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- PDB-7rlc: Cryo-EM structure of human p97-A232E mutant bound to ATPgS. -

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Basic information

Entry
Database: PDB / ID: 7rlc
TitleCryo-EM structure of human p97-A232E mutant bound to ATPgS.
ComponentsTransitional endoplasmic reticulum ATPase
KeywordsHYDROLASE / p97 / VCP / TERA / Inhibitor / CB-5083
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 / BAT3 complex binding / cellular response to arsenite ion / cytoplasmic ubiquitin ligase complex / Derlin-1 retrotranslocation complex / positive regulation of protein K63-linked deubiquitination / ATPase complex ...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 / cellular response to arsenite ion / cytoplasmic ubiquitin ligase complex / Derlin-1 retrotranslocation complex / positive regulation of protein K63-linked deubiquitination / ATPase complex / protein-DNA covalent cross-linking repair / deubiquitinase activator activity / positive regulation of oxidative phosphorylation / cytoplasm protein quality control / regulation of protein localization to chromatin / ubiquitin-modified protein reader activity / 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 / regulation of aerobic respiration / NAD+ metabolic process / retrograde protein transport, ER to cytosol / stress granule disassembly / ubiquitin-specific protease binding / regulation of synapse organization / positive regulation of ATP biosynthetic process / ubiquitin-like protein ligase binding / RHOH GTPase cycle / intracellular membrane-bounded organelle / MHC class I protein binding / autophagosome maturation / negative regulation of hippo signaling / HSF1 activation / endoplasmic reticulum to Golgi vesicle-mediated transport / polyubiquitin modification-dependent protein binding / mitophagy / interstrand cross-link repair / ATP metabolic process / protein unfolding / proteasome complex / ribosome-associated ubiquitin-dependent protein catabolic process / Attachment and Entry / endoplasmic reticulum unfolded protein response / Protein methylation / ERAD pathway / negative regulation of smoothened signaling pathway / viral genome replication / translesion synthesis / negative regulation of protein localization to chromatin / rescue of stalled cytosolic ribosome / macroautophagy / lipid droplet / Josephin domain DUBs / establishment of protein localization / proteasomal protein catabolic process / N-glycan trimming in the ER and Calnexin/Calreticulin cycle / positive regulation of protein-containing complex assembly / Hh mutants are degraded by ERAD / positive regulation of non-canonical NF-kappaB signal transduction / ADP binding / Dengue Virus Genome Translation and Replication / Translesion Synthesis by POLH / Hedgehog ligand biogenesis / Defective CFTR causes cystic fibrosis / AMPK-induced ERAD and lysosome mediated degradation of PD-L1(CD274) / ABC-family protein mediated transport / autophagy / cytoplasmic stress granule / Ribosome Quality Control (RQC) complex extracts and degrades nascent peptide / positive regulation of protein catabolic process / Aggrephagy / positive regulation of canonical Wnt signaling pathway / double-strand break repair / azurophil granule lumen / Ovarian tumor domain proteases / positive regulation of proteasomal ubiquitin-dependent protein catabolic process / KEAP1-NFE2L2 pathway / site of double-strand break / cellular response to heat / E3 ubiquitin ligases ubiquitinate target proteins / Neddylation / secretory granule lumen / protein phosphatase binding / ubiquitin-dependent protein catabolic process / ficolin-1-rich granule lumen / proteasome-mediated ubiquitin-dependent protein catabolic process / regulation of apoptotic process / Attachment and Entry / protein ubiquitination / protein domain specific binding / ubiquitin protein ligase binding / DNA repair / DNA damage response / Neutrophil degranulation / lipid binding / endoplasmic reticulum membrane / perinuclear region of cytoplasm
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
PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / Transitional endoplasmic reticulum ATPase
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsCaffrey, B. / Zhu, X. / Berezuk, A. / Tuttle, K. / Chittori, S. / Subramaniam, S.
CitationJournal: J Biol Chem / Year: 2021
Title: AAA+ ATPase p97/VCP mutants and inhibitor binding disrupt inter-domain coupling and subsequent allosteric activation.
Authors: Brian Caffrey / Xing Zhu / Alison Berezuk / Katharine Tuttle / Sagar Chittori / Sriram Subramaniam /
Abstract: The human AAA+ ATPase p97, also known as valosin-containing protein, a potential target for cancer therapeutics, plays a vital role in the clearing of misfolded proteins. p97 dysfunction is also ...The human AAA+ ATPase p97, also known as valosin-containing protein, a potential target for cancer therapeutics, plays a vital role in the clearing of misfolded proteins. p97 dysfunction is also known to play a crucial role in several neurodegenerative disorders, such as MultiSystem Proteinopathy 1 (MSP-1) and Familial Amyotrophic Lateral Sclerosis (ALS). However, the structural basis of its role in such diseases remains elusive. Here, we present cryo-EM structural analyses of four disease mutants p97, p97, p97, p97, as well as p97, implicated in resistance to the drug CB-5083, a potent p97 inhibitor. Our cryo-EM structures demonstrate that these mutations affect nucleotide-driven allosteric activation across the three principal p97 domains (N, D1, and D2) by predominantly interfering with either (1) the coupling between the D1 and N-terminal domains (p97 and p97), (2) the interprotomer interactions (p97), or (3) the coupling between D1 and D2 nucleotide domains (p97, p97). We also show that binding of the competitive inhibitor, CB-5083, to the D2 domain prevents conformational changes similar to those seen for mutations that affect coupling between the D1 and D2 domains. Our studies enable tracing of the path of allosteric activation across p97 and establish a common mechanistic link between active site inhibition and defects in allosteric activation by disease-causing mutations and have potential implications for the design of novel allosteric compounds that can modulate p97 function.
History
DepositionJul 23, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 22, 2021Provider: repository / Type: Initial release
Revision 1.1Sep 29, 2021Group: Database references / Category: citation / citation_author
Item: _citation.page_first / _citation.page_last ..._citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.2Nov 17, 2021Group: Database references / Category: citation / Item: _citation.journal_volume / _citation.title
Revision 1.3Jun 5, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Structure visualization

