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- PDB-7z3p: Crystal structure of the mouse leptin:LepR-CRH2 encounter complex... -

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Basic information

Entry
Database: PDB / ID: 7z3p
TitleCrystal structure of the mouse leptin:LepR-CRH2 encounter complex to 1.95 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
DI(HYDROXYETHYL)ETHER / Leptin / Leptin receptor
Similarity search - Component
Biological speciesMus musculus (house mouse)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.943 Å
AuthorsTsirigotaki, A. / Verschueren, K. / Savvides, S.N. / Verstraete, K.
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)43,5254
Polymers43,3792
Non-polymers1462
Water2,774154
1
A: Leptin
hetero molecules


Theoretical massNumber of molelcules
Total (without water)17,3812
Polymers17,3411
Non-polymers401
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
B: Leptin receptor
hetero molecules


Theoretical massNumber of molelcules
Total (without water)26,1452
Polymers26,0391
Non-polymers1061
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)96.727, 52.811, 89.055
Angle α, β, γ (deg.)90, 90, 90
Int Tables number18
Space group name H-MP21212

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Components

#1: Protein Leptin / / Obesity factor


Mass: 17340.684 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Mouse leptin:CRH2 complex was produced in HEK293 FreeStyle cells in the presence of kifunensine.
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Lep, Ob / Plasmid: pTWIST-CMV-BetaGlobin / Production host: Homo sapiens (human) / Strain (production host): Hek293 / Variant (production host): FreeStyle / References: UniProt: P41160
#2: Protein Leptin receptor / / LEP-R / B219 / OB receptor / OB-R


Mass: 26038.537 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Prior the crystallisation, the N-terminal His-tag was removed by a proteolytic digest with TEV protease.
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Lepr, Db, Obr / Plasmid: pCAGS / Cell line (production host): HEK293 / Production host: Homo sapiens (human) / Variant (production host): FreeStyle / References: UniProt: P48356
#3: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Ca
#4: Chemical ChemComp-PEG / DI(HYDROXYETHYL)ETHER / Diethylene glycol


Mass: 106.120 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C4H10O3
#5: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 154 / 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: 2.62 Å3/Da / Density % sol: 53.08 %
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop / pH: 8
Details: BCS screen, condition H1 0.04 M Calcium chloride dihydrate 0.04 M sodium formate 0.1 M Tris pH 8.0 25% PEG Smear Low cryo: 20 % PEG 400
Temp details: Temperature-controlled cabinet

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Data collection

DiffractionMean temperature: 100 K / Ambient temp details: cold nitrogen gas stream / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SOLEIL / Beamline: PROXIMA 2 / Wavelength: 0.98011 Å
DetectorType: DECTRIS EIGER X 9M / Detector: PIXEL / Date: Feb 1, 2020 / Details: HPM and KB mirrors
RadiationMonochromator: Si[111] monochromator / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.98011 Å / Relative weight: 1
ReflectionResolution: 1.94→46.35 Å / Num. obs: 34115 / % possible obs: 99.2 % / Redundancy: 13.2 % / Biso Wilson estimate: 44.417 Å2 / CC1/2: 0.999 / Rrim(I) all: 0.127 / Net I/σ(I): 16.08
Reflection shellResolution: 1.94→1.99 Å / Mean I/σ(I) obs: 0.8 / Num. unique obs: 2017 / CC1/2: 0.346 / Rrim(I) all: 3.271 / % possible all: 88.9

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Processing

Software
NameVersionClassification
BUSTER2.10.3refinement
XDSversion March 15, 2019data reduction
Aimless0.7.3data scaling
PHASER2.7.0phasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 1ax8, 3v6o

