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- PDB-6ud0: Solution-state NMR structural ensemble of human Tsg101 UEV in com... -

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基本情報

登録情報
データベース: PDB / ID: 6ud0
タイトルSolution-state NMR structural ensemble of human Tsg101 UEV in complex with K63-linked diubiquitin
要素
  • (Ubiquitinユビキチン) x 2
  • Tumor susceptibility gene 101 proteinTSG101
キーワードCELL CYCLE (細胞周期) / ESCRT-I (ESCRT) / viral budding (ウイルス排出) / ubiquitin E2 variant
機能・相同性
機能・相同性情報


positive regulation of viral budding via host ESCRT complex / positive regulation of ubiquitin-dependent endocytosis / extracellular transport / ESCRT I complex / negative regulation of epidermal growth factor-activated receptor activity / regulation of extracellular exosome assembly / ウイルス排出 / regulation of MAP kinase activity / exosomal secretion / protein transport to vacuole involved in ubiquitin-dependent protein catabolic process via the multivesicular body sorting pathway ...positive regulation of viral budding via host ESCRT complex / positive regulation of ubiquitin-dependent endocytosis / extracellular transport / ESCRT I complex / negative regulation of epidermal growth factor-activated receptor activity / regulation of extracellular exosome assembly / ウイルス排出 / regulation of MAP kinase activity / exosomal secretion / protein transport to vacuole involved in ubiquitin-dependent protein catabolic process via the multivesicular body sorting pathway / membrane fission / positive regulation of exosomal secretion / ubiquitin-dependent protein catabolic process via the multivesicular body sorting pathway / multivesicular body assembly / Flemming body / hypothalamus gonadotrophin-releasing hormone neuron development / virion binding / female meiosis I / positive regulation of protein monoubiquitination / mitochondrion transport along microtubule / fat pad development / endosome to lysosome transport / female gonad development / negative regulation of epidermal growth factor receptor signaling pathway / seminiferous tubule development / male meiosis I / viral budding via host ESCRT complex / autophagosome maturation / positive regulation of intrinsic apoptotic signaling pathway by p53 class mediator / viral release from host cell / energy homeostasis / regulation of neuron apoptotic process / regulation of proteasomal protein catabolic process / Maturation of protein E / Maturation of protein E / keratinocyte differentiation / ER Quality Control Compartment (ERQC) / Myoclonic epilepsy of Lafora / FLT3 signaling by CBL mutants / Prevention of phagosomal-lysosomal fusion / IRAK2 mediated activation of TAK1 complex / Alpha-protein kinase 1 signaling pathway / グリコーゲン合成 / IRAK1 recruits IKK complex / IRAK1 recruits IKK complex upon TLR7/8 or 9 stimulation / Membrane binding and targetting of GAG proteins / Constitutive Signaling by NOTCH1 HD Domain Mutants / Endosomal Sorting Complex Required For Transport (ESCRT) / NOTCH2 Activation and Transmission of Signal to the Nucleus / IRAK2 mediated activation of TAK1 complex upon TLR7/8 or 9 stimulation / PTK6 Regulates RTKs and Their Effectors AKT1 and DOK1 / Negative regulation of FLT3 / Regulation of FZD by ubiquitination / TICAM1,TRAF6-dependent induction of TAK1 complex / TICAM1-dependent activation of IRF3/IRF7 / APC/C:Cdc20 mediated degradation of Cyclin B / p75NTR recruits signalling complexes / Downregulation of ERBB4 signaling / TRAF6 mediated IRF7 activation in TLR7/8 or 9 signaling / APC-Cdc20 mediated degradation of Nek2A / PINK1-PRKN Mediated Mitophagy / TRAF6-mediated induction of TAK1 complex within TLR4 complex / Pexophagy / InlA-mediated entry of Listeria monocytogenes into host cells / Regulation of innate immune responses