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- PDB-8vku: Structure of VCP in complex with an ATPase activator (D2 domains ... -

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基本情報

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データベース: PDB / ID: 8vku
タイトルStructure of VCP in complex with an ATPase activator (D2 domains only, hexameric form)
要素Transitional endoplasmic reticulum ATPase
キーワードHYDROLASE/ACTIVATOR / activator / complex / ATPase / AAA protein / HYDROLASE / HYDROLASE-ACTIVATOR complex
機能・相同性
機能・相同性情報


flavin adenine dinucleotide catabolic process / VCP-NSFL1C complex / endoplasmic reticulum stress-induced pre-emptive quality control / endosome to lysosome transport via multivesicular body sorting pathway / BAT3 complex binding / cytoplasmic ubiquitin ligase complex / cellular response to arsenite ion / protein-DNA covalent cross-linking repair / Derlin-1 retrotranslocation complex / positive regulation of protein K63-linked deubiquitination ...flavin adenine dinucleotide catabolic process / VCP-NSFL1C complex / endoplasmic reticulum stress-induced pre-emptive quality control / endosome to lysosome transport via multivesicular body sorting pathway / BAT3 complex binding / cytoplasmic ubiquitin ligase complex / cellular response to arsenite ion / protein-DNA covalent cross-linking repair / Derlin-1 retrotranslocation complex / positive regulation of protein K63-linked deubiquitination / deubiquitinase activator activity / cytoplasm protein quality control / positive regulation of oxidative phosphorylation / aggresome assembly / ubiquitin-modified protein reader activity / regulation of protein localization to chromatin / cellular response to misfolded protein / mitotic spindle disassembly / VCP-NPL4-UFD1 AAA ATPase complex / positive regulation of mitochondrial membrane potential / vesicle-fusing ATPase / K48-linked polyubiquitin modification-dependent protein binding / NAD+ metabolic process / regulation of aerobic respiration / retrograde protein transport, ER to cytosol / stress granule disassembly / ATPase complex / ubiquitin-specific protease binding / regulation of synapse organization / ciliary transition zone / positive regulation of ATP biosynthetic process / ubiquitin-like protein ligase binding / intracellular membrane-bounded organelle / RHOH GTPase cycle / MHC class I protein binding / autophagosome maturation / endoplasmic reticulum to Golgi vesicle-mediated transport / negative regulation of hippo signaling / HSF1 activation / polyubiquitin modification-dependent protein binding / interstrand cross-link repair / ATP metabolic process / translesion synthesis / Attachment and Entry / Protein methylation / endoplasmic reticulum unfolded protein response / ERAD pathway / negative regulation of protein localization to chromatin / proteasomal protein catabolic process / lipid droplet / ciliary tip / proteasome complex / viral genome replication / Josephin domain DUBs / macroautophagy / negative regulation of smoothened signaling pathway / N-glycan trimming in the ER and Calnexin/Calreticulin cycle / establishment of protein localization / positive regulation of protein-containing complex assembly / Hh mutants are degraded by ERAD / Hedgehog ligand biogenesis / Translesion Synthesis by POLH / Defective CFTR causes cystic fibrosis / ADP binding / positive regulation of non-canonical NF-kappaB signal transduction / ABC-family protein mediated transport / autophagy / cytoplasmic stress granule / Aggrephagy / positive regulation of protein catabolic process / azurophil granule lumen / Ovarian tumor domain proteases / KEAP1-NFE2L2 pathway / positive regulation of canonical Wnt signaling pathway / double-strand break repair / positive regulation of proteasomal ubiquitin-dependent protein catabolic process / cellular response to heat / E3 ubiquitin ligases ubiquitinate target proteins / site of double-strand break / Neddylation / secretory granule lumen / protein phosphatase binding / regulation of apoptotic process / ficolin-1-rich granule lumen / ubiquitin-dependent protein catabolic process / Attachment and Entry / proteasome-mediated ubiquitin-dependent protein catabolic process / ciliary basal body / protein ubiquitination / protein domain specific binding / DNA repair / DNA damage response / Neutrophil degranulation / ubiquitin protein ligase binding / lipid binding / endoplasmic reticulum membrane / perinuclear region of cytoplasm / glutamatergic synapse / endoplasmic reticulum / ATP hydrolysis activity
類似検索 - 分子機能
AAA ATPase, CDC48 family / Cell division protein 48 (CDC48), N-terminal domain / : / CDC48, N-terminal subdomain / Cell division protein 48 (CDC48) N-terminal domain / CDC48, domain 2 / Cell division protein 48 (CDC48), domain 2 / Cell division protein 48 (CDC48) domain 2 / CDC48 domain 2-like superfamily / Aspartate decarboxylase-like domain superfamily ...AAA ATPase, CDC48 family / Cell division protein 48 (CDC48), N-terminal domain / : / CDC48, N-terminal subdomain / Cell division protein 48 (CDC48) N-terminal domain / CDC48, domain 2 / Cell division protein 48 (CDC48), domain 2 / Cell division protein 48 (CDC48) domain 2 / CDC48 domain 2-like superfamily / Aspartate decarboxylase-like domain superfamily / AAA ATPase, AAA+ lid domain / AAA+ lid domain / ATPase, AAA-type, conserved site / AAA-protein family signature. / ATPase family associated with various cellular activities (AAA) / ATPase, AAA-type, core / 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
