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

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基本情報

登録情報
データベース: PDB / ID: 8ave
タイトルHuman leptin in complex with the human LEP-R ectodomain fused to a C-terminal trimeric isoleucine GCN4 zipper (2:2 model)
要素
  • Leptin
  • Leptin receptor
キーワードCYTOKINE / leptin / LEP-R / obesity / metabolism / energy balance
機能・相同性
機能・相同性情報


multicellular organism development / leptin receptor activity / regulation of lipoprotein lipid oxidation / cellular response to L-ascorbic acid / positive regulation of fat cell apoptotic process / negative regulation of glutamine transport / activation of protein kinase C activity / negative regulation of appetite by leptin-mediated signaling pathway / negative regulation of glucagon secretion / regulation of endothelial cell proliferation ...multicellular organism development / leptin receptor activity / regulation of lipoprotein lipid oxidation / cellular response to L-ascorbic acid / positive regulation of fat cell apoptotic process / negative regulation of glutamine transport / activation of protein kinase C activity / negative regulation of appetite by leptin-mediated signaling pathway / negative regulation of glucagon secretion / regulation of endothelial cell proliferation / leptin receptor binding / sexual reproduction / regulation of natural killer cell mediated cytotoxicity / regulation of bone remodeling / regulation of natural killer cell proliferation / positive regulation of luteinizing hormone secretion / regulation of natural killer cell activation / glycerol biosynthetic process / regulation of steroid biosynthetic process / elastin metabolic process / regulation of transport / leptin-mediated signaling pathway / positive regulation of follicle-stimulating hormone secretion / positive regulation of monoatomic ion transport / bone growth / regulation of intestinal cholesterol absorption / regulation of brown fat cell differentiation / positive regulation of hepatic stellate cell activation / positive regulation of peroxisome proliferator activated receptor signaling pathway / regulation of nitric-oxide synthase activity / regulation of feeding behavior / adult feeding behavior / bone mineralization involved in bone maturation / response to leptin / negative regulation of cartilage development / negative regulation of D-glucose import across plasma membrane / negative regulation of appetite / ovulation from ovarian follicle / positive regulation of developmental growth / energy reserve metabolic process / leukocyte tethering or rolling / prostaglandin secretion / cellular response to leptin stimulus / cardiac muscle hypertrophy / bile acid metabolic process / Signaling by Leptin / hormone metabolic process / positive regulation of p38MAPK cascade / cell surface receptor signaling pathway via STAT / cytokine receptor activity / intestinal absorption / insulin secretion / regulation of gluconeogenesis / negative regulation of vasoconstriction / aorta development / eating behavior / response to vitamin E / glycogen metabolic process / peptide hormone receptor binding / fatty acid beta-oxidation / regulation of cytokine production involved in inflammatory response / response to dietary excess / cytokine binding / central nervous system neuron development / regulation of insulin secretion / peptide hormone binding / T cell differentiation / negative regulation of lipid storage / Synthesis, secretion, and deacylation of Ghrelin / regulation of angiogenesis / negative regulation of gluconeogenesis / positive regulation of TOR signaling / adipose tissue development / positive regulation of insulin receptor signaling pathway / phagocytosis / cell surface receptor signaling pathway via JAK-STAT / glial cell proliferation / cholesterol metabolic process / transport across blood-brain barrier / cellular response to retinoic acid / energy homeostasis / positive regulation of interleukin-12 production / positive regulation of T cell proliferation / placenta development / response to activity / negative regulation of autophagy / gluconeogenesis / determination of adult lifespan / positive regulation of interleukin-8 production / positive regulation of receptor signaling pathway via JAK-STAT / lipid metabolic process / female pregnancy / hormone activity / circadian rhythm / Transcriptional regulation of white adipocyte differentiation / response to insulin / positive regulation of protein import into nucleus / positive regulation of interleukin-6 production / regulation of blood pressure / glucose metabolic process
類似検索 - 分子機能
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
類似検索 - ドメイン・相同性
Leptin / Leptin receptor
類似検索 - 構成要素
生物種Homo sapiens (ヒト)
手法電子顕微鏡法 / 単粒子再構成法 / クライオ電子顕微鏡法 / 解像度: 5.62 Å
データ登録者Verstraete, K. / Savvides, S.N. / Verschueren, K.G. / Tsirigotaki, A.
資金援助 ベルギー, 1件
組織認可番号国
Research Foundation - Flanders (FWO)G0G0619N ベルギー
引用ジャーナル: Nat Struct Mol Biol / 年: 2023
タイトル: Mechanism of receptor assembly via the pleiotropic adipokine Leptin.
著者: Alexandra Tsirigotaki / Ann Dansercoer / Koen H G Verschueren / Iva Marković / Christoph Pollmann / Maximillian Hafer / Jan Felix / Catherine Birck / Wouter Van Putte / Dominiek Catteeuw / ...著者: 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 /
要旨: 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.
履歴
登録2022年8月26日登録サイト: PDBE / 処理サイト: PDBE
改定 1.02023年4月5日Provider: repository / タイプ: Initial release
改定 1.12023年4月26日Group: Database references / カテゴリ: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
改定 1.22024年10月23日Group: Data collection / Refinement description / Structure summary
カテゴリ: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _em_admin.last_update