to cytosolic DNA / VLDLR internalisation and degradation / Downregulation of ERBB2:ERBB3 signaling / NRIF signals cell death from the nucleus / Activated NOTCH1 Transmits Signal to the Nucleus / NF-kB is activated and signals survival / Regulation of PTEN localization / Regulation of BACH1 activity / Translesion synthesis by REV1 / エンドソーム / Synthesis of active ubiquitin: roles of E1 and E2 enzymes / Translesion synthesis by POLK / MAP3K8 (TPL2)-dependent MAPK1/3 activation / TICAM1, RIP1-mediated IKK complex recruitment / Gap-filling DNA repair synthesis and ligation in GG-NER / Downregulation of TGF-beta receptor signaling / Translesion synthesis by POLI / Josephin domain DUBs / Activation of IRF3, IRF7 mediated by TBK1, IKKε (IKBKE) / Regulation of activated PAK-2p34 by proteasome mediated degradation / neuron projection morphogenesis / InlB-mediated entry of Listeria monocytogenes into host cell / IKK complex recruitment mediated by RIP1 / JNK (c-Jun kinases) phosphorylation and activation mediated by activated human TAK1 / regulation of mitochondrial membrane potential / TGF-beta receptor signaling in EMT (epithelial to mesenchymal transition) / HCMV Late Events / N-glycan trimming in the ER and Calnexin/Calreticulin cycle / Autodegradation of Cdh1 by Cdh1:APC/C / TNFR1-induced NF-kappa-B signaling pathway / ubiquitin binding / APC/C:Cdc20 mediated degradation of Securin / positive regulation of protein ubiquitination / Asymmetric localization of PCP proteins / TCF dependent signaling in response to WNT / SCF-beta-TrCP mediated degradation of Emi1
類似検索 - 分子機能
Steadiness box (SB) domain / Vps23 core domain / Steadiness box (SB) domain profile. / Ubiquitin E2 variant, N-terminal / UEV domain / UEV domain profile. / ESCRT assembly domain / Ubiquitin-conjugating enzyme E2, catalytic domain homologues / Ubiquitin-conjugating enzyme/RWD-like / Ubiquitin conserved site ...Steadiness box (SB) domain / Vps23 core domain / Steadiness box (SB) domain profile. / Ubiquitin E2 variant, N-terminal / UEV domain / UEV domain profile. / ESCRT assembly domain / Ubiquitin-conjugating enzyme E2, catalytic domain homologues / Ubiquitin-conjugating enzyme/RWD-like / Ubiquitin conserved site / Ubiquitin domain / Ubiquitin domain signature. / Ubiquitin family / Ubiquitin homologues / ユビキチン様タンパク質 / Ubiquitin domain profile. / Ubiquitin-like domain superfamily
類似検索 - ドメイン・相同性
Polyubiquitin-B / Tumor susceptibility gene 101 protein
類似検索 - 構成要素
生物種Homo sapiens (ヒト)
手法溶液NMR / simulated annealing
データ登録者Strickland, M. / Watanabe, S. / Bonn, S.M. / Camara, C.M. / Fushman, D. / Carter, C.A. / Tjandra, N.
引用ジャーナル: Structure / 年: 2021
タイトル: Tsg101/ESCRT-I Recruitment Regulated by the Dual Binding Modes of K63-Linked Diubiquitin
著者: Strickland, M. / Watanabe, S. / Bonn, S.M. / Camara, C.M. / Fushman, D. / Carter, C.A. / Tjandra, N.
履歴
登録2019年9月18日登録サイト: RCSB / 処理サイト: RCSB
改定 1.02021年3月17日Provider: repository / タイプ: Initial release
改定 1.12021年9月22日Group: Database references / カテゴリ: citation / database_2
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.title / _citation.year / _database_2.pdbx_DOI / _database_2.pdbx_database_accession
改定 1.22023年6月14日Group: Other / カテゴリ: pdbx_database_status / Item: _pdbx_database_status.status_code_nmr_data

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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: Ubiquitin
B: Ubiquitin
C: Tumor susceptibility gene 101 protein


分子量 (理論値)分子数
合計 (水以外)33,9303
ポリマ－33,9303
非ポリマー00
水0
1


  • 登録構造と同一
  • 登録者が定義した集合体
  • 根拠: NMR spectroscopy was used to determine the quaternary structure.
タイプ名称対称操作数
identity operation1_5551
NMR アンサンブル
データ基準
コンフォーマー数 (登録 / 計算)20 / 100structures with the lowest energy
代表モデルモデル #1target function