: / Transitional endoplasmic reticulum ATPase
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: 3.5 Å
データ登録者Jones, N.H. / Urnivicius, L. / Kapoor, T.M.
資金援助 米国, 1件
組織認可番号国
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM130234 米国
引用
ジャーナル: Proc Natl Acad Sci U S A / 年: 2024
タイトル: Allosteric activation of VCP, an AAA unfoldase, by small molecule mimicry.
著者: Natalie H Jones / Qiwen Liu / Linas Urnavicius / Noa E Dahan / Lauren E Vostal / Tarun M Kapoor /
要旨: The loss of function of AAA (ATPases associated with diverse cellular activities) mechanoenzymes has been linked to diseases, and small molecules that activate these proteins can be powerful tools to ...The loss of function of AAA (ATPases associated with diverse cellular activities) mechanoenzymes has been linked to diseases, and small molecules that activate these proteins can be powerful tools to probe mechanisms and test therapeutic hypotheses. Unlike chemical inhibitors that can bind a single conformational state to block enzyme function, activator binding must be permissive to different conformational states needed for mechanochemistry. However, we do not know how AAA proteins can be activated by small molecules. Here, we focus on valosin-containing protein (VCP)/p97, an AAA unfoldase whose loss of function has been linked to protein aggregation-based disorders, to identify druggable sites for chemical activators. We identified VCP ATPase Activator 1 (VAA1), a compound that dose-dependently stimulates VCP ATPase activity up to ~threefold. Our cryo-EM studies resulted in structures (ranging from ~2.9 to 3.7 Å-resolution) of VCP in apo and ADP-bound states and revealed that VAA1 binds an allosteric pocket near the C-terminus in both states. Engineered mutations in the VAA1-binding site confer resistance to VAA1, and furthermore, modulate VCP activity. Mutation of a phenylalanine residue in the VCP C-terminal tail that can occupy the VAA1 binding site also stimulates ATPase activity, suggesting that VAA1 acts by mimicking this interaction. Together, our findings uncover a druggable allosteric site and a mechanism of enzyme regulation that can be tuned through small molecule mimicry.
#1: ジャーナル: bioRxiv / 年: 2023
タイトル: Allosteric activation of VCP, a AAA unfoldase, by small molecule mimicry.
著者: N H Jones / Q Liu / L Urnavicius / N E Dahan / L E Vostal / T M Kapoor
要旨: The loss of function of AAA (ATPases associated with diverse cellular activities) mechanoenzymes has been linked to diseases, and small molecules that activate these proteins can be powerful tools to ...The loss of function of AAA (ATPases associated with diverse cellular activities) mechanoenzymes has been linked to diseases, and small molecules that activate these proteins can be powerful tools to probe mechanisms and test therapeutic hypotheses. Unlike chemical inhibitors that can bind a single conformational state to block enzyme activity, activator binding must be permissive to different conformational states needed for enzyme function. However, we do not know how AAA proteins can be activated by small molecules. Here, we focus on valosin-containing protein (VCP)/p97, a AAA unfoldase whose loss of function has been linked to protein aggregation-based disorders, to identify druggable sites for chemical activators. We identified VCP Activator 1 (VA1), a compound that dose-dependently stimulates VCP ATPase activity up to ∼3-fold. Our cryo-EM studies resulted in structures (∼2.9-3.5 Å-resolution) of VCP in apo and ADP-bound states, and revealed VA1 binding an allosteric pocket near the C-terminus in both states. Engineered mutations in the VA1 binding site confer resistance to VA1, and furthermore, modulate VCP activity to a similar level as VA1-mediated activation. The VA1 binding site can alternatively be occupied by a phenylalanine residue in the VCP C-terminal tail, a motif that is post-translationally modified and interacts with cofactors. Together, our findings uncover a druggable allosteric site and a mechanism of enzyme regulation that can be tuned through small molecule mimicry.
SIGNIFICANCE: The loss of function of valosin-containing protein (VCP/p97), a mechanoenzyme from the AAA superfamily that hydrolyzes ATP and uses the released energy to extract or unfold substrate ...SIGNIFICANCE: The loss of function of valosin-containing protein (VCP/p97), a mechanoenzyme from the AAA superfamily that hydrolyzes ATP and uses the released energy to extract or unfold substrate proteins, is linked to protein aggregation-based disorders. However, druggable allosteric sites to activate VCP, or any AAA mechanoenzyme, have not been identified. Here, we report cryo-EM structures of VCP in two states in complex with VA1, a compound we identified that dose-dependently stimulates VCP's ATP hydrolysis activity. The VA1 binding site can also be occupied by a phenylalanine residue in the VCP C-terminal tail, suggesting that VA1 acts through mimicry of this interaction. Our study reveals a druggable allosteric site and a mechanism of enzyme regulation.
履歴
登録2024年1月9日登録サイト: RCSB / 処理サイト: RCSB
改定 1.02024年6月19日Provider: repository / タイプ: Initial release

