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- PDB-8vku: Structure of VCP in complex with an ATPase activator (D2 domains ... -
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Basic information
Entry | Database: PDB / ID: 8vku | ||||||
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Title | Structure of VCP in complex with an ATPase activator (D2 domains only, hexameric form) | ||||||
![]() | Transitional endoplasmic reticulum ATPase | ||||||
![]() | HYDROLASE/ACTIVATOR / activator / complex / ATPase / AAA protein / HYDROLASE / HYDROLASE-ACTIVATOR complex | ||||||
Function / homology | ![]() positive regulation of Lys63-specific deubiquitinase activity / flavin adenine dinucleotide catabolic process / positive regulation of oxidative phosphorylation / VCP-NSFL1C complex / endosome to lysosome transport via multivesicular body sorting pathway / protein-DNA covalent cross-linking repair / endoplasmic reticulum stress-induced pre-emptive quality control / cellular response to arsenite ion / Derlin-1 retrotranslocation complex / BAT3 complex binding ...positive regulation of Lys63-specific deubiquitinase activity / flavin adenine dinucleotide catabolic process / positive regulation of oxidative phosphorylation / VCP-NSFL1C complex / endosome to lysosome transport via multivesicular body sorting pathway / protein-DNA covalent cross-linking repair / endoplasmic reticulum stress-induced pre-emptive quality control / cellular response to arsenite ion / Derlin-1 retrotranslocation complex / BAT3 complex binding / positive regulation of protein K63-linked deubiquitination / deubiquitinase activator activity / aggresome assembly / regulation of protein localization to chromatin / vesicle-fusing ATPase / mitotic spindle disassembly / VCP-NPL4-UFD1 AAA ATPase complex / NADH metabolic process / cellular response to misfolded protein / stress granule disassembly / K48-linked polyubiquitin modification-dependent protein binding / positive regulation of mitochondrial membrane potential / negative regulation of protein localization to chromatin / ubiquitin-modified protein reader activity / retrograde protein transport, ER to cytosol / regulation of aerobic respiration / positive regulation of ATP biosynthetic process / regulation of synapse organization / ATPase complex / ubiquitin-specific protease binding / ubiquitin-like protein ligase binding / MHC class I protein binding / RHOH GTPase cycle / autophagosome maturation / HSF1 activation / polyubiquitin modification-dependent protein binding / proteasomal protein catabolic process / Protein methylation / endoplasmic reticulum to Golgi vesicle-mediated transport / translesion synthesis / interstrand cross-link repair / negative regulation of smoothened signaling pathway / ERAD pathway / ATP metabolic process / Attachment and Entry / endoplasmic reticulum unfolded protein response / viral genome replication / lipid droplet / proteasome complex / Josephin domain DUBs / N-glycan trimming in the ER and Calnexin/Calreticulin cycle / Hh mutants are degraded by ERAD / macroautophagy / Defective CFTR causes cystic fibrosis / Hedgehog ligand biogenesis / Translesion Synthesis by POLH / positive regulation of protein-containing complex assembly / ABC-family proteins mediated transport / establishment of protein localization / ADP binding / autophagy / Aggrephagy / cytoplasmic stress granule / positive regulation of non-canonical NF-kappaB signal transduction / positive regulation of protein catabolic process / activation of cysteine-type endopeptidase activity involved in apoptotic process / KEAP1-NFE2L2 pathway / azurophil granule lumen / double-strand break repair / Ovarian tumor domain proteases / positive regulation of proteasomal ubiquitin-dependent protein catabolic process / positive regulation of canonical Wnt signaling pathway / E3 ubiquitin ligases ubiquitinate target proteins / Neddylation / site of double-strand break / cellular response to heat / ubiquitin-dependent protein catabolic process / regulation of apoptotic process / protein phosphatase binding / proteasome-mediated ubiquitin-dependent protein catabolic process / secretory granule lumen / ficolin-1-rich granule lumen / Attachment and Entry / protein ubiquitination / protein domain specific binding / intracellular membrane-bounded organelle / DNA repair / glutamatergic synapse / lipid binding / ubiquitin protein ligase binding / DNA damage response / Neutrophil degranulation / endoplasmic reticulum membrane / perinuclear region of cytoplasm / endoplasmic reticulum / ATP hydrolysis activity / protein-containing complex / RNA binding / extracellular exosome / extracellular region Similarity search - Function | ||||||
Biological species | ![]() | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.5 Å | ||||||
![]() | Jones, N.H. / Urnivicius, L. / Kapoor, T.M. | ||||||
Funding support | ![]()
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![]() | ![]() Title: Allosteric activation of VCP, an AAA unfoldase, by small molecule mimicry. Authors: Natalie H Jones / Qiwen Liu / Linas Urnavicius / Noa E Dahan / Lauren E Vostal / Tarun M Kapoor / ![]() Abstract: 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: Journal: bioRxiv / Year: 2023 Title: Allosteric activation of VCP, a AAA unfoldase, by small molecule mimicry. Authors: N H Jones / Q Liu / L Urnavicius / N E Dahan / L E Vostal / T M Kapoor Abstract: 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. | ||||||
History |
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Structure visualization
Structure viewer | Molecule: ![]() ![]() |
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-Validation report
Summary document | ![]() | 1.5 MB | Display | ![]() |
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Full document | ![]() | 1.5 MB | Display | |
Data in XML | ![]() | 63.9 KB | Display | |
Data in CIF | ![]() | 88.4 KB | Display | |
Arichive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 43329MC ![]() 8vlsC ![]() 8vovC M: map data used to model this data C: citing same article ( |
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Similar structure data | Similarity search - Function & homology ![]() |
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Links
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Assembly
Deposited unit | ![]()
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Components
#1: Protein | Mass: 89436.820 Da / Num. of mol.: 6 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() #2: Chemical | ChemComp-A1AC1 / ( Mass: 354.466 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C20H22N2O2S / Feature type: SUBJECT OF INVESTIGATION Has ligand of interest | Y | |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
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Sample preparation
Component | Name: Complex of VCP with ATPase activator small molecule VAA1 Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT | |||||||||||||||||||||||||||||||||||
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Source (natural) | Organism: ![]() | |||||||||||||||||||||||||||||||||||
Source (recombinant) | Organism: ![]() ![]() | |||||||||||||||||||||||||||||||||||
Buffer solution | pH: 7.5 Details: 50 mM K.HEPES pH 7.5, 25 mM KCl, 2.5 mM MgCl2, 2.5 mM GSH, 0.5% DMSO, 0.01% FOM | |||||||||||||||||||||||||||||||||||
Buffer component |
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Specimen | Conc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES | |||||||||||||||||||||||||||||||||||
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 298 K |
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Electron microscopy imaging
Experimental equipment | ![]() Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: ![]() |
Electron lens | Mode: BRIGHT FIELD / Nominal defocus max: 4000 nm / Nominal defocus min: 1000 nm |
Image recording | Electron dose: 49 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) |
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Processing
EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 5933232 / Details: Autopicking | ||||||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: C6 (6 fold cyclic) | ||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 84156 / Symmetry type: POINT | ||||||||||||||||||||||||||||||||||||
Atomic model building | Space: REAL | ||||||||||||||||||||||||||||||||||||
Atomic model building | PDB-ID: 5FTL Accession code: 5FTL / Source name: PDB / Type: experimental model | ||||||||||||||||||||||||||||||||||||
Refinement | Cross valid method: NONE Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2 | ||||||||||||||||||||||||||||||||||||
Displacement parameters | Biso mean: 81.62 Å2 | ||||||||||||||||||||||||||||||||||||
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