oxidation-dependent protein catabolic process / PH domain binding / endopeptidase La / mitochondrial protein catabolic process / G-quadruplex DNA binding / mitochondrial DNA metabolic process / mitochondrial genome maintenance / ATP-dependent peptidase activity / protein quality control for misfolded or incompletely synthesized proteins / mitochondrial nucleoid ...oxidation-dependent protein catabolic process / PH domain binding / endopeptidase La / mitochondrial protein catabolic process / G-quadruplex DNA binding / mitochondrial DNA metabolic process / mitochondrial genome maintenance / ATP-dependent peptidase activity / protein quality control for misfolded or incompletely synthesized proteins / mitochondrial nucleoid / insulin receptor substrate binding / chaperone-mediated protein complex assembly / DNA polymerase binding / regulation of peptidyl-tyrosine phosphorylation / Mitochondrial protein degradation / negative regulation of insulin receptor signaling pathway / mitochondrion organization / proteolysis involved in protein catabolic process / protein catabolic process / ADP binding / single-stranded DNA binding / cellular response to oxidative stress / sequence-specific DNA binding / single-stranded RNA binding / response to hypoxia / mitochondrial matrix / serine-type endopeptidase activity / ATP hydrolysis activity / mitochondrion / nucleoplasm / ATP binding / identical protein binding / membrane / cytosol 類似検索 - 分子機能
Lon protease homologue, chloroplastic/mitochondrial / Lon protease, bacterial/eukaryotic-type / Peptidase S16, active site / ATP-dependent serine proteases, lon family, serine active site. / Lon proteolytic domain profile. / Peptidase S16, Lon proteolytic domain / Lon protease / Lon protease (S16) C-terminal proteolytic domain / Lon protease, N-terminal domain superfamily / Lon N-terminal domain profile. ...Lon protease homologue, chloroplastic/mitochondrial / Lon protease, bacterial/eukaryotic-type / Peptidase S16, active site / ATP-dependent serine proteases, lon family, serine active site. / Lon proteolytic domain profile. / Peptidase S16, Lon proteolytic domain / Lon protease / Lon protease (S16) C-terminal proteolytic domain / Lon protease, N-terminal domain superfamily / Lon N-terminal domain profile. / Lon protease, N-terminal domain / ATP-dependent protease La (LON) substrate-binding domain / Found in ATP-dependent protease La (LON) / PUA-like superfamily / ATPase family associated with various cellular activities (AAA) / ATPase, AAA-type, core / Ribosomal protein S5 domain 2-type fold, subgroup / Ribosomal protein S5 domain 2-type fold / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase 類似検索 - ドメイン・相同性
National Institutes of Health/National Institute on Aging (NIH/NIA)
AG067594
米国
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)
NS095892
米国
National Institutes of Health/National Institute on Aging (NIH/NIA)
AG061697
米国
National Institutes of Health/Office of the Director
S10OD021634
米国
引用
ジャーナル: Nat Commun / 年: 2021 タイトル: Structures of the human LONP1 protease reveal regulatory steps involved in protease activation. 著者: Mia Shin / Edmond R Watson / Albert S Song / Jeffrey T Mindrebo / Scott J Novick / Patrick R Griffin / R Luke Wiseman / Gabriel C Lander / 要旨: The human mitochondrial AAA+ protein LONP1 is a critical quality control protease involved in regulating diverse aspects of mitochondrial biology including proteostasis, electron transport chain ...The human mitochondrial AAA+ protein LONP1 is a critical quality control protease involved in regulating diverse aspects of mitochondrial biology including proteostasis, electron transport chain activity, and mitochondrial transcription. As such, genetic or aging-associated imbalances in LONP1 activity are implicated in pathologic mitochondrial dysfunction associated with numerous human diseases. Despite this importance, the molecular basis for LONP1-dependent proteolytic activity remains poorly defined. Here, we solved cryo-electron microscopy structures of human LONP1 to reveal the underlying molecular mechanisms governing substrate proteolysis. We show that, like bacterial Lon, human LONP1 adopts both an open and closed spiral staircase orientation dictated by the presence of substrate and nucleotide. Unlike bacterial Lon, human LONP1 contains a second spiral staircase within its ATPase domain that engages substrate as it is translocated toward the proteolytic chamber. Intriguingly, and in contrast to its bacterial ortholog, substrate binding within the central ATPase channel of LONP1 alone is insufficient to induce the activated conformation of the protease domains. To successfully induce the active protease conformation in substrate-bound LONP1, substrate binding within the protease active site is necessary, which we demonstrate by adding bortezomib, a peptidomimetic active site inhibitor of LONP1. These results suggest LONP1 can decouple ATPase and protease activities depending on whether AAA+ or both AAA+ and protease domains bind substrate. Importantly, our structures provide a molecular framework to define the critical importance of LONP1 in regulating mitochondrial proteostasis in health and disease.