1ax8

3v6o


Resolution: 1.943→46.35 Å / Cor.coef. Fo:Fc: 0.947 / Cor.coef. Fo:Fc free: 0.935 / SU R Cruickshank DPI: 0.125 / Cross valid method: THROUGHOUT / SU R Blow DPI: 0.128 / SU Rfree Blow DPI: 0.121 / SU Rfree Cruickshank DPI: 0.12
RfactorNum. reflection% reflectionSelection details
Rfree0.221 2865 -RANDOM
Rwork0.191 ---
obs0.1937 34115 99.2 %-
Displacement parametersBiso mean: 54.97 Å2
Baniso -1Baniso -2Baniso -3
1--11.833 Å20 Å20 Å2
2--7.3322 Å20 Å2
3---4.5008 Å2
Refine analyzeLuzzati coordinate error obs: 0.27 Å
Refinement stepCycle: LAST / Resolution: 1.943→46.35 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2485 0 8 154 2647
Refine LS restraints
Refine-IDTypeDev idealNumberRestraint functionWeight
X-RAY DIFFRACTIONt_bond_d0.0082548HARMONIC2
X-RAY DIFFRACTIONt_angle_deg0.973470HARMONIC2
X-RAY DIFFRACTIONt_dihedral_angle_d873SINUSOIDAL2
X-RAY DIFFRACTIONt_gen_planes415HARMONIC5
X-RAY DIFFRACTIONt_it2548HARMONIC10
X-RAY DIFFRACTIONt_chiral_improper_torsion342SEMIHARMONIC5
X-RAY DIFFRACTIONt_ideal_dist_contact2082SEMIHARMONIC4
X-RAY DIFFRACTIONt_omega_torsion3.65
X-RAY DIFFRACTIONt_other_torsion15.88
LS refinement shellResolution: 1.943→1.96 Å
RfactorNum. reflection% reflection
Rfree0.2358 66 -
Rwork0.2265 --
obs--72.75 %
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)
110.81730.80162.072111.57172.66967.11050.04420.229-0.90830.229-0.01680.0262-0.90830.0262-0.02730.04250.06970.0297-0.0676-0.027-0.0213-23.66957.442-10.2269
213.315-1.43183.61598.237914.5529.3333-0.4098-1.12980.1871-1.12980.23440.62320.18710.62320.17540.3679-0.1405-0.0070.4330.01320.2228-33.964215.0942-1.646
328.495812.932112.965827.18119.06416.1071-0.17140.6020.55990.6020.735-0.52580.5599-0.5258-0.56360.00840.1230.24710.06490.02090.1915-33.1259-2.4514-0.8712
411.328-0.16195.53726.77634.732812.2694-0.21720.33690.06440.33690.08-0.25580.0644-0.25580.1372-0.05080.06750.0775-0.019-0.0167-0.0354-24.1416-0.9483-4.7102
519.2541.6595-0.331110.9391.616810.88270.02292.10480.03232.10481.34760.28070.03230.2807-1.37050.80190.58530.26370.42440.13760.3234-26.3677-0.52934.9156
610.813211.5361.115222.54686.06589.81720.1078-0.1823-1.2852-0.1823-0.5395-0.9252-1.2852-0.92520.43170.0280.19170.08730.0883-0.08420.0114-35.87936.9707-4.9432
725.24412.274-2.40110.5459-2.554331.6958-1.2557-0.7309-2.0723-0.73091.25322.6036-2.07232.60360.00250.5815-0.14770.08740.1271-0.38120.3097-22.002612.933.9426
84.0464-2.1658-0.46566.53190.76772.79670.0869-0.37790.4184-0.3779-0.07710.33090.41840.3309-0.0098-0.01780.05550.0384-0.06540.0093-0.0198-11.7557-12.8691-20.9015
92.4053-0.2864-0.0315.69060.8622.8234-0.0366-0.3090.27-0.3090.00820.09990.270.09990.0283-0.04050.04570.0115-0.13210.0315-0.0743-14.0963-14.2973-21.2168
101.0978-1.25740.84279.1075-4.69716.0538-0.071-0.4547-0.5398-0.4547-0.0376-0.0239-0.5398-0.02390.10860.13940.0236-0.0826-0.1394-0.0119-0.0856-21.206417.4418-34.17
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1{ A|22 - A|44 }A23 - 44
2X-RAY DIFFRACTION2{ A|45 - A|71 }A45 - 71
3X-RAY DIFFRACTION3{ A|72 - A|91 }A72 - 91
4X-RAY DIFFRACTION4{ A|92 - A|115 }A92 - 115
5X-RAY DIFFRACTION5{ A|116 - A|142 }A116 - 142
6X-RAY DIFFRACTION6{ A|143 - A|160 }A143 - 160
7X-RAY DIFFRACTION7{ A|161 - A|167 }A161 - 167
8X-RAY DIFFRACTION8{ B|426 - B|481 }B426 - 481
9X-RAY DIFFRACTION9{ B|482 - B|531 }B482 - 531
10X-RAY DIFFRACTION10{ B|532 - B|630 }B532 - 630

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