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構造の表示

構造ビューア分子:
MolmilJmol/JSmol

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集合体

登録構造単位
A: Leptin
B: Leptin receptor
C: Leptin
D: Leptin receptor


分子量 (理論値)分子数
合計 (水以外)234,8544
ポリマ－234,8544
非ポリマー00
水00
1


  • 登録構造と同一
  • 登録者が定義した集合体
  • 根拠: light scattering, The molecular weight of the trimeric human leptin:LEP-R assembly was confirmed by SEC-MALLS
タイプ名称対称操作数
identity operation1_5551
非結晶学的対称性 (NCS)NCSドメイン:
IDEns-ID詳細
d_1ens_1chain "C"
d_2ens_1chain "A"
d_1ens_2chain "B"
d_2ens_2chain "D"

NCSドメイン領域:
Dom-IDComponent-IDEns-IDBeg label comp-IDEnd label comp-IDLabel asym-IDLabel seq-ID
d_11ens_1VALCYSC1 - 146
d_21ens_1VALCYSA1 - 146
d_11ens_2LYSASPB1 - 596
d_21ens_2LYSASPD1 - 596

NCSアンサンブル:
ID
ens_1
ens_2

NCS oper:
IDCodeMatrixベクター
1given(-0.339856162415, 0.925591767857, 0.166666337762), (-0.689049708833, -0.365669189504, 0.625696845608), (0.640084594116, 0.0978055372767, 0.762053665599)48.009553301, 300.298613223, -109.053112904
2given(-0.305058035114, -0.687959244415, 0.658522340727), (0.940363649278, -0.326892247472, 0.0941151723072), (0.150518445143, 0.647961060996, 0.746652972341)281.946652637, 65.8513117364, -109.977741298

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要素

#1: タンパク質 Leptin / Obese protein / Obesity factor


分子量: 18605.061 Da / 分子数: 2 / 由来タイプ: 組換発現 / 詳細: pTwist-N-His-hLEP / 由来: (組換発現) Homo sapiens (ヒト) / Cell: pTwist / 遺伝子: LEP, OB, OBS / プラスミド: pTwist / 詳細 (発現宿主): pTwist-N-His-hLEP / 細胞株 (発現宿主): HEK293 / 発現宿主: Homo sapiens (ヒト) / Variant (発現宿主): FreeStyle / 参照: UniProt: P41159
#2: タンパク質 Leptin receptor / LEP-R / HuB219 / OB receptor / OB-R


分子量: 98822.062 Da / 分子数: 2 / 由来タイプ: 組換発現
詳細: 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 ...詳細: 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.
由来: (組換発現) Homo sapiens (ヒト) / 遺伝子: LEPR, DB, OBR / プラスミド: pTwist / 詳細 (発現宿主): pTwist-hLEPR-5xGGS-tGCN4 / 発現宿主: Homo sapiens (ヒト) / 株 (発現宿主): FreeStyle / 参照: UniProt: P48357
Has protein modificationY