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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)554,30930
Polymers547,7386
Non-polymers6,57124
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: native gel electrophoresis
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: 91289.680 Da / Num. of mol.: 6 / Mutation: A232E
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: VCP / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P55072, vesicle-fusing ATPase
#2: Chemical
ChemComp-AGS / PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / ATP-GAMMA-S / ADENOSINE 5'-(3-THIOTRIPHOSPHATE) / ADENOSINE 5'-(GAMMA-THIOTRIPHOSPHATE) / ADENOSINE-5'-DIPHOSPHATE MONOTHIOPHOSPHATE


Mass: 523.247 Da / Num. of mol.: 12 / Source method: obtained synthetically / Formula: C10H16N5O12P3S / Comment: ATP-gamma-S, energy-carrying molecule analogue*YM
#3: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 12 / Source method: obtained synthetically / Formula: Mg
Has ligand of interestN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: 2D ARRAY / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Full-length Hexameric p97-A232E mutant. / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.540 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: BL21 DE3
Buffer solutionpH: 8 / Details: Protein Storage Buffer with ADP
Buffer component
IDConc.NameFormulaBuffer-ID
1150 mMSodium ChlorideNaCl1
250 mMTris HydrochlorideTris-HCl1
35 mMMagnesium ChlorideMgCl21
40.5 uMOctyl GlucosideC14H28O61
51 mMADPC10H15N5O10P21
61 mMTCEPC9H15O6P1
SpecimenConc.: 2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
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: FLOOD BEAM
Electron lensMode: BRIGHT FIELD
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of real images: 640

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 310167
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 163041 / Symmetry type: POINT

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