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要素

#1: タンパク質 Ubiquitin / ユビキチン


分子量: 8604.845 Da / 分子数: 1 / 変異: K63R / 由来タイプ: 組換発現
詳細: Distal domain of K63-linked diubiquitin. The K63R mutation is made to prevent formation of polyubiquitin during enzymatic linkage to the proximal domain. Joined to the proximal domain of ...詳細: Distal domain of K63-linked diubiquitin. The K63R mutation is made to prevent formation of polyubiquitin during enzymatic linkage to the proximal domain. Joined to the proximal domain of diubiquitin at residue G76 using an isopeptide bond.
由来: (組換発現) Homo sapiens (ヒト) / 遺伝子: UBB / 発現宿主: Escherichia coli BL21 (大腸菌) / 参照: UniProt: P0CG47
#2: タンパク質 Ubiquitin / ユビキチン


分子量: 8691.918 Da / 分子数: 1 / 変異: +D77 / 由来タイプ: 組換発現
詳細: Proximal domain of K63-linked diubiquitin. The additional aspartic acid on the C-terminus (+D77) is made to prevent formation of polyubiquitin during enzymatic linkage to the distal domain. ...詳細: Proximal domain of K63-linked diubiquitin. The additional aspartic acid on the C-terminus (+D77) is made to prevent formation of polyubiquitin during enzymatic linkage to the distal domain. Joined to the distal domain of diubiquitin at residue K63 using an isopeptide bond.
由来: (組換発現) Homo sapiens (ヒト) / 遺伝子: UBB / 発現宿主: Escherichia coli BL21 (大腸菌) / 参照: UniProt: P0CG47
#3: タンパク質 Tumor susceptibility gene 101 protein / TSG101 / ESCRT-I complex subunit TSG101


分子量: 16633.352 Da / 分子数: 1 / 由来タイプ: 組換発現 / 由来: (組換発現) Homo sapiens (ヒト) / 遺伝子: TSG101 / プラスミド: pET-28B / 発現宿主: Escherichia coli BL21(DE3) (大腸菌) / Variant (発現宿主): Rosetta2 / 参照: UniProt: Q99816

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実験情報

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実験

実験手法: 溶液NMR
NMR実験
Conditions-IDExperiment-IDSolution-IDSample stateSpectrometer-IDタイプ
111isotropic22D 1H-15N HSQC
188isotropic22D 1H-15N HSQC
122isotropic12D 1H-15N HSQC
199isotropic12D 1H-15N HSQC
133isotropic12D 1H-15N HSQC
11010isotropic12D 1H-15N HSQC
144isotropic12D 1H-15N HSQC
155anisotropic12D 1H-15N HSQC
166isotropic12D 1H-15N HSQC
177anisotropic12D 1H-15N HSQC

