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- PDB-7bp8: Human AAA+ ATPase VCP mutant - T76A, ADP-bound form -

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Basic information

Entry
Database: PDB / ID: 7bp8
TitleHuman AAA+ ATPase VCP mutant - T76A, ADP-bound form
ComponentsTransitional endoplasmic reticulum ATPase
KeywordsCELL CYCLE / Complex / ATPase / Unfoldase / Protein Transportation
Function / homology
Function and homology information


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 / BAT3 complex binding / Derlin-1 retrotranslocation complex ...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 / BAT3 complex binding / Derlin-1 retrotranslocation complex / positive regulation of protein K63-linked deubiquitination / deubiquitinase activator activity / mitotic spindle disassembly / VCP-NPL4-UFD1 AAA ATPase complex / aggresome assembly / regulation of protein localization to chromatin / NADH metabolic process / vesicle-fusing ATPase / 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 / ERAD pathway / ubiquitin-modified protein reader activity / retrograde protein transport, ER to cytosol / regulation of aerobic respiration / ATPase complex / regulation of synapse organization / ubiquitin-specific protease binding / positive regulation of ATP biosynthetic process / ubiquitin-like protein ligase binding / autophagosome maturation / RHOH GTPase cycle / polyubiquitin modification-dependent protein binding / HSF1 activation / translesion synthesis / endoplasmic reticulum to Golgi vesicle-mediated transport / MHC class I protein binding / Protein methylation / negative regulation of smoothened signaling pathway / interstrand cross-link repair / Attachment and Entry / : / ATP metabolic process / endoplasmic reticulum unfolded protein response / proteasome complex / lipid droplet / viral genome replication / Josephin domain DUBs / N-glycan trimming in the ER and Calnexin/Calreticulin cycle / ADP binding / proteasomal protein catabolic process / Hh mutants are degraded by ERAD / positive regulation of protein-containing complex assembly / Defective CFTR causes cystic fibrosis / Hedgehog ligand biogenesis / macroautophagy / Translesion Synthesis by POLH / ABC-family proteins mediated transport / establishment of protein localization / autophagy / Aggrephagy / cytoplasmic stress granule / positive regulation of non-canonical NF-kappaB signal transduction / positive regulation of canonical Wnt signaling pathway / 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 / E3 ubiquitin ligases ubiquitinate target proteins / site of double-strand break / cellular response to heat / Neddylation / ubiquitin-dependent protein catabolic process / proteasome-mediated ubiquitin-dependent protein catabolic process / protein phosphatase binding / regulation of apoptotic process / secretory granule lumen / ficolin-1-rich granule lumen / Attachment and Entry / protein ubiquitination / protein domain specific binding / DNA repair / intracellular membrane-bounded organelle / lipid binding / glutamatergic synapse / DNA damage response / ubiquitin protein ligase binding / Neutrophil degranulation / endoplasmic reticulum membrane / perinuclear region of cytoplasm / endoplasmic reticulum / ATP hydrolysis activity / protein-containing complex / RNA binding
Similarity search - Function
Vps4 C terminal oligomerisation domain / 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 ...Vps4 C terminal oligomerisation domain / 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
Similarity search - Domain/homology
ADENOSINE-5'-DIPHOSPHATE / Transitional endoplasmic reticulum ATPase
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsYang, C. / Zhang, H.
CitationJournal: Cell Death Differ / Year: 2022
Title: The phosphorylation and dephosphorylation switch of VCP/p97 regulates the architecture of centrosome and spindle.
Authors: Kaiyuan Zhu / Yang Cai / Xiaotong Si / Zuodong Ye / Yuanzhu Gao / Chuang Liu / Rui Wang / Zhibin Ma / Huazhang Zhu / Liang Zhang / Shengjin Li / Hongmin Zhang / Jianbo Yue /
Abstract: The proper orientation of centrosome and spindle is essential for genome stability; however, the mechanism that governs these processes remains elusive. Here, we demonstrated that polo-like kinase 1 ...The proper orientation of centrosome and spindle is essential for genome stability; however, the mechanism that governs these processes remains elusive. Here, we demonstrated that polo-like kinase 1 (Plk1), a key mitotic kinase, phosphorylates residue Thr76 in VCP/p97 (an AAA-ATPase), at the centrosome from prophase to anaphase. This phosphorylation process recruits VCP to the centrosome and in this way, it regulates centrosome orientation. VCP exhibits strong co-localization with Eg5 (a mitotic kinesin motor), at the mitotic spindle, and the dephosphorylation of Thr76 in VCP is required for the enrichment of both VCP and Eg5 at the spindle, thus ensuring proper spindle architecture and chromosome segregation. We also showed that the phosphatase, PTEN, is responsible for the dephosphorylation of Thr76 in VCP; when PTEN was knocked down, the normal spread of VCP from the centrosome to the spindle was abolished. Cryo-EM structures of VCP and VCP, which represent dephosphorylated and phosphorylated states of VCP, respectively, revealed that the Thr76 phosphorylation modulates VCP by altering the inter-domain and inter-subunit interactions, and ultimately the nucleotide-binding pocket conformation. Interestingly, the tumor growth in nude mice implanted with VCP-reconstituted cancer cells was significantly slower when compared with those implanted with VCP-reconstituted cancer cells. Collectively, our findings demonstrate that the phosphorylation and dephosphorylation switch of VCP regulates the architecture of centrosome and spindle for faithful chromosome segregation.
History
DepositionMar 21, 2020Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Mar 31, 2021Provider: repository / Type: Initial release
Revision 1.1Apr 27, 2022Group: Database references / Category: citation / citation_author / database_2
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Assembly

