assembly was confirmed by SEC-MALLS
TypeNameSymmetry operationNumber
identity operation1_5551
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
d_1ens_1chain "C"
d_2ens_1chain "A"
d_3ens_1chain "E"
d_1ens_2chain "D"
d_2ens_2chain "B"
d_3ens_2chain "F"

NCS domain segments:
Dom-IDComponent-IDEns-IDBeg label comp-IDEnd label comp-IDLabel asym-IDLabel seq-ID
d_11ens_1VALCYSC1 - 146
d_21ens_1VALCYSA1 - 146
d_31ens_1VALCYSE1 - 146
d_11ens_2LYSASPD1 - 596
d_21ens_2LYSASPB1 - 596
d_31ens_2LYSASPF1 - 596

NCS ensembles :
ID
ens_1
ens_2

NCS oper:
IDCodeMatrixVector
1given(-0.225408661967, 0.967864623813, 0.111485447846), (-0.834322667314, -0.25085253545, 0.490895805915), (0.503087091781, 0.0176373305625, 0.864055728905)20.940539977, 328.882667524, -86.2307478692
2given(-0.719454982442, -0.501626489483, 0.480370058693), (0.636243234765, -0.753374651642, 0.166196210783), (0.278530203847, 0.425202891938, 0.861175607082)352.600913911, 200.612949574, -129.97252997
3given(-0.21737033937, 0.972514587742, 0.0834596440834), (-0.842949712712, -0.230142748675, 0.486281911109), (0.492123884201, 0.0353509810441, 0.869807099728)23.4815696262, 326.234827775, -89.0170779643
4given(-0.729837746741, -0.466909722557, 0.499331727825), (0.625627075433, -0.750603311896, 0.21256864928), (0.275549679581, 0.467536072568, 0.839929994065)342.228009652, 193.359351742, -137.541780645

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Components

#1: Protein Leptin / Obese protein / Obesity factor


Mass: 18605.061 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
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.
Source: (gene. exp.) Homo sapiens (human) / Gene: LEP, OB, OBS / Plasmid: pTwist / Details (production host): pTwist-N-His-hLEP / Cell line (production host): HEK293 / Production host: Homo sapiens (human) / Variant (production host): FreeStyle / References: UniProt: P41159
#2: Protein Leptin receptor / LEP-R / HuB219 / OB receptor / OB-R


Mass: 98822.062 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
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: (gene. exp.) Homo sapiens (human) / Gene: LEPR, DB, OBR / Plasmid: pTwist / Details (production host): pTwist-hLEPR-5xGGS-tGCN4 / Production host: Homo sapiens (human) / Strain (production host): Freestyle / References: UniProt: P48357

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Human leptin in complex with the human LEP-R ectodomain C-terminally fused to a trimeric GCN4 isoleucine zipper tag
Type: COMPLEX
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.
Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.350 MDa / Experimental value: YES
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human) / Strain: HEK293 FreeStyle / Plasmid: pTwist
Buffer solutionpH: 7.4 / Details: 20 mM HEPES, 150 mM NaCl, pH 7.4
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPESC8H18N2O4S1
2150 mMsodium chlorideNaCl1
SpecimenConc.: 0.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: This sample was monodisperse.
Specimen supportDetails: 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
Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R0.6/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 295 K

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Electron microscopy imaging

MicroscopyModel: JEOL CRYO ARM 300
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: OTHER / Nominal defocus max: 3000 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 62.4 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 13755

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Processing

Software
NameVersionClassificationNB
phenix.real_space_refine1.19.2_4158refinement
PHENIX1.19.2_4158refinement
EM software
IDNameVersionCategoryDetails
1cryoSPARCv3.1.0particle selectionTOPAZ, template picking
4cryoSPARCv3.1.0CTF correctionpatch CTF
7UCSF Chimera1.17model fitting
9PHENIX1.19.2model refinement
10cryoSPARCv3.1.0initial Euler assignmentAb initio
12cryoSPARCv3.1.0classificationAb initio
13cryoSPARCv3.1.0classificationHetero-refinement
14cryoSPARCv3.3.23D reconstructionNon-uniform refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles 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.
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 6.84 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 30353 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
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- ...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.
Atomic model building
IDPDB-ID 3D fitting-ID
17Z3R

7z3r

1
27Z3Q

7z3q

1
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 92.4 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.002418291
ELECTRON MICROSCOPYf_angle_d0.645924924
ELECTRON MICROSCOPYf_chiral_restr0.04672814
ELECTRON MICROSCOPYf_plane_restr0.00643117
ELECTRON MICROSCOPYf_dihedral_angle_d11.00776693
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDRefine-IDTypeRms dev position (Å)
ens_1d_2CELECTRON MICROSCOPYNCS constraints0.00071283147411
ens_1d_3CELECTRON MICROSCOPYNCS constraints0.0948332883736
ens_2d_2DELECTRON MICROSCOPYNCS constraints0.000712835216255
ens_2d_3DELECTRON MICROSCOPYNCS constraints0.000705046210652

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