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試料調製

詳細
タイプSolution-ID内容詳細Label溶媒系
solution1200 uM [U-98% 15N] Tsg101 UEV domain, 200 uM K63-linked diubiquitin, distal domain, 200 uM K63-linked diubiquitin, proximal domain, 20 mM potassium phosphate, 50 mM sodium chloride, 93% H2O/7% D2OSample used for measuring chemical shift perturbations of Tsg101 UEV domain (bound form)15NTsg_CSPs_bound93% H2O/7% D2O
solution8200 uM [U-98% 15N] Tsg101 UEV domain, 20 mM potassium phosphate, 50 mM sodium chloride, 93% H2O/7% D2OSample used for measuring chemical shift perturbations of Tsg101 UEV domain (free form)15NTsg_CSPs_free93% H2O/7% D2O
solution2200 uM Tsg101 UEV domain, 200 uM [U-98% 15N] K63-linked diubiquitin, distal domain, 200 uM K63-linked diubiquitin, proximal domain, 20 mM potassium phosphate, 50 mM sodium chloride, 93% H2O/7% D2OSample used for measuring chemical shift perturbations of 15N-distal domain of diubiquitin (bound form)15NDistal_CSPs_bound93% H2O/7% D2O
solution9200 uM [U-98% 15N] K63-linked diubiquitin, distal domain, 200 uM K63-linked diubiquitin, proximal domain, 20 mM potassium phosphate, 50 mM sodium chloride, 93% H2O/7% D2OSample used for measuring chemical shift perturbations of 15N-distal domain of diubiquitin (free form)15NDistal_CSPs_free93% H2O/7% D2O
solution3200 uM Tsg101 UEV domain, 200 uM K63-linked diubiquitin, distal domain, 200 uM [U-98% 15N] K63-linked diubiquitin, proximal domain, 20 mM potassium phosphate, 50 mM sodium chloride, 93% H2O/7% D2OSample used for measuring chemical shift perturbations of 15N-proximal domain of diubiquitin (bound form)15NProx_CSPs_bound93% H2O/7% D2O
solution10200 uM K63-linked diubiquitin, distal domain, 200 uM [U-98% 15N] K63-linked diubiquitin, proximal domain, 20 mM potassium phosphate, 50 mM sodium chloride, 93% H2O/7% D2OSample used for measuring chemical shift perturbations of 15N-proximal domain of diubiquitin (free form)15N_Prox_CSPs_free93% H2O/7% D2O
solution4125 uM [U-98% 15N] Tsg101 UEV domain, 125 uM K63-linked diubiquitin, distal domain, 125 uM K63-linked diubiquitin, proximal domain, 20 mM potassium phosphate, 50 mM sodium chloride, 93% H2O/7% D2OThe distal domain of diubiquitin was mutated (T14C) to incorporate a covalently bound Lu-M8-DOTA-SPy tag for the measurement of pseudocontact shifts in Tsg101 UEV domain (reference).15NTsg_PCS_Dist_Lu93% H2O/7% D2O
solution5125 uM [U-98% 15N] Tsg101 UEV domain, 125 uM K63-linked diubiquitin, distal domain, 125 uM K63-linked diubiquitin, proximal domain, 20 mM potassium phosphate, 50 mM sodium chloride, 93% H2O/7% D2OThe distal domain of diubiquitin was mutated (T14C) to incorporate a covalently bound Tm-M8-DOTA-SPy tag for the measurement of pseudocontact shifts in Tsg101 UEV domain (paramagnetic).15NTsg_PCS_Dist_Tm93% H2O/7% D2O
solution6125 uM [U-98% 15N] Tsg101 UEV domain, 125 uM K63-linked diubiquitin, distal domain, 125 uM K63-linked diubiquitin, proximal domain, 20 mM potassium phosphate, 50 mM sodium chloride, 93% H2O/7% D2OThe proximal domain of diubiquitin was mutated (T14C) to incorporate a covalently bound Lu-M8-DOTA-SPy tag for the measurement of pseudocontact shifts in Tsg101 UEV domain (reference).15NTsg_PCS_Prox_Lu93% H2O/7% D2O
solution7125 uM [U-98% 15N] Tsg101 UEV domain, 125 uM K63-linked diubiquitin, distal domain, 125 uM K63-linked diubiquitin, proximal domain, 20 mM potassium phosphate, 50 mM sodium chloride, 93% H2O/7% D2OThe proximal domain of diubiquitin was mutated (T14C) to incorporate a covalently bound Tm-M8-DOTA-SPy tag for the measurement of pseudocontact shifts in Tsg101 UEV domain (paramagnetic).15NTsg_PCS_Prox_Tm93% H2O/7% D2O
試料
濃度 (mg/ml)構成要素Isotopic labelingSolution-ID
200 uMTsg101 UEV domain[U-98% 15N]1
200 uMK63-linked diubiquitin, distal domainnatural abundance1
200 uMK63-linked diubiquitin, proximal domainnatural abundance1
20 mMpotassium phosphatenatural abundance1
50 mMsodium chloridenatural abundance1
200 uMTsg101 UEV domain[U-98% 15N]8
20 mMpotassium phosphatenatural abundance8
50 mMsodium chloridenatural abundance8
200 uMTsg101 UEV domainnatural abundance2
200 uMK63-linked diubiquitin, distal domain[U-98% 15N]2
200 uMK63-linked diubiquitin, proximal domainnatural abundance2
20 mMpotassium phosphatenatural abundance2
50 mMsodium chloridenatural abundance2
200 uMK63-linked diubiquitin, distal domain[U-98% 15N]9
200 uMK63-linked diubiquitin, proximal domainnatural abundance9
20 mMpotassium phosphatenatural abundance9
50 mMsodium chloridenatural abundance9
200 uMTsg101 UEV domainnatural abundance3
200 uMK63-linked diubiquitin, distal domainnatural abundance3
200 uMK63-linked diubiquitin, proximal domain[U-98% 15N]3
20 mMpotassium phosphatenatural abundance3
50 mMsodium chloridenatural abundance3
200 uMK63-linked diubiquitin, distal domainnatural abundance10
200 uMK63-linked diubiquitin, proximal domain[U-98% 15N]10
20 mMpotassium phosphatenatural abundance10
50 mMsodium chloridenatural abundance10
125 uMTsg101 UEV domain[U-98% 15N]4
125 uMK63-linked diubiquitin, distal domainnatural abundance4
125 uMK63-linked diubiquitin, proximal domainnatural abundance4
20 mMpotassium phosphatenatural abundance4
50 mMsodium chloridenatural abundance4
125 uMTsg101 UEV domain[U-98% 15N]5
125 uMK63-linked diubiquitin, distal domainnatural abundance5
125 uMK63-linked diubiquitin, proximal domainnatural abundance5
20 mMpotassium phosphatenatural abundance5
50 mMsodium chloridenatural abundance5
125 uMTsg101 UEV domain[U-98% 15N]6
125 uMK63-linked diubiquitin, distal domainnatural abundance6
125 uMK63-linked diubiquitin, proximal domainnatural abundance6
20 mMpotassium phosphatenatural abundance6
50 mMsodium chloridenatural abundance6
125 uMTsg101 UEV domain[U-98% 15N]7
125 uMK63-linked diubiquitin, distal domainnatural abundance7
125 uMK63-linked diubiquitin, proximal domainnatural abundance7
20 mMpotassium phosphatenatural abundance7
50 mMsodium chloridenatural abundance7
試料状態イオン強度: 0.1 M / Label: Conditions_1 / pH: 5.8 / 圧: 1 atm / 温度: 300 K