Deposited unit
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
hetero molecules


Theoretical massNumber of molelcules
Total (without water)541,56718
Polymers536,4416
Non-polymers5,12612
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Transitional endoplasmic reticulum ATPase / TER ATPase / 15S Mg(2+)-ATPase p97 subunit / Valosin-containing protein / VCP


Mass: 89406.789 Da / Num. of mol.: 6 / Mutation: T76A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: VCP / Plasmid: pET / Production host: Escherichia coli (E. coli) / Variant (production host): T7 SHuffle / References: UniProt: P55072, vesicle-fusing ATPase
#2: Chemical
ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE / Adenosine diphosphate


Mass: 427.201 Da / Num. of mol.: 12 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: ADP, energy-carrying molecule*YM
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Transitional endoplasmic reticulum ATPase, VCP. / Type: COMPLEX / Details: T76A mutant of VCP of ADP-bound form / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 97 kDa/nm / Experimental value: YES
Source (natural)Organism: Homo sapiens (human) / Cellular location: cytoplasm nucleus ER
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: T7 SHuffle (NEB C3026) / Plasmid: pET
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
125 mMHEPESHEPES1
250 mMSodium ChlorideNaClSodium chloride1
35 mMMagnesium ChlorideMgCl21
40.001 %2-Mercaptoethanol2-ME1
SpecimenConc.: 2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELDBright-field microscopy
Image recordingElectron dose: 50 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.16_3549: / Classification: refinement
EM software
IDNameVersionCategory
2EPUimage acquisition
4RELION3.0.6CTF correction
7PHENIX1.14model fitting
9RELION3.0.6initial Euler assignment
10RELION3.0.6final Euler assignment
11RELION3.0.6classification
12RELION3.0.63D reconstruction
13PHENIX1.14model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C6 (6 fold cyclic)
3D reconstructionResolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 53858 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT
RefinementHighest resolution: 3.9 Å
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00635772
ELECTRON MICROSCOPYf_angle_d0.84648354
ELECTRON MICROSCOPYf_dihedral_angle_d9.33122218
ELECTRON MICROSCOPYf_chiral_restr0.0535418
ELECTRON MICROSCOPYf_plane_restr0.0056372

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