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- PDB-8b5r: p97-p37-SPI substrate complex -

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Basic information

Entry
Database: PDB / ID: 8b5r
Titlep97-p37-SPI substrate complex
Components
  • (UBX domain-containing protein ...) x 2
  • I3 sequence being threaded through the p97 channel
  • Protein phosphatase 1 regulatory subunit 7
  • Serine/threonine-protein phosphatase PP1-gamma catalytic subunit
  • Transitional endoplasmic reticulum ATPase
KeywordsCHAPERONE / AAA+ ATPase / p97 / VCP / Cdc48 / unfoldase / protein phosphatase-1 / protein maturation
Function / homology
Function and homology information


positive regulation of mitotic centrosome separation / negative regulation of protein localization to centrosome / PTW/PP1 phosphatase complex / regulation of nucleocytoplasmic transport / nuclear membrane reassembly / protein phosphatase 1 binding / protein phosphatase regulator activity / lamin binding / positive regulation of Lys63-specific deubiquitinase activity / flavin adenine dinucleotide catabolic process ...positive regulation of mitotic centrosome separation / negative regulation of protein localization to centrosome / PTW/PP1 phosphatase complex / regulation of nucleocytoplasmic transport / nuclear membrane reassembly / protein phosphatase 1 binding / protein phosphatase regulator activity / lamin binding / positive regulation of Lys63-specific deubiquitinase activity / flavin adenine dinucleotide catabolic process / positive regulation of oxidative phosphorylation / VCP-NSFL1C complex / protein-DNA covalent cross-linking repair / endoplasmic reticulum stress-induced pre-emptive quality control / endosome to lysosome transport via multivesicular body sorting pathway / cellular response to arsenite ion / Derlin-1 retrotranslocation complex / BAT3 complex binding / SHOC2 M1731 mutant abolishes MRAS complex function / Gain-of-function MRAS complexes activate RAF signaling / spindle pole centrosome / positive regulation of protein K63-linked deubiquitination / deubiquitinase activator activity / mitotic spindle disassembly / VCP-NPL4-UFD1 AAA ATPase complex / aggresome assembly / NADH metabolic process / regulation of protein localization to chromatin / 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 / microtubule organizing center / myosin phosphatase activity / regulation of aerobic respiration / protein serine/threonine phosphatase activity / ATPase complex / regulation of synapse organization / glycogen metabolic process / ubiquitin-specific protease binding / protein-serine/threonine phosphatase / positive regulation of ATP biosynthetic process / entrainment of circadian clock by photoperiod / Golgi organization / Triglyceride catabolism / ubiquitin-like protein ligase binding / phosphatase activity / autophagosome maturation / establishment of mitotic spindle orientation / phosphoprotein phosphatase activity / cleavage furrow / blastocyst development / RHOH GTPase cycle / autophagosome assembly / polyubiquitin modification-dependent protein binding / HSF1 activation / translesion synthesis / endoplasmic reticulum to Golgi vesicle-mediated transport / MHC class I protein binding / Protein methylation / enzyme regulator activity / Amplification of signal from unattached kinetochores via a MAD2 inhibitory signal / interstrand cross-link repair / negative regulation of smoothened signaling pathway / : / Attachment and Entry / ATP metabolic process / endoplasmic reticulum unfolded protein response / Mitotic Prometaphase / EML4 and NUDC in mitotic spindle formation / positive regulation of glial cell proliferation / positive regulation of protein dephosphorylation / Resolution of Sister Chromatid Cohesion / lipid droplet / proteasome complex / viral genome replication / protein dephosphorylation / Downregulation of TGF-beta receptor signaling / Josephin domain DUBs / N-glycan trimming in the ER and Calnexin/Calreticulin cycle / ubiquitin binding / RHO GTPases Activate Formins / proteasomal protein catabolic process / ADP binding / Hh mutants are degraded by ERAD / positive regulation of protein-containing complex assembly / Defective CFTR causes cystic fibrosis / macroautophagy / Hedgehog ligand biogenesis / Translesion Synthesis by POLH / RAF activation / circadian regulation of gene expression / ABC-family proteins mediated transport / establishment of protein localization
Similarity search - Function
SEP domain / NSFL1 cofactor p47, SEP domain superfamily / SEP domain / SEP domain profile. / Domain present in Saccharomyces cerevisiae Shp1, Drosophila melanogaster eyes closed gene (eyc), and vertebrate p47. / Domain present in ubiquitin-regulatory proteins / UBX domain / UBX domain / UBX domain profile. / Serine-threonine protein phosphatase, N-terminal ...SEP domain / NSFL1 cofactor p47, SEP domain superfamily / SEP domain / SEP domain profile. / Domain present in Saccharomyces cerevisiae Shp1, Drosophila melanogaster eyes closed gene (eyc), and vertebrate p47. / Domain present in ubiquitin-regulatory proteins / UBX domain / UBX domain / UBX domain profile. / Serine-threonine protein phosphatase, N-terminal / Serine-threonine protein phosphatase N-terminal domain / U2A'/phosphoprotein 32 family A, C-terminal / occurring C-terminal to leucine-rich repeats / Leucine rich repeat 4 / Leucine-rich repeat / Leucine Rich repeats (2 copies) / Serine/threonine specific protein phosphatases signature. / Protein phosphatase 2A homologues, catalytic domain. / Serine/threonine-specific protein phosphatase/bis(5-nucleosyl)-tetraphosphatase / 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 / Leucine-rich repeat, SDS22-like subfamily / Calcineurin-like phosphoesterase domain, ApaH type / Calcineurin-like phosphoesterase / Aspartate decarboxylase-like domain superfamily / Metallo-dependent phosphatase-like / Leucine-rich repeat, typical subtype / Leucine-rich repeats, typical (most populated) subfamily / AAA ATPase, AAA+ lid domain / AAA+ lid domain / ATPase, AAA-type, conserved site / AAA-protein family signature. / Leucine-rich repeat profile. / Leucine-rich repeat / ATPase family associated with various cellular activities (AAA) / ATPase, AAA-type, core / Leucine-rich repeat domain superfamily / Ubiquitin-like domain superfamily / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Serine/threonine-protein phosphatase PP1-gamma catalytic subunit / Transitional endoplasmic reticulum ATPase / UBX domain-containing protein 2B / Protein phosphatase 1 regulatory subunit 7
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 6.1 Å
Authorsvan den Boom, J. / Marini, G. / Meyer, H. / Saibil, H.
Funding support United Kingdom, 3items
OrganizationGrant numberCountry
Wellcome Trust106249/Z/14/Z United Kingdom
Wellcome Trust202679/Z/16/Z United Kingdom
Wellcome Trust206166/Z/17/Z United Kingdom
CitationJournal: EMBO J / Year: 2023
Title: Structural basis of ubiquitin-independent PP1 complex disassembly by p97.
Authors: Johannes van den Boom / Guendalina Marini / Hemmo Meyer / Helen R Saibil /
Abstract: The AAA+-ATPase p97 (also called VCP or Cdc48) unfolds proteins and disassembles protein complexes in numerous cellular processes, but how substrate complexes are loaded onto p97 and disassembled is ...The AAA+-ATPase p97 (also called VCP or Cdc48) unfolds proteins and disassembles protein complexes in numerous cellular processes, but how substrate complexes are loaded onto p97 and disassembled is unclear. Here, we present cryo-EM structures of p97 in the process of disassembling a protein phosphatase-1 (PP1) complex by extracting an inhibitory subunit from PP1. We show that PP1 and its partners SDS22 and inhibitor-3 (I3) are loaded tightly onto p97, surprisingly via a direct contact of SDS22 with the p97 N-domain. Loading is assisted by the p37 adapter that bridges two adjacent p97 N-domains underneath the substrate complex. A stretch of I3 is threaded into the central channel of the spiral-shaped p97 hexamer, while other elements of I3 are still attached to PP1. Thus, our data show how p97 arranges a protein complex between the p97 N-domain and central channel, suggesting a hold-and-extract mechanism for p97-mediated disassembly.
History
DepositionSep 24, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jun 7, 2023Provider: repository / Type: Initial release
Revision 2.0Jun 14, 2023Group: Atomic model / Database references / Category: atom_site / citation / citation_author
Item: _atom_site.Cartn_x / _atom_site.Cartn_y ..._atom_site.Cartn_x / _atom_site.Cartn_y / _atom_site.Cartn_z / _atom_site.auth_atom_id / _atom_site.label_atom_id / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title
Revision 2.1Jul 26, 2023Group: Database references / Category: citation / Item: _citation.journal_volume