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NMR測定

NMRスペクトロメーター
タイプ製造業者モデル磁場強度 (MHz)Spectrometer-ID詳細
Bruker AVANCEBrukerAVANCE6001Cryoprobe
Bruker AVANCEBrukerAVANCE8002Cryoprobe

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解析

NMR software
名称バージョン開発者分類
NMRPipe9.5Delaglio, Grzesiek, Vuister, Zhu, Pfeifer and Bax解析
TopSpin3Bruker Biospincollection
CcpNmr Analysis2.4.2CCPNpeak picking
CcpNmr Analysis2.4.2CCPNchemical shift assignment
X-PLOR NIH2.51Schwieters, Kuszewski, Tjandra and Clore精密化
X-PLOR NIH2.51Schwieters, Kuszewski, Tjandra and Clorestructure calculation
精密化手法: simulated annealing / ソフトェア番号: 7
詳細: Docking was carried out between the two proteins using rigid body simulated annealing with a flexible linker between diubiquitin domains. Initial structures were 3H7P and 2MJB for ubiquitin, ...詳細: Docking was carried out between the two proteins using rigid body simulated annealing with a flexible linker between diubiquitin domains. Initial structures were 3H7P and 2MJB for ubiquitin, and 1S1Q for Tsg101 UEV. Distal domain and Tsg101 backbone were fixed to 1S1Q position. Following initial docking, the lowest energy structure in each of the two clusters was used as input for refinement, with an ensemble number of 2.
代表構造選択基準: target function
NMRアンサンブルコンフォーマー選択の基準: structures with the lowest energy
計算したコンフォーマーの数: 100 / 登録したコンフォーマーの数: 20

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