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データベース: PDB / ID: 8avb
タイトルCryo-EM structure for mouse leptin in complex with the mouse LEP-R ectodomain (1:2 mLEP:mLEPR 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


negative regulation of metabolic process / negative regulation of locomotor rhythm / negative regulation of eating behavior / Synthesis, secretion, and deacylation of Ghrelin / 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 metabolic process / negative regulation of locomotor rhythm / negative regulation of eating behavior / Synthesis, secretion, and deacylation of Ghrelin / 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 / 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 / leptin receptor binding / regulation of natural killer cell mediated cytotoxicity / protein-hormone receptor activity / bone growth / positive regulation of luteinizing hormone secretion / regulation of natural killer cell activation / glycerol biosynthetic process / positive regulation of monoatomic ion transport / 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 / positive regulation of hepatic stellate cell activation / adult feeding behavior / regulation of nitric-oxide synthase activity / response to leptin / regulation of lipid biosynthetic process / bone mineralization involved in bone maturation / regulation of feeding behavior / sexual reproduction / activation of protein kinase C activity / fatty acid catabolic process / negative regulation of cartilage development / ovulation from ovarian follicle / negative regulation of appetite / positive regulation of developmental growth / leukocyte tethering or rolling / cellular response to leptin stimulus / energy reserve metabolic process / bile acid metabolic process / negative regulation of D-glucose import / tyrosine phosphorylation of STAT protein / prostaglandin secretion / regulation of protein localization to nucleus / cardiac muscle hypertrophy / regulation of metabolic process / hormone metabolic process / aorta development / intestinal absorption / cytokine receptor activity / insulin secretion / regulation of fat cell differentiation / positive regulation of p38MAPK cascade / negative regulation of vasoconstriction / eating behavior / peptide hormone receptor binding / regulation of gluconeogenesis / glycogen metabolic process / fatty acid beta-oxidation / regulation of cytokine production involved in inflammatory response / cytokine binding / central nervous system neuron development / positive regulation of insulin secretion involved in cellular response to glucose stimulus / peptide hormone binding / response to dietary excess / negative regulation of lipid storage / T cell differentiation / positive regulation of TOR signaling / response to vitamin E / glial cell proliferation / negative regulation of gluconeogenesis / regulation of angiogenesis / adipose tissue development / phagocytosis / positive regulation of insulin receptor signaling pathway / positive regulation of T cell proliferation / cellular response to retinoic acid / positive regulation of tyrosine phosphorylation of STAT protein / energy homeostasis / positive regulation of interleukin-12 production / cholesterol metabolic process / regulation of insulin secretion / negative regulation of autophagy / determination of adult lifespan / gluconeogenesis / response to activity / positive regulation of interleukin-8 production / female pregnancy / positive regulation of receptor signaling pathway via JAK-STAT / response to insulin / regulation of protein phosphorylation / placenta development
類似検索 - 分子機能
Leptin / Leptin / Leptin receptor, immunoglobulin-like domain / 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. / : ...Leptin / Leptin / Leptin receptor, immunoglobulin-like domain / 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Mus musculus (ハツカネズミ)
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: 4.43 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 1.22023年5月24日Group: Structure summary / カテゴリ: struct / Item: _struct.title
改定 1.32023年7月26日Group: Other / カテゴリ: pdbx_database_status / Item: _pdbx_database_status.pdb_format_compatible
改定 1.42024年10月16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
F: Leptin receptor


分子量 (理論値)分子数
合計 (水以外)204,5973
ポリマ－204,5973
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, SEC-MALLS experiments show that mouse leptin and the mouse LEP-R ectodomain form a higher-order complex in a concentration-dependent and reversible manner. At 5 mg/mL (40 ...根拠: light scattering, SEC-MALLS experiments show that mouse leptin and the mouse LEP-R ectodomain form a higher-order complex in a concentration-dependent and reversible manner. At 5 mg/mL (40 micromolar) the leptin:LEP-R complex elutes as complex with an apparent protein molecular weight of 229 kDa, which would correspond to a 2:2 stoichiometry.
タイプ名称対称操作数
identity operation1_5551

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要素

#1: タンパク質 Leptin / Obesity factor


分子量: 16434.676 Da / 分子数: 1 / 由来タイプ: 組換発現
詳細: 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
由来: (組換発現) Mus musculus (ハツカネズミ) / 遺伝子: Lep, Ob / プラスミド: pET15b / 詳細 (発現宿主): pET15b_His_TEV_mLEP / 発現宿主: Escherichia coli BL21(DE3) (大腸菌) / 参照: UniProt: P41160
#2: タンパク質 Leptin receptor / LEP-R / B219 / OB receptor / OB-R


分子量: 94081.344 Da / 分子数: 2 / 由来タイプ: 組換発現
詳細: 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.
由来: (組換発現) Mus musculus (ハツカネズミ) / 遺伝子: Lepr, Db, Obr / プラスミド: pCAGGS / 詳細 (発現宿主): pCAGGS_ss_His_Casp_mLepR / 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: P48356
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: Complex between mouse leptin and the mouse LEP-R ectodomain.
タイプ: COMPLEX
詳細: 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, ...詳細: 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.
Entity ID: all / 由来: RECOMBINANT
分子量値: 0.230 MDa / 実験値: YES
由来(天然)生物種: Mus musculus (ハツカネズミ)
由来(組換発現)生物種: Homo sapiens (ヒト) / 株: Freestyle / プラスミド: pCAGGS
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
1150 mMsodium chlorideNaCl1
220 mMHepesC8H18N2O4S1
試料濃度: 0.2 mg/ml / 包埋: NO / シャドウイング: NO / 染色: NO / 凍結: YES
詳細: 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, ...詳細: 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.
試料支持グリッドの材料: 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 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: 7100

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解析

ソフトウェア
名称バージョン分類NB
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
1cryoSPARCv3.3.2粒子像選択Blob picker, TOPAZ, template picking
4cryoSPARCv3.3.2CTF補正Patch CTF
7UCSF Chimera1.17モデルフィッティング
9PHENIX1.19.2モデル精密化Phenix real space refine
10cryoSPARCv3.3.2初期オイラー角割当Ab initio
11cryoSPARCv3.3.2最終オイラー角割当Non-uniform refinement
13cryoSPARCv3.3.2３次元再構成Non-uniform refinement
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: 28684
詳細: 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 ...詳細: 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.
対称性点対称性: C1 (非対称)
3次元再構成解像度: 4.43 Å / 解像度の算出法: FSC 0.143 CUT-OFF / 粒子像の数: 28296 / 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: 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 ...詳細: 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 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.
原子モデル構築PDB-ID: 7Z3R

7z3r


Accession code: 7Z3R / Source name: PDB / タイプ: experimental 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: 509.52 Å2
拘束条件
Refine-IDタイプDev ideal数
ELECTRON MICROSCOPYf_bond_d0.004710869
ELECTRON MICROSCOPYf_angle_d0.777714817
ELECTRON MICROSCOPYf_chiral_restr0.04961678
ELECTRON MICROSCOPYf_plane_restr0.00611863
ELECTRON MICROSCOPYf_dihedral_angle_d5.47291433

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