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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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
I: I3 sequence being threaded through the p97 channel
P: Serine/threonine-protein phosphatase PP1-gamma catalytic subunit
S: Protein phosphatase 1 regulatory subunit 7
X: UBX domain-containing protein 2B
Y: UBX domain-containing protein 2B


Theoretical massNumber of molelcules
Total (without water)633,01411
Polymers633,01411
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 3 types, 8 molecules ABCDEFPS

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


Mass: 90265.695 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: VCP / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P55072, vesicle-fusing ATPase
#3: Protein Serine/threonine-protein phosphatase PP1-gamma catalytic subunit / PP-1G / Protein phosphatase 1C catalytic subunit


Mass: 37030.777 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: PPP1CC / Production host: Trichoplusia ni (cabbage looper)
References: UniProt: P36873, protein-serine/threonine phosphatase
#4: Protein Protein phosphatase 1 regulatory subunit 7 / Protein phosphatase 1 regulatory subunit 22


Mass: 41616.129 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: PPP1R7, SDS22 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q15435

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Protein/peptide , 1 types, 1 molecules I

#2: Protein/peptide I3 sequence being threaded through the p97 channel


Mass: 1890.321 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Trichoplusia ni (cabbage looper)

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UBX domain-containing protein ... , 2 types, 2 molecules XY

#5: Protein/peptide UBX domain-containing protein 2B / NSFL1 cofactor p37 / p97 cofactor p37


Mass: 1123.238 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: UBXN2B
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: Q14CS0
#6: Protein UBX domain-containing protein 2B / NSFL1 cofactor p37 / p97 cofactor p37


Mass: 9759.220 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: UBXN2B
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: Q14CS0

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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: p97 complex with p37 adaptor and SPI substrate / Type: COMPLEX / Entity ID: #1-#4, #6, #5 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Trichoplusia ni (cabbage looper)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: C-flat-1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 85 % / Chamber temperature: 277 K

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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: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 81000 X / Nominal defocus max: 3300 nm / Nominal defocus min: 1800 nm / Alignment procedure: BASIC
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 3 sec. / Electron dose: 48 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

EM softwareName: PHENIX / Version: 1.20.1_4487: / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 6.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 191477 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00226454
ELECTRON MICROSCOPYf_angle_d0.50737038
ELECTRON MICROSCOPYf_dihedral_angle_d4.8555196
ELECTRON MICROSCOPYf_chiral_restr0.0444868
ELECTRON MICROSCOPYf_plane_restr0.0055462

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