|Entry||Database: EMDB / ID: EMD-24523|
|Title||Cryo-EM structure of human p97-R191Q mutant bound to ATPgS.|
|Sample||Full-length Hexameric p97-R191Q mutant.|
|Function / homology|
Function and homology information
flavin adenine dinucleotide catabolic process / positive regulation of Lys63-specific deubiquitinase activity / ATPase complex / positive regulation of protein K63-linked deubiquitination / protein-DNA covalent cross-linking repair / positive regulation of oxidative phosphorylation / VCP-NSFL1C complex / deubiquitinase activator activity / endoplasmic reticulum stress-induced pre-emptive quality control / endosome to lysosome transport via multivesicular body sorting pathway ...flavin adenine dinucleotide catabolic process / positive regulation of Lys63-specific deubiquitinase activity / ATPase complex / positive regulation of protein K63-linked deubiquitination / protein-DNA covalent cross-linking repair / positive regulation of oxidative phosphorylation / VCP-NSFL1C complex / deubiquitinase activator activity / endoplasmic reticulum stress-induced pre-emptive quality control / endosome to lysosome transport via multivesicular body sorting pathway / cellular response to arsenite ion / BAT3 complex binding / Derlin-1 retrotranslocation complex / ERAD pathway / mitotic spindle disassembly / VCP-NPL4-UFD1 AAA ATPase complex / NADH metabolic process / aggresome assembly / ER-associated misfolded protein catabolic process / vesicle-fusing ATPase / regulation of protein localization to chromatin / K48-linked polyubiquitin modification-dependent protein binding / stress granule disassembly / positive regulation of mitochondrial membrane potential / retrograde protein transport, ER to cytosol / regulation of aerobic respiration / regulation of synapse organization / autophagosome maturation / positive regulation of ATP biosynthetic process / MHC class I protein binding / ubiquitin-specific protease binding / negative regulation of smoothened signaling pathway / ubiquitin-like protein ligase binding / Attachment and Entry / ATP metabolic process / proteasomal protein catabolic process / polyubiquitin modification-dependent protein binding / Protein methylation / HSF1 activation / proteasome complex / endoplasmic reticulum unfolded protein response / lipid droplet / ubiquitin-dependent ERAD pathway / Josephin domain DUBs / N-glycan trimming in the ER and Calnexin/Calreticulin cycle / ADP binding / Hh mutants are degraded by ERAD / Defective CFTR causes cystic fibrosis / Hedgehog ligand biogenesis / Translesion Synthesis by POLH / positive regulation of protein-containing complex assembly / ABC-family proteins mediated transport / positive regulation of protein catabolic process / error-free translesion synthesis / macroautophagy / interstrand cross-link repair / autophagy / Aggrephagy / establishment of protein localization / translesion synthesis / cytoplasmic stress granule / Attachment and Entry / site of double-strand break / azurophil granule lumen / positive regulation of proteasomal ubiquitin-dependent protein catabolic process / positive regulation of canonical Wnt signaling pathway / viral genome replication / activation of cysteine-type endopeptidase activity involved in apoptotic process / Ovarian tumor domain proteases / E3 ubiquitin ligases ubiquitinate target proteins / proteasome-mediated ubiquitin-dependent protein catabolic process / endoplasmic reticulum to Golgi vesicle-mediated transport / cellular response to heat / double-strand break repair / transmembrane transport / secretory granule lumen / protein folding / ficolin-1-rich granule lumen / protein phosphatase binding / regulation of apoptotic process / lipid binding / protein ubiquitination / protein deubiquitination / ATP hydrolysis activity / glutamatergic synapse / DNA repair / protein domain specific binding / intracellular membrane-bounded organelle / cellular response to DNA damage stimulus / ubiquitin protein ligase binding / endoplasmic reticulum membrane / Neutrophil degranulation / neutrophil degranulation / perinuclear region of cytoplasm / endoplasmic reticulum / protein-containing complex / RNA binding / extracellular exosome / extracellular region / nucleoplasm
Similarity search - Function
AAA ATPase, CDC48 family / Cell division protein 48 (CDC48), N-terminal domain / Cell division protein 48 (CDC48) N-terminal domain / CDC48, N-terminal subdomain / Cell division protein 48 (CDC48), domain 2 / CDC48, domain 2 / Cell division protein 48 (CDC48) domain 2 / CDC48 domain 2-like superfamily / Vps4 C terminal oligomerisation domain / Vps4 oligomerisation, C-terminal ...AAA ATPase, CDC48 family / Cell division protein 48 (CDC48), N-terminal domain / Cell division protein 48 (CDC48) N-terminal domain / CDC48, N-terminal subdomain / Cell division protein 48 (CDC48), domain 2 / CDC48, domain 2 / Cell division protein 48 (CDC48) domain 2 / CDC48 domain 2-like superfamily / Vps4 C terminal oligomerisation domain / Vps4 oligomerisation, C-terminal / Aspartate decarboxylase-like domain superfamily / AAA+ lid domain / AAA ATPase, 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 species||Homo sapiens (human)|
|Method||single particle reconstruction / cryo EM / Resolution: 3.1 Å|
|Authors||Caffrey B / Zhu X / Berezuk A / Tuttle K / Chittori S / Subramaniam S|
|Citation||Journal: J Biol Chem / Year: 2021|
Title: Common mutations of AAA ATPase p97 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, 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 ...The human AAA ATPase p97, 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 remain elusive. Here, we present cryo-EM structural analyses of four disease mutants p97R155H, p97R191Q, p97A232E, p97D592N, as well as p97E470D, 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 (p97R155H and p97R191Q), 2) the inter-protomer interactions (p97A232E), or 3) the coupling between D1 and D2 nucleotide domains (p97D592N, p97E470D). 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.
|Structure viewer||EM map: |
Downloads & links
|File||Download / File: emd_24523.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)|
|Projections & slices|
Images are generated by Spider.
|Voxel size||X=Y=Z: 1.102 Å|
|Symmetry||Space group: 1|
CCP4 map header:
-Entire Full-length Hexameric p97-R191Q mutant.
|Entire||Name: Full-length Hexameric p97-R191Q mutant. / Number of Components: 4|
-Component #1: protein, Full-length Hexameric p97-R191Q mutant.
|Protein||Name: Full-length Hexameric p97-R191Q mutant. / Recombinant expression: No|
|Mass||Theoretical: 540 kDa|
|Source||Species: Homo sapiens (human)|
|Source (engineered)||Expression System: Escherichia coli (E. coli) / Strain: BL21 DE3|
-Component #2: protein, Transitional endoplasmic reticulum ATPase
|Protein||Name: Transitional endoplasmic reticulum ATPase / Number of Copies: 6 / Recombinant expression: No|
|Mass||Theoretical: 91.202586 kDa|
|Source||Species: Homo sapiens (human)|
|Source (engineered)||Expression System: Escherichia coli BL21(DE3) (bacteria)|
-Component #3: ligand, PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER
|Ligand||Name: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / Number of Copies: 12 / Recombinant expression: No|
|Mass||Theoretical: 0.523247 kDa|
-Component #4: ligand, MAGNESIUM ION
|Ligand||Name: MAGNESIUM