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- PDB-8uvp: Human p97/VCP R155H mutant structure with a triazole inhibitor (N... -

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Basic information

Entry
Database: PDB / ID: 8uvp
TitleHuman p97/VCP R155H mutant structure with a triazole inhibitor (NSC819701/up)
ComponentsTransitional endoplasmic reticulum ATPase
KeywordsMOTOR PROTEIN/INHIBITOR / AAA+ ATPase / p97 / VCP / triazole / allosteric inhibition / MOTOR PROTEIN-INHIBITOR complex
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 / cytoplasm protein quality control / endosome to lysosome transport via multivesicular body sorting pathway / 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 / cytoplasm protein quality control / endosome to lysosome transport via multivesicular body sorting pathway / endoplasmic reticulum stress-induced pre-emptive quality control / cellular response to arsenite ion / Derlin-1 retrotranslocation complex / BAT3 complex binding / protein-DNA covalent cross-linking repair / positive regulation of protein K63-linked deubiquitination / deubiquitinase activator activity / ubiquitin-modified protein reader activity / regulation of protein localization to chromatin / aggresome assembly / NADH metabolic process / mitotic spindle disassembly / VCP-NPL4-UFD1 AAA ATPase complex / vesicle-fusing ATPase / cellular response to misfolded protein / stress granule disassembly / negative regulation of protein localization to chromatin / positive regulation of mitochondrial membrane potential / retrograde protein transport, ER to cytosol / K48-linked polyubiquitin modification-dependent protein binding / regulation of aerobic respiration / regulation of synapse organization / positive regulation of ATP biosynthetic process / ATPase complex / ubiquitin-specific protease binding / MHC class I protein binding / ubiquitin-like protein ligase binding / RHOH GTPase cycle / polyubiquitin modification-dependent protein binding / autophagosome maturation / HSF1 activation / negative regulation of hippo signaling / endoplasmic reticulum to Golgi vesicle-mediated transport / translesion synthesis / proteasomal protein catabolic process / Protein methylation / interstrand cross-link repair / ATP metabolic process / negative regulation of smoothened signaling pathway / endoplasmic reticulum unfolded protein response / ERAD pathway / Attachment and Entry / proteasome complex / viral genome replication / lipid droplet / Josephin domain DUBs / N-glycan trimming in the ER and Calnexin/Calreticulin cycle / Hh mutants are degraded by ERAD / macroautophagy / Hedgehog ligand biogenesis / Defective CFTR causes cystic fibrosis / positive regulation of protein-containing complex assembly / ADP binding / Translesion Synthesis by POLH / establishment of protein localization / ABC-family proteins mediated transport / : / autophagy / Aggrephagy / cytoplasmic stress granule / positive regulation of non-canonical NF-kappaB signal transduction / positive regulation of protein catabolic process / azurophil granule lumen / KEAP1-NFE2L2 pathway / positive regulation of canonical Wnt signaling pathway / Ovarian tumor domain proteases / double-strand break repair / positive regulation of proteasomal ubiquitin-dependent protein catabolic process / E3 ubiquitin ligases ubiquitinate target proteins / site of double-strand break / Neddylation / cellular response to heat / ubiquitin-dependent protein catabolic process / protein phosphatase binding / secretory granule lumen / regulation of apoptotic process / proteasome-mediated ubiquitin-dependent protein catabolic process / ficolin-1-rich granule lumen / Attachment and Entry / protein ubiquitination / protein domain specific binding / intracellular membrane-bounded organelle / DNA repair / lipid binding / DNA damage response / glutamatergic synapse / 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
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
ADENOSINE-5'-DIPHOSPHATE / : / Transitional endoplasmic reticulum ATPase
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsNandi, P. / DeVore, K. / Chiu, P.-L.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI) United States
CitationJournal: Commun Chem / Year: 2024
Title: Mechanism of allosteric inhibition of human p97/VCP ATPase and its disease mutant by triazole inhibitors.
Authors: Purbasha Nandi / Kira DeVore / Feng Wang / Shan Li / Joel D Walker / Thanh Tung Truong / Matthew G LaPorte / Peter Wipf / Heidi Schlager / John McCleerey / William Paquette / Rod Carlo A ...Authors: Purbasha Nandi / Kira DeVore / Feng Wang / Shan Li / Joel D Walker / Thanh Tung Truong / Matthew G LaPorte / Peter Wipf / Heidi Schlager / John McCleerey / William Paquette / Rod Carlo A Columbres / Taiping Gan / Yu-Ping Poh / Petra Fromme / Andrew J Flint / Mark Wolf / Donna M Huryn / Tsui-Fen Chou / Po-Lin Chiu /
Abstract: Human p97 ATPase is crucial in various cellular processes, making it a target for inhibitors to treat cancers, neurological, and infectious diseases. Triazole allosteric p97 inhibitors have been ...Human p97 ATPase is crucial in various cellular processes, making it a target for inhibitors to treat cancers, neurological, and infectious diseases. Triazole allosteric p97 inhibitors have been demonstrated to match the efficacy of CB-5083, an ATP-competitive inhibitor, in cellular models. However, the mechanism is not well understood. This study systematically investigates the structures of new triazole inhibitors bound to  both wild-type and disease mutant forms of p97 and measures their effects on function. These inhibitors bind at the interface of the D1 and D2 domains of each p97 subunit, shifting surrounding helices and altering the loop structures near the C-terminal α2 G helix to modulate domain-domain communications. A key structural moiety of the inhibitor affects the rotameric conformations of interacting side chains, indirectly modulating the N-terminal domain conformation in p97 R155H mutant. The differential effects of inhibitor binding to wild-type and mutant p97 provide insights into drug design with enhanced specificity, particularly for oncology applications.
History
DepositionNov 3, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 21, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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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)545,38124
Polymers536,5076
Non-polymers8,87518
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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


