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- PDB-7z3r: Crystal structure of the mouse leptin:LepR-IgCRH2 complex to 2.95... -

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Basic information

Entry
Database: PDB / ID: 7z3r
TitleCrystal structure of the mouse leptin:LepR-IgCRH2 complex to 2.95 A resolution.
Components
  • Leptin receptor
  • Leptin
KeywordsCYTOKINE / leptin / obesity / leptin receptor / LepR / adipose tissue / hypothalamus / immune system
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 / regulation of transport ...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 / regulation of transport / 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 feeding behavior / bone mineralization involved in bone maturation / sexual reproduction / activation of protein kinase C activity / regulation of lipid biosynthetic process / 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 / bile acid metabolic process / negative regulation of glucose import / tyrosine phosphorylation of STAT protein / cellular response to leptin stimulus / hormone metabolic process / cardiac muscle hypertrophy / regulation of protein localization to nucleus / insulin secretion / cytokine receptor activity / intestinal absorption / aorta development / regulation of fat cell differentiation / positive regulation of p38MAPK cascade / peptide hormone receptor binding / negative regulation of vasoconstriction / regulation of nitric-oxide synthase activity / regulation of gluconeogenesis / eating behavior / fatty acid beta-oxidation / glycogen metabolic process / cytokine binding / 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 / response to dietary excess / regulation of insulin secretion / negative regulation of lipid storage / T cell differentiation / positive regulation of TOR signaling / response to vitamin E / glial cell proliferation / adipose tissue development / regulation of angiogenesis / negative regulation of gluconeogenesis / phagocytosis / energy homeostasis / positive regulation of insulin receptor signaling pathway / cellular response to retinoic acid / positive regulation of tyrosine phosphorylation of STAT protein / positive regulation of T cell proliferation / positive regulation of interleukin-12 production / cholesterol metabolic process / negative regulation of autophagy / response to activity / placenta development / female pregnancy / gluconeogenesis / determination of adult lifespan / positive regulation of interleukin-8 production / regulation of protein phosphorylation / positive regulation of receptor signaling pathway via JAK-STAT
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)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.951 Å
AuthorsVerstraete, K. / Verschueren, K. / Savvides, S.N. / 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
DepositionMar 2, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Mar 22, 2023Provider: repository / Type: Initial release
Revision 1.1Apr 5, 2023Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Apr 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
Revision 1.3Feb 7, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Leptin
B: Leptin receptor
hetero molecules


Theoretical massNumber of molelcules
Total (without water)53,8713
Polymers53,6502
Non-polymers2211
Water1267
1
A: Leptin
B: Leptin receptor
hetero molecules

A: Leptin
B: Leptin receptor
hetero molecules

A: Leptin
B: Leptin receptor
hetero molecules


Theoretical massNumber of molelcules
Total (without water)161,6149
Polymers160,9506
Non-polymers6643
Water543
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_445-y-1,x-y-1,z1
crystal symmetry operation3_545-x+y,-x-1,z1
Buried area10130 Å2
ΔGint-44 kcal/mol
Surface area66370 Å2
MethodPISA
Unit cell
Length a, b, c (Å)132.678, 132.678, 249.191
Angle α, β, γ (deg.)90, 90, 120
Int Tables number155
Space group name H-MH32

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Components

#1: Protein Leptin / / Obesity factor


Mass: 16220.559 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Mouse leptin (Uniprot ID P41160) was refolded from inclusion bodies produced in E.coli.
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Lep, Ob / Plasmid: pET11d / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P41160
#2: Protein Leptin receptor / / LEP-R / B219 / OB receptor / OB-R


Mass: 37429.477 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: The mouse LepR-IgCRH2 fragment was produced in HEK293 Freestyle cells in the presence of kifunensine. Prior to crystallisation, the N-terminal His-tag was removed with TEV protease and the N- ...Details: The mouse LepR-IgCRH2 fragment was produced in HEK293 Freestyle cells in the presence of kifunensine. Prior to crystallisation, the N-terminal His-tag was removed with TEV protease and the N-terminal glycans were trimmed with EndoH.
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Lepr, Db, Obr / Plasmid: pTwist CMV BetaGlobin / Production host: Homo sapiens (human) / Strain (production host): FreeStyle / References: UniProt: P48356
#3: Sugar ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE / N-Acetylglucosamine


