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- EMDB-30148: Human AAA+ ATPase VCP mutant - T76E, ADP-bound form -

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Basic information

Entry
Database: EMDB / ID: EMD-30148
TitleHuman AAA+ ATPase VCP mutant - T76E, ADP-bound form
Map dataCryo-EM structure of VCP mutant-T76E, ADP bound form
Sample
  • Complex: Transitional endoplasmic reticulum ATPase, VCP.
    • Protein or peptide: Transitional endoplasmic reticulum ATPase
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
KeywordsComplex / ATPase / Unfoldase / Protein Transportation / CELL CYCLE
Function / homology
Function and homology information


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 / cytoplasmic ubiquitin ligase complex / BAT3 complex binding / 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 / cytoplasmic ubiquitin ligase complex / BAT3 complex binding / 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 / RHOH GTPase cycle / intracellular membrane-bounded organelle / MHC class I protein binding / polyubiquitin modification-dependent protein binding / autophagosome maturation / endoplasmic reticulum to Golgi vesicle-mediated transport / negative regulation of hippo signaling / HSF1 activation / 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 / N-glycan trimming in the ER and Calnexin/Calreticulin cycle / negative regulation of smoothened signaling pathway / establishment of protein localization / positive regulation of protein-containing complex assembly / Hh mutants are degraded by ERAD / Hedgehog ligand biogenesis / Defective CFTR causes cystic fibrosis / Translesion Synthesis by POLH / positive regulation of non-canonical NF-kappaB signal transduction / ADP binding / ABC-family proteins 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 / proteasome-mediated ubiquitin-dependent protein catabolic process / Attachment and Entry / 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
Similarity search - Function
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
Similarity search - Domain/homology
Transitional endoplasmic reticulum ATPase
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.6 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, 2020-
Header (metadata) releaseMar 31, 2021-
Map releaseMar 31, 2021-
UpdateMay 29, 2024-
Current statusMay 29, 2024Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.205
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.205
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7bp9
  • Surface level: 0.205
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_30148.png

Downloads & links

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Map

FileDownload / File: emd_30148.map.gz / Format: CCP4 / Size: 27 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM structure of VCP mutant-T76E, ADP bound form
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.07 Å/pix.
x 192 pix.
= 205.44 Å		1.07 Å/pix.
x 192 pix.
= 205.44 Å		1.07 Å/pix.
x 192 pix.
= 205.44 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.07 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.205 / Movie #1: 0.205
Minimum - Maximum-0.47931385 - 1.2170985
Average (Standard dev.)0.0000019345891 (±0.06984084)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions192192192
Spacing192192192
CellA=B=C: 205.44 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.071.071.07
M x/y/z192192192
origin x/y/z0.0000.0000.000
length x/y/z205.440205.440205.440
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ336336336
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS192192192
D min/max/mean-0.4791.2170.000

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Supplemental data

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Sample components

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Entire : Transitional endoplasmic reticulum ATPase, VCP.

EntireName: Transitional endoplasmic reticulum ATPase, VCP.
Components
  • Complex: Transitional endoplasmic reticulum ATPase, VCP.
    • Protein or peptide: Transitional endoplasmic reticulum ATPase
  • Ligand: ADENOSINE-5'-DIPHOSPHATE

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Supramolecule #1: Transitional endoplasmic reticulum ATPase, VCP.

SupramoleculeName: Transitional endoplasmic reticulum ATPase, VCP. / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1 / Details: T76E mutant of VCP of ADP-bound form
Source (natural)Organism: Homo sapiens (human) / Location in cell: cytoplasm nucleus ER
Molecular weightTheoretical: 97 kDa/nm

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Macromolecule #1: Transitional endoplasmic reticulum ATPase

MacromoleculeName: Transitional endoplasmic reticulum ATPase / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO / EC number: vesicle-fusing ATPase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 89.46482 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MASGADSKGD DLSTAILKQK NRPNRLIVDE AINEDNSVVS LSQPKMDELQ LFRGDTVLLK GKKRREAVCI VLSDDECSDE KIRMNRVVR NNLRVRLGDV ISIQPCPDVK YGKRIHVLPI DDTVEGITGN LFEVYLKPYF LEAYRPIRKG DIFLVRGGMR A VEFKVVET ...String:
MASGADSKGD DLSTAILKQK NRPNRLIVDE AINEDNSVVS LSQPKMDELQ LFRGDTVLLK GKKRREAVCI VLSDDECSDE KIRMNRVVR NNLRVRLGDV ISIQPCPDVK YGKRIHVLPI DDTVEGITGN LFEVYLKPYF LEAYRPIRKG DIFLVRGGMR A VEFKVVET DPSPYCIVAP DTVIHCEGEP IKREDEEESL NEVGYDDIGG CRKQLAQIKE MVELPLRHPA LFKAIGVKPP RG ILLYGPP GTGKTLIARA VANETGAFFF LINGPEIMSK LAGESESNLR KAFEEAEKNA PAIIFIDELD AIAPKREKTH GEV ERRIVS QLLTLMDGLK QRAHVIVMAA TNRPNSIDPA LRRFGRFDRE VDIGIPDATG RLEILQIHTK NMKLADDVDL EQVA NETHG HVGADLAALC SEAALQAIRK KMDLIDLEDE TIDAEVMNSL AVTMDDFRWA LSQSNPSALR ETVVEVPQVT WEDIG GLED VKRELQELVQ YPVEHPDKFL KFGMTPSKGV LFYGPPGCGK TLLAKAIANE CQANFISIKG PELLTMWFGE SEANVR EIF DKARQAAPCV LFFDELDSIA KARGGNIGDG GGAADRVINQ ILTEMDGMST KKNVFIIGAT NRPDIIDPAI LRPGRLD QL IYIPLPDEKS RVAILKANLR KSPVAKDVDL EFLAKMTNGF SGADLTEICQ RACKLAIRES IESEIRRERE RQTNPSAM E VEEDDPVPEI RRDHFEEAMR FARRSVSDND IRKYEMFAQT LQQSRGFGSF RFPSGNQGGA GPSQGSGGGT GGSVYTEDN DDDLYG

UniProtKB: Transitional endoplasmic reticulum ATPase

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Macromolecule #2: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 2 / Number of copies: 12 / Formula: ADP
Molecular weightTheoretical: 427.201 Da
Chemical component information

ChemComp-ADP:
ADENOSINE-5'-DIPHOSPHATE / ADP, energy-carrying molecule*YM

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration2 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
25.0 mMHEPESHEPES
50.0 mMNaClSodium Chloride
5.0 mMMgCl2Magnesium Chloride
0.001 %2-ME2-Mercaptoethanol
VitrificationCryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: EMDB MAP
EMDB ID:

3297

Final reconstructionApplied symmetry - Point group: C6 (6 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0.6) / Number images used: 243820
Initial angle assignmentType: RANDOM ASSIGNMENT / Software - Name: RELION (ver. 3.0.6)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 3.0.6)

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Atomic model buiding 1

RefinementProtocol: RIGID BODY FIT
Output model

PDB-7bp9:
Human AAA+ ATPase VCP mutant - T76E, ADP-bound form

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