超分子 #1: Human mitochondrial LONP1 in complex with Bortezomib
超分子
名称: Human mitochondrial LONP1 in complex with Bortezomib タイプ: complex / ID: 1 / 親要素: 0 / 含まれる分子: #1-#2 詳細: Complexes consisting of homohexameric LONP1 protease from Homo sapiens bound to endogenous co-purified substrate and Bortezomib.
由来(天然)
生物種: Homo sapiens (ヒト) / Organelle: Mitochondria / 細胞中の位置: Matrix
詳細: Solutions were made fresh from concentrated and filtered using a 0.1 um syringe filter to avoid microbial contamination. Samples were mixed on ice and incubated at 37 degrees C for 30 minutes ...詳細: Solutions were made fresh from concentrated and filtered using a 0.1 um syringe filter to avoid microbial contamination. Samples were mixed on ice and incubated at 37 degrees C for 30 minutes to ensure Bortezomib binding. Additional ATP was added to the sample mix on ice 5 minutes prior to vitrification.
凍結剤: ETHANE / チャンバー内湿度: 95 % / チャンバー内温度: 277 K / 装置: HOMEMADE PLUNGER 詳細: 4 uL of sample was applied per grid and manually blotted for 4 seconds followed by immediately plunge-freezing in liquid ethane cooled by liquid nitrogen..
詳細
This sample was monodisperse.
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電子顕微鏡法
顕微鏡
FEI TALOS ARCTICA
温度
最低: 80.0 K / 最高: 90.0 K
アライメント法
Coma free - Residual tilt: 0.14 mrad
詳細
Coma-free alignment procedure from Herzik & Wu, Nature Methods (2017). Preliminary grid screening was performed manually prior to data collection.
選択した数: 2736565 / 詳細: Template-based particle selection in RELION
CTF補正
ソフトウェア - 名称: RELION (ver. 3.1) ソフトウェア - 詳細: CTF correction was done using RELION's implementation of gCTF during refinement 詳細: CTF parameters were estimated with gCTF
初期モデル
モデルのタイプ: OTHER 詳細: A low-resolution negative stain reconstruction of Human mitochondrial LONP1 was used as an initial model.
最終 再構成
使用したクラス数: 1 / 解像度のタイプ: BY AUTHOR / 解像度: 3.2 Å / 解像度の算出法: FSC 0.143 CUT-OFF / ソフトウェア - 名称: RELION (ver. 3.1) ソフトウェア - 詳細: RELION 3.1 was used to perform final reconstruction 使用した粒子像数: 532298
初期 角度割当
タイプ: MAXIMUM LIKELIHOOD / ソフトウェア - 名称: RELION (ver. 3.1) ソフトウェア - 詳細: RELION 3.1 was used to assign initial euler angles
最終 角度割当
タイプ: MAXIMUM LIKELIHOOD / ソフトウェア - 名称: RELION (ver. 3.1) ソフトウェア - 詳細: RELION 3.1 was used to assign final euler angles 詳細: RELION 3.1 was used to assign initial angles
最終 3次元分類
クラス数: 2 / 平均メンバー数/クラス: 500000 / ソフトウェア - 名称: RELION (ver. 3.1) ソフトウェア - 詳細: RELION 3.1 was used to perform final classification 詳細: The final 3D classification had an somewhat even distribution owing to preferred specimen orientation due to differential interactions with the air-water interface
FSC曲線 (解像度の算出)
-
原子モデル構築 1
詳細
Initial homology model was built using SWISS-MODEL and initial rigid body docking was done using UCSF Chimera.
精密化
空間: REAL / プロトコル: AB INITIO MODEL / 温度因子: 52 / 当てはまり具合の基準: Correlation coefficient
得られたモデル
PDB-7krz: Human mitochondrial LONP1 in complex with Bortezomib