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0
#4: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 7 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestN

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 3.93 Å3/Da / Density % sol: 68.73 %
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop / pH: 7.5
Details: Proplex HT #G7 3.0 M formate 0.1 M Tris pH 7.5 cryo: 4 M Na-formate + 10% ethylene glycol
Temp details: Temperature-controlled cabinet

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Data collection

DiffractionMean temperature: 100 K / Ambient temp details: N2 cryo-stream / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: PETRA III, EMBL c/o DESY / Beamline: P14 (MX2) / Wavelength: 0.9762 Å
DetectorType: DECTRIS EIGER2 X 16M / Detector: PIXEL / Date: Oct 31, 2020
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9762 Å / Relative weight: 1
ReflectionResolution: 2.95→83.06 Å / Num. obs: 18075 / % possible obs: 100 % / Redundancy: 20 % / Biso Wilson estimate: 95.3 Å2 / CC1/2: 0.999 / Rrim(I) all: 0.151 / Net I/σ(I): 17.7
Reflection shellResolution: 2.95→3.13 Å / Redundancy: 20.6 % / Mean I/σ(I) obs: 1.1 / Num. unique obs: 2876 / CC1/2: 0.417 / Rrim(I) all: 3.4 / % possible all: 100

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Processing

Software
NameVersionClassification
BUSTER2.10.4 (3-FEB-2022)refinement
XDSversiondata reduction
Aimless0.7.3data scaling
PHASER2.7.0phasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 1ax8

1ax8


Resolution: 2.951→66.34 Å / Cor.coef. Fo:Fc: 0.943 / Cor.coef. Fo:Fc free: 0.927 / SU R Cruickshank DPI: 0.633 / Cross valid method: THROUGHOUT / SU R Blow DPI: 0.597 / SU Rfree Blow DPI: 0.32 / SU Rfree Cruickshank DPI: 0.328
RfactorNum. reflection% reflectionSelection details
Rfree0.2512 1194 -RANDOM
Rwork0.2227 ---
obs0.2245 18073 100 %-
Displacement parametersBiso mean: 111.92 Å2
Baniso -1Baniso -2Baniso -3
1--0.7229 Å20 Å20 Å2
2---0.7229 Å20 Å2
3---1.4458 Å2
Refine analyzeLuzzati coordinate error obs: 0.42 Å
Refinement stepCycle: LAST / Resolution: 2.951→66.34 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3542 0 14 7 3563
Refine LS restraints
Refine-IDTypeDev idealNumberRestraint functionWeight
X-RAY DIFFRACTIONt_bond_d0.0063642HARMONIC2
X-RAY DIFFRACTIONt_angle_deg0.864965HARMONIC2
X-RAY DIFFRACTIONt_dihedral_angle_d1252SINUSOIDAL2
X-RAY DIFFRACTIONt_gen_planes602HARMONIC5
X-RAY DIFFRACTIONt_it3641HARMONIC10
X-RAY DIFFRACTIONt_chiral_improper_torsion489SEMIHARMONIC5
X-RAY DIFFRACTIONt_ideal_dist_contact2440SEMIHARMONIC4
X-RAY DIFFRACTIONt_omega_torsion2.71
X-RAY DIFFRACTIONt_other_torsion17.26
LS refinement shellResolution: 2.951→2.97 Å
RfactorNum. reflection% reflection
Rfree0.4504 22 -
Rwork0.3412 --
obs--100 %
Refinement TLS params.

Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
114.08940.4457-6.01130.1321-0.1456.6770.5409-0.0221-0.5796-0.0221-0.1727-0.171-0.5796-0.171-0.3682-0.03340.03370.0695-0.11180.0512-0.0639-12.4881-32.6682-16.2672
217.51414.2912.1306-0.88337.95147.46790.35151.0297-1.36381.02970.41940.4695-1.36380.4695-0.77090.33110.09940.19150.19120.17740.26688.3523-32.2483-19.5645
311.8152-8.6011-2.13068.3044-7.951400.31250.7442-1.20590.74420.4669-0.0884-1.2059-0.0884-0.77940.01340.0120.13920.15580.36690.09857.8233-25.771-31.5933
411.58992.91042.910421.6719-5.0410-0.0917-0.8493-0.1458-0.84930.14351.1727-0.14581.1727-0.05180.45860.03280.26450.69560.14980.6079-4.1463-19.0631-27.4411
521.3174-3.115-6.396911.4718-0.561813.24490.4275-0.7523-0.4615-0.75230.0393-0.0787-0.4615-0.0787-0.4668-0.31980.01250.02990.12760.3422-0.1262-5.2425-31.7192-31.1504
66.11851.4686-3.7951.70443.77826.86380.3603-0.62590.349-0.6259-0.0306-0.68470.349-0.6847-0.3297-0.12610.07750.0544-0.01760.2448-0.134-13.2246-34.26-25.7863
74.9002-5.83330.94545.5064-2.681313.4770.1112-0.9055-1.2471-0.9055-0.1708-0.0169-1.2471-0.01690.05960.8712-0.152-0.17680.34470.08760.5754-10.5361-26.971-37.4564
810.52248.3276-2.51446.1085-5.808416.63090.2201-0.6711.4869-0.671-0.2619-0.39841.4869-0.39840.04180.0296-0.0299-0.0110.7662-0.1407-0.21358.48-36.8452-38.288
912.06671.1459-3.1554-2.6991-0.32061.65990.62790.0326-0.55320.03260.13060.5757-0.55320.5757-0.7584-0.018-0.01630.155-0.0690.1450.07670.5189-29.3569-23.2256
1015.5051.9501-5.217513.2533-2.60474.2537-0.5016-0.20481.0414-0.20481.2975-0.30941.0414-0.3094-0.79590.47190.2028-0.05460.31360.0690.6079-15.8373-17.5645-21.4914
1112.7487-1.52732.11599.7711-0.34119.09870.5030.09530.06420.0953-0.33-0.62110.0642-0.6211-0.173-0.08780.0070.094-0.1461-0.0192-0.3815-49.9364-77.6568-28.6648
125.03633.35810.33085.04071.80873.93-0.0366-0.08430.8507-0.08430.1083-0.40010.8507-0.4001-0.07170.18760.0117-0.0790.0221-0.03720.11-21.6619-56.4577-22.626
135.2180.73934.47194.01561.919415.01660.17190.61130.51170.61130.08670.49760.51170.4976-0.2585-0.2340.1004-0.0114-0.18220.0113-0.0607-8.3847-45.16745.9069
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1{ A|22 - A|44 }A22 - 44
2X-RAY DIFFRACTION2{ A|45 - A|54 }A45 - 54
3X-RAY DIFFRACTION3{ A|55 - A|64 }A55 - 64
4X-RAY DIFFRACTION4{ A|65 - A|71 }A65 - 71
5X-RAY DIFFRACTION5{ A|72 - A|91 }A72 - 91
6X-RAY DIFFRACTION6{ A|92 - A|115 }A92 - 115
7X-RAY DIFFRACTION7{ A|116 - A|130 }A116 - 130
8X-RAY DIFFRACTION8{ A|131 - A|138 }A131 - 138
9X-RAY DIFFRACTION9{ A|139 - A|161 }A139 - 161
10X-RAY DIFFRACTION10{ A|162 - A|167 }A162 - 167
11X-RAY DIFFRACTION11{ B|327 - B|426 }B327 - 426
12X-RAY DIFFRACTION12{ B|427 - B|524 }B427 - 524
13X-RAY DIFFRACTION13{ B|525 - B|632 }B525 - 632

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