Mass: 89417.773 Da / Num. of mol.: 6 / Mutation: R155H
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: VCP / Production host: Escherichia coli (E. coli) / References: UniProt: P55072, vesicle-fusing ATPase
#2: Chemical
ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 12 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Comment: ADP, energy-carrying molecule*YM
#3: Chemical
ChemComp-XO8 / 2-[(4P)-4-(4-{[(4P)-5-(cyclohexylsulfanyl)-4-(pyridin-3-yl)-4H-1,2,4-triazol-3-yl]methoxy}-2,5-difluorophenyl)-2H-1,2,3-triazol-2-yl]-1-[(2R,6S)-2,6-dimethylmorpholin-4-yl]ethan-1-one


Mass: 624.705 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C30H34F2N8O3S / Feature type: SUBJECT OF INVESTIGATION
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: p97/VCP AAA+ ATPase R155H mutant/NSC819701 / Type: COMPLEX
Details: p97/VCP AAA+ ATPase R155H mutant bound with the triazole NSC819701.
Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.536 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.4
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPESHEPES1
2150 mMPotassium chlorideKCl1
31 mMMagnesium chlorideMgCl21
41 nMAdenosine diphosphateADP1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid type: C-flat-2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 90 % / Chamber temperature: 298 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 FIELD / Nominal magnification: 48077 X / Nominal defocus max: 2500 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 8 sec. / Electron dose: 47.84 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)
Image scansSampling size: 5 µm

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Processing

EM software
IDNameVersionCategory
2SerialEM3.8image acquisition
4cryoSPARC2.14.2CTF correction
7Coot0.9-premodel fitting
9cryoSPARC2.14.2initial Euler assignment
10cryoSPARC2.14.2final Euler assignment
11cryoSPARC2.14.2classification
12cryoSPARC2.14.23D reconstruction
13PHENIX1.20.1-4487model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C6 (6 fold cyclic)
3D reconstructionResolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 31543 / Algorithm: BACK PROJECTION / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model buildingPDB-ID: 5FTN

5ftn


Accession code: 5FTN / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00227030
ELECTRON MICROSCOPYf_angle_d0.62236558
ELECTRON MICROSCOPYf_dihedral_angle_d12.7953810
ELECTRON MICROSCOPYf_chiral_restr0.0394056
ELECTRON MICROSCOPYf_plane_restr0.0044776

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