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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: Transitional endoplasmic reticulum ATPase
B: Transitional endoplasmic reticulum ATPase
C: Transitional endoplasmic reticulum ATPase
D: Transitional endoplasmic reticulum ATPase
E: Transitional endoplasmic reticulum ATPase
F: Transitional endoplasmic reticulum ATPase
ヘテロ分子


分子量 (理論値)分子数
合計 (水以外)538,74812
ポリマ－536,6216
非ポリマー2,1276
水00
1


  • 登録構造と同一
  • 登録者が定義した集合体
  • 根拠: 電子顕微鏡法, not applicable
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
Transitional endoplasmic reticulum ATPase / TER ATPase / 15S Mg(2+)-ATPase p97 subunit / Valosin-containing protein / VCP


分子量: 89436.820 Da / 分子数: 6 / 由来タイプ: 組換発現 / 由来: (組換発現) Homo sapiens (ヒト) / 遺伝子: VCP / 発現宿主: Escherichia coli (大腸菌) / 参照: UniProt: P55072, vesicle-fusing ATPase
#2: 化合物
ChemComp-A1AC1 / (3R)-N-[2-(ethylsulfanyl)phenyl]-3-(1-oxo-1,3-dihydro-2H-isoindol-2-yl)butanamide


分子量: 354.466 Da / 分子数: 6 / 由来タイプ: 合成 / 式: C20H22N2O2S / タイプ: SUBJECT OF INVESTIGATION
研究の焦点であるリガンドがあるかY

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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 of VCP with ATPase activator small molecule VAA1
タイプ: COMPLEX / Entity ID: #1 / 由来: RECOMBINANT
由来(天然)生物種: Homo sapiens (ヒト)
由来(組換発現)生物種: Escherichia coli (大腸菌)
緩衝液pH: 7.5
詳細: 50 mM K.HEPES pH 7.5, 25 mM KCl, 2.5 mM MgCl2, 2.5 mM GSH, 0.5% DMSO, 0.01% FOM
緩衝液成分
ID濃度名称式Buffer-ID
150 mMHEPESC8H18N2O4S1
225 mMpotassium chlorideKCl1
32.5 mMmagnesium chlorideMgCl21
42.5 mMglutathioneC10H17N3O6S1
50.5 %DMSO(CH3)2SO1
60.01 %FOMC20H25F13O111
試料濃度: 1 mg/ml / 包埋: NO / シャドウイング: NO / 染色: NO / 凍結: YES
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: 298 K

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電子顕微鏡撮影

実験機器
モデル: Titan Krios / 画像提供: FEI Company
顕微鏡モデル: FEI TITAN KRIOS
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: BRIGHT FIELD / 最大 デフォーカス（公称値）: 4000 nm / 最小 デフォーカス（公称値）: 1000 nm
撮影電子線照射量: 49 e/Å2 / フィルム・検出器のモデル: GATAN K3 (6k x 4k)

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解析

EMソフトウェア
ID名称バージョンカテゴリ
1RELION粒子像選択
4CTFFIND4CTF補正
7UCSF Chimeraモデルフィッティング
8Cootモデルフィッティング
10Cootモデル精密化
11PHENIXモデル精密化
14RELION分類
15RELION３次元再構成
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: 5933232 / 詳細: Autopicking
対称性点対称性: C6 (6回回転対称)
3次元再構成解像度: 3.5 Å / 解像度の算出法: FSC 0.143 CUT-OFF / 粒子像の数: 84156 / 対称性のタイプ: POINT
原子モデル構築空間: REAL
原子モデル構築PDB-ID: 5FTL

5ftl


Accession code: 5FTL / 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: 81.62 Å2
拘束条件
Refine-IDタイプDev ideal数
ELECTRON MICROSCOPYf_bond_d0.003413416
ELECTRON MICROSCOPYf_angle_d0.57818084
ELECTRON MICROSCOPYf_chiral_restr0.04051992
ELECTRON MICROSCOPYf_plane_restr0.00572364
ELECTRON MICROSCOPYf_dihedral_angle_d4.4521794

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