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基本情報
登録情報 | ![]() | ||||||||||||||||||
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タイトル | Lysosomal transporting complex of beta-glucocerebrosidase (GCase) and lysosomal integral membrane protein 2 (LIMP-2) with bound Pro-macrobodies (Combined focus map) | ||||||||||||||||||
![]() | Composite Map of GCase/LIMP-2/PMbs tetramer, generated in Phenix. Obtained from Consensus map and focused local refinements of GCase and LIMP-2. | ||||||||||||||||||
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![]() | Parkinson / lysosome / Gaucher / GCase / SCARB2 / Pro-macrobody / Glucosylceramide / complex / TRANSPORT PROTEIN | ||||||||||||||||||
機能・相同性 | ![]() regulation of glucosylceramide catabolic process / regulation of carbohydrate catabolic process / regulation of endosome organization / steryl-beta-glucosidase activity / positive regulation of neuronal action potential / beta-glucoside catabolic process / cerebellar Purkinje cell layer formation / aminophospholipid transport / termination of signal transduction / galactosylceramidase ...regulation of glucosylceramide catabolic process / regulation of carbohydrate catabolic process / regulation of endosome organization / steryl-beta-glucosidase activity / positive regulation of neuronal action potential / beta-glucoside catabolic process / cerebellar Purkinje cell layer formation / aminophospholipid transport / termination of signal transduction / galactosylceramidase / galactosylceramidase activity / glucosylceramidase / scavenger receptor binding / lymphocyte migration / glucosylceramide catabolic process / regulation of lysosomal protein catabolic process / response to thyroid hormone / glucosylceramidase activity / sphingosine biosynthetic process / autophagosome organization / lysosomal protein catabolic process / microglial cell proliferation / glucosyltransferase activity / endosome to plasma membrane protein transport / regulation of TOR signaling / regulation of lysosome organization / protein targeting to lysosome / Glycosphingolipid catabolism / lipid storage / microglia differentiation / ceramide biosynthetic process / phosphatidylcholine binding / positive regulation of type 2 mitophagy / : / brain morphogenesis / response to pH / 加水分解酵素; 糖加水分解酵素; 配糖体結合加水分解酵素または糖加水分解酵素 / pyramidal neuron differentiation / cargo receptor activity / negative regulation of protein metabolic process / scavenger receptor activity / lysosome organization / motor behavior / 転移酵素; グリコシル基を移すもの; 六炭糖残基を移すもの / neuromuscular process / cholesterol binding / phosphatidylserine binding / response to dexamethasone / hematopoietic stem cell proliferation / antigen processing and presentation / response to testosterone / Association of TriC/CCT with target proteins during biosynthesis / negative regulation of interleukin-6 production / homeostasis of number of cells / regulation of macroautophagy / establishment of skin barrier / negative regulation of protein-containing complex assembly / negative regulation of MAPK cascade / cell maturation / mitophagy / receptor-mediated endocytosis / cholesterol metabolic process / lysosomal lumen / cellular response to starvation / respiratory electron transport chain / determination of adult lifespan / sensory perception of sound / trans-Golgi network / clathrin-coated endocytic vesicle membrane / positive regulation of neuron projection development / autophagy / negative regulation of inflammatory response / response to estrogen / endocytic vesicle membrane / late endosome membrane / transmembrane signaling receptor activity / positive regulation of proteasomal ubiquitin-dependent protein catabolic process / cellular response to tumor necrosis factor / Cargo recognition for clathrin-mediated endocytosis / Clathrin-mediated endocytosis / protein-folding chaperone binding / T cell differentiation in thymus / virus receptor activity / neuron apoptotic process / proteasome-mediated ubiquitin-dependent protein catabolic process / negative regulation of neuron apoptotic process / lysosome / endosome membrane / Golgi membrane / signaling receptor binding / lysosomal membrane / focal adhesion / endoplasmic reticulum membrane / enzyme binding / endoplasmic reticulum / Golgi apparatus / protein homodimerization activity / extracellular exosome / membrane / plasma membrane 類似検索 - 分子機能 | ||||||||||||||||||
生物種 | ![]() | ||||||||||||||||||
手法 | 単粒子再構成法 / クライオ電子顕微鏡法 / 解像度: 3.7 Å | ||||||||||||||||||
![]() | Dobert JP / Schaefer JHS / Dal Maso T / Socher E / Versees W / Moeller A / Zunke F / Arnold P | ||||||||||||||||||
資金援助 | ![]()
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![]() | ![]() タイトル: Cryo-TEM structure of β-glucocerebrosidase in complex with its transporter LIMP-2. 著者: Jan Philipp Dobert / Jan-Hannes Schäfer / Thomas Dal Maso / Priyadarshini Ravindran / Dustin J E Huard / Eileen Socher / Lisa A Schildmeyer / Raquel L Lieberman / Wim Versées / Arne Moeller ...著者: Jan Philipp Dobert / Jan-Hannes Schäfer / Thomas Dal Maso / Priyadarshini Ravindran / Dustin J E Huard / Eileen Socher / Lisa A Schildmeyer / Raquel L Lieberman / Wim Versées / Arne Moeller / Friederike Zunke / Philipp Arnold / ![]() ![]() ![]() 要旨: Targeting proteins to their final cellular destination requires transport mechanisms and nearly all lysosomal enzymes reach the lysosome via the mannose-6-phosphate receptor pathway. One of the few ...Targeting proteins to their final cellular destination requires transport mechanisms and nearly all lysosomal enzymes reach the lysosome via the mannose-6-phosphate receptor pathway. One of the few known exceptions is the enzyme β-glucocerebrosidase (GCase) that requires the lysosomal integral membrane protein type-2 (LIMP-2) as a proprietary lysosomal transporter. Genetic variations in the GCase encoding gene GBA1 cause Gaucher's disease (GD) and present the highest genetic risk factor to develop Parkinson's disease (PD). Activators targeting GCase emerge as a promising therapeutic approach to treat GD and PD, with pre-clinical and clinical trials ongoing. In this study, we resolve the complex of GCase and LIMP-2 using cryo-electron microscopy with the aid of an engineered LIMP-2 shuttle and two GCase-targeted pro-macrobodies. We identify helix 5 and helix 7 of LIMP-2 to interact with a binding pocket in GCase, forming a mostly hydrophobic interaction interface supported by one essential salt bridge. Understanding the interplay of GCase and LIMP-2 on a structural level is crucial to identify potential activation sites and conceptualizing novel therapeutic approaches targeting GCase. Here, we unveil the protein structure of a mannose-6-phosphate-independent lysosomal transport complex and provide fundamental knowledge for translational clinical research to overcome GD and PD. | ||||||||||||||||||
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画像 | ![]() | 146.5 KB | ||
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-検証レポート
文書・要旨 | ![]() | 189.7 KB | 表示 | ![]() |
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文書・詳細版 | ![]() | 189.3 KB | 表示 | |
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-関連構造データ
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EMDBのページ | ![]() ![]() |
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「今月の分子」の関連する項目 |
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マップ
ファイル | ![]() | ||||||||||||||||||||||||||||||||||||
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注釈 | Composite Map of GCase/LIMP-2/PMbs tetramer, generated in Phenix. Obtained from Consensus map and focused local refinements of GCase and LIMP-2. | ||||||||||||||||||||||||||||||||||||
投影像・断面図 | 画像のコントロール
画像は Spider により作成 | ||||||||||||||||||||||||||||||||||||
ボクセルのサイズ | X=Y=Z: 0.925 Å | ||||||||||||||||||||||||||||||||||||
密度 |
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対称性 | 空間群: 1 | ||||||||||||||||||||||||||||||||||||
詳細 | EMDB XML:
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-添付データ
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試料の構成要素
-全体 : Lysosomal transporting complex of beta-glucocerebrosidase and lys...
全体 | 名称: Lysosomal transporting complex of beta-glucocerebrosidase and lysosomal integral membrane protein 2 (LIMP-2) with two bound Pro-macrobodies. |
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要素 |
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-超分子 #1: Lysosomal transporting complex of beta-glucocerebrosidase and lys...
超分子 | 名称: Lysosomal transporting complex of beta-glucocerebrosidase and lysosomal integral membrane protein 2 (LIMP-2) with two bound Pro-macrobodies. タイプ: complex / ID: 1 / 親要素: 0 / 含まれる分子: #2, #4, #1, #3 詳細: Maltose-binding protein (MBP) domains of Pro-macrobodies were not resolved, only nanobody domains are included in the model. |
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由来(天然) | 生物種: ![]() |
分子量 | 理論値: 250 KDa |
-分子 #1: Lysosome membrane protein 2
分子 | 名称: Lysosome membrane protein 2 / タイプ: protein_or_peptide / ID: 1 / コピー数: 1 / 光学異性体: LEVO |
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由来(天然) | 生物種: ![]() |
分子量 | 理論値: 44.862691 KDa |
組換発現 | 生物種: ![]() |
配列 | 文字列: KKIVLRNGTE AFDSWEKPPL PVYTQFYFFN VTNPEEILRG ETPRVEEVGP YTYRELRNKA NIQFGDNGTT ISAVSNKAYV FERDQSVGD PKIDLIRTLN IPVLTVIEWS QVHFLREIIE AMLKAYQQKL FVTHTVDELL WGYKDEILSL IHVFRPDISP Y FGLFYEKN ...文字列: KKIVLRNGTE AFDSWEKPPL PVYTQFYFFN VTNPEEILRG ETPRVEEVGP YTYRELRNKA NIQFGDNGTT ISAVSNKAYV FERDQSVGD PKIDLIRTLN IPVLTVIEWS QVHFLREIIE AMLKAYQQKL FVTHTVDELL WGYKDEILSL IHVFRPDISP Y FGLFYEKN GTNDGDYVFL TGEDSYLNFT KIVEWNGKTS LDWWITDKCN MINGTDGDSF HPLITKDEVL YVFPSDFCRS VY ITFSDYE SVQGLPAFRY KVPAEILANT SDNAGFCIPE GNCLGSGVLN VSICKNGAPI IMSFPHFYQA DERFVSAIEG MHP NQEDHE TFVDINPLTG IILKAAKRFQ INIYVKKLDD FVETGDIRTM VFPVMYLNES VHIDKETASR LKSMI UniProtKB: Lysosome membrane protein 2 |
-分子 #2: Lysosomal acid glucosylceramidase
分子 | 名称: Lysosomal acid glucosylceramidase / タイプ: protein_or_peptide / ID: 2 / コピー数: 1 / 光学異性体: LEVO / EC番号: glucosylceramidase |
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由来(天然) | 生物種: ![]() |
分子量 | 理論値: 55.659219 KDa |
組換発現 | 生物種: ![]() |
配列 | 文字列: ARPCIPKSFG YSSVVCVCNA TYCDSFDPPT FPALGTFSRY ESTRSGRRME LSMGPIQANH TGTGLLLTLQ PEQKFQKVKG FGGAMTDAA ALNILALSPP AQNLLLKSYF SEEGIGYNII RVPMASCDFS IRTYTYADTP DDFQLHNFSL PEEDTKLKIP L IHRALQLA ...文字列: ARPCIPKSFG YSSVVCVCNA TYCDSFDPPT FPALGTFSRY ESTRSGRRME LSMGPIQANH TGTGLLLTLQ PEQKFQKVKG FGGAMTDAA ALNILALSPP AQNLLLKSYF SEEGIGYNII RVPMASCDFS IRTYTYADTP DDFQLHNFSL PEEDTKLKIP L IHRALQLA QRPVSLLASP WTSPTWLKTN GAVNGKGSLK GQPGDIYHQT WARYFVKFLD AYAEHKLQFW AVTAENEPSA GL LSGYPFQ CLGFTPEHQR DFIARDLGPT LANSTHHNVR LLMLDDQRLL LPHWAKVVLT DPEAAKYVHG IAVHWYLDFL APA KATLGE THRLFPNTML FASEACVGSK FWEQSVRLGS WDRGMQYSHS IITNLLYHVV GWTDWNLALN PEGGPNWVRN FVDS PIIVD ITKDTFYKQP MFYHLGHFSK FIPEGSQRVG LVASQKNDLD AVALMHPDGS AVVVVLNRSS KDVPLTIKDP AVGFL ETIS PGYSIHTYLW RRQ UniProtKB: Lysosomal acid glucosylceramidase |
-分子 #3: Nanobody Nb1
分子 | 名称: Nanobody Nb1 / タイプ: protein_or_peptide / ID: 3 詳細: Pro-Macrobody; nanobody fused to maltose-binding protein (MBP) via Pro-Pro linker; MBP not resolved and not modeled. コピー数: 1 / 光学異性体: LEVO |
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由来(天然) | 生物種: synthetic construct (人工物) |
分子量 | 理論値: 14.409816 KDa |
組換発現 | 生物種: ![]() ![]() |
配列 | 文字列: QVQLVESGGG LVQPGGSLRL SCAASGFTLD YYAIGWFRQA PGKEREGVSC ISSSDGSTYY ADSAKGRFTI SRDNAKNTVY LQMNSLKPE DTAVYYCATD RGQCTYYSSG YYRDLRWYDY WGQGTQVTVP P |
-分子 #4: Nanobody Nb6
分子 | 名称: Nanobody Nb6 / タイプ: protein_or_peptide / ID: 4 詳細: Pro-Macrobody; nanobody fused to maltose-binding protein (MBP) via Pro-Pro linker; MBP not resolved and not modeled. コピー数: 1 / 光学異性体: LEVO |
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由来(天然) | 生物種: synthetic construct (人工物) |
分子量 | 理論値: 13.145612 KDa |
組換発現 | 生物種: ![]() ![]() |
配列 | 文字列: QVQLVESGGG LVQPGGSLRL SCAASGSIFS INTMGWYRQA PGKEREMVAY IITFGSTNYA DSVKGRFTIS GDNANNTMWL QMNSLKPED TAVYYCYAAI RPTDSSTYTS YWGQGTQVTV PP |
-分子 #8: 2-acetamido-2-deoxy-beta-D-glucopyranose
分子 | 名称: 2-acetamido-2-deoxy-beta-D-glucopyranose / タイプ: ligand / ID: 8 / コピー数: 7 / 式: NAG |
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分子量 | 理論値: 221.208 Da |
Chemical component information | ![]() ChemComp-NAG: |
-分子 #9: 2-(N-MORPHOLINO)-ETHANESULFONIC ACID
分子 | 名称: 2-(N-MORPHOLINO)-ETHANESULFONIC ACID / タイプ: ligand / ID: 9 / コピー数: 4 / 式: MES |
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分子量 | 理論値: 195.237 Da |
Chemical component information | ![]() ChemComp-MES: |
-分子 #10: water
分子 | 名称: water / タイプ: ligand / ID: 10 / コピー数: 27 / 式: HOH |
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分子量 | 理論値: 18.015 Da |
Chemical component information | ![]() ChemComp-HOH: |
-実験情報
-構造解析
手法 | クライオ電子顕微鏡法 |
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![]() | 単粒子再構成法 |
試料の集合状態 | particle |
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試料調製
濃度 | 0.6 mg/mL | |||||||||
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緩衝液 | pH: 7.4 構成要素:
詳細: 20 mM MES; 150 mM NaCl, pH: 7.4 | |||||||||
グリッド | モデル: C-flat-1.2/1.3 / 材質: COPPER / メッシュ: 300 / 支持フィルム - 材質: CARBON / 支持フィルム - トポロジー: HOLEY / 支持フィルム - Film thickness: 2 / 前処理 - タイプ: GLOW DISCHARGE / 前処理 - 時間: 90 sec. / 詳細: Protochips; CF-1.2/1.3-3Cu-50 | |||||||||
凍結 | 凍結剤: ETHANE / チャンバー内湿度: 100 % / チャンバー内温度: 277 K / 装置: FEI VITROBOT MARK IV | |||||||||
詳細 | Monodisperse, complex seperated from monomers via size exclusion chhromatography. |
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電子顕微鏡法
顕微鏡 | TFS GLACIOS |
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特殊光学系 | エネルギーフィルター - 名称: TFS Selectris / エネルギーフィルター - スリット幅: 10 eV |
撮影 | フィルム・検出器のモデル: FEI FALCON IV (4k x 4k) 検出モード: COUNTING / 撮影したグリッド数: 1 / 実像数: 10550 / 平均電子線量: 50.0 e/Å2 |
電子線 | 加速電圧: 200 kV / 電子線源: ![]() |
電子光学系 | 照射モード: FLOOD BEAM / 撮影モード: BRIGHT FIELD / 最大 デフォーカス(公称値): 2.0 µm / 最小 デフォーカス(公称値): 0.6 µm / 倍率(公称値): 130000 |
試料ステージ | ホルダー冷却材: NITROGEN |
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画像解析
-原子モデル構築 1
初期モデル | PDB ID: Chain - Chain ID: AAA / Chain - Residue range: 1-497 / Chain - Source name: PDB / Chain - Initial model type: experimental model / 詳細: GCase |
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詳細 | Initial fitting with ChimeraX, manual flexible fitting with Coot, refinement with Phenix |
精密化 | 空間: REAL / プロトコル: FLEXIBLE FIT |
得られたモデル | ![]() PDB-9fjf: |
-原子モデル構築 2
初期モデル | PDB ID: Chain - Chain ID: A / Chain - Residue range: 37-430 / Chain - Source name: PDB / Chain - Initial model type: experimental model / 詳細: LIMP-2 |
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詳細 | Initial fitting with ChimeraX, manual flexible fitting with Coot, refinement with Phenix |
精密化 | 空間: REAL / プロトコル: FLEXIBLE FIT |
得られたモデル | ![]() PDB-9fjf: |
-原子モデル構築 3
初期モデル | PDB ID: Chain - Source name: PDB / Chain - Initial model type: experimental model / 詳細: Nanobody Nb1; Unpublished |
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詳細 | Initial fitting with ChimeraX, manual flexible fitting with Coot, refinement with Phenix |
精密化 | 空間: REAL / プロトコル: RIGID BODY FIT |
得られたモデル | ![]() PDB-9fjf: |