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実験情報

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実験

実験手法: 電子顕微鏡法
EM実験試料の集合状態: PARTICLE / ３次元再構成法: 単粒子再構成法

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試料調製

構成要素名称: Human leptin in complex with the human LEP-R ectodomain C-terminally fused to a trimeric GCN4 isoleucine zipper tag
タイプ: COMPLEX
詳細: 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 ...詳細: 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 / 由来: RECOMBINANT
分子量値: 0.350 MDa / 実験値: YES
由来(天然)生物種: Homo sapiens (ヒト)
由来(組換発現)生物種: Homo sapiens (ヒト) / 株: HEK293 FreeStyle / プラスミド: pTwist
緩衝液pH: 7.4 / 詳細: 20 mM HEPES, 150 mM NaCl, pH 7.4
緩衝液成分
ID濃度名称式Buffer-ID
120 mMHEPESC8H18N2O4S1
2150 mMsodium chlorideNaCl1
試料濃度: 0.3 mg/ml / 包埋: NO / シャドウイング: NO / 染色: NO / 凍結: YES / 詳細: This sample was monodisperse.
試料支持詳細: 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 ...詳細: 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
グリッドの材料: GOLD / グリッドのサイズ: 300 divisions/in. / グリッドのタイプ: Quantifoil R0.6/1
急速凍結装置: FEI VITROBOT MARK IV / 凍結剤: ETHANE / 湿度: 100 % / 凍結前の試料温度: 295 K

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電子顕微鏡撮影

顕微鏡モデル: JEOL CRYO ARM 300
電子銃電子線源: FIELD EMISSION GUN / 加速電圧: 300 kV / 照射モード: FLOOD BEAM
電子レンズモード: OTHER / 最大 デフォーカス（公称値）: 3000 nm / 最小 デフォーカス（公称値）: 800 nm
撮影電子線照射量: 62.4 e/Å2 / フィルム・検出器のモデル: GATAN K3 (6k x 4k) / 撮影したグリッド数: 1 / 実像数: 13755

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解析

ソフトウェア
名称バージョン分類
phenix.real_space_refine1.19.2_4158精密化
PHENIX1.19.2_4158精密化
EMソフトウェア
ID名称バージョンカテゴリ詳細
4cryoSPARCv3.1.0CTF補正patch CTF
7UCSF Chimera1.17モデルフィッティング
9cryoSPARCv3.1.0初期オイラー角割当Ab initio
12cryoSPARCv3.3.2３次元再構成Non-uniform refinement
13PHENIX1.19.2モデル精密化
CTF補正タイプ: PHASE FLIPPING AND AMPLITUDE CORRECTION
粒子像の選択選択した粒子像数: 206218
詳細: 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 ...詳細: 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.
対称性点対称性: C1 (非対称)
3次元再構成解像度: 5.62 Å / 解像度の算出法: FSC 0.143 CUT-OFF / 粒子像の数: 91899 / 詳細: Non-uniform refinement in cryoSPARC / クラス平均像の数: 1 / 対称性のタイプ: POINT
原子モデル構築プロトコル: FLEXIBLE FIT / 空間: REAL
詳細: 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 mouse 3:3 Leptin:LEP-RIgCRH2 complex (pdb 7z3r) and ...詳細: 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 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 and hydrogen-bonding restraints across the site II and site III hLeptin:hLEP-R interface regions.
原子モデル構築
IDPDB-ID 3D fitting-IDAccession codeInitial refinement model-IDSource nameタイプ
17Z3R

7z3r

17Z3R1PDBexperimental model
27Z3Q

7z3q

17Z3Q2PDBexperimental model
精密化交差検証法: NONE
立体化学のターゲット値: GeoStd + Monomer Library + CDL v1.2
原子変位パラメータBiso mean: 140.2 Å2
拘束条件
Refine-IDタイプDev ideal数
ELECTRON MICROSCOPYf_bond_d0.003912194
ELECTRON MICROSCOPYf_angle_d0.933716616
ELECTRON MICROSCOPYf_chiral_restr0.05561876
ELECTRON MICROSCOPYf_plane_restr0.01012078
ELECTRON MICROSCOPYf_dihedral_angle_d13.15824462
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDRefine-IDタイプRms dev position (Å)
ens_1d_2CELECTRON MICROSCOPYNCS constraints0.000693532035869
ens_2d_2BELECTRON MICROSCOPYNCS constraints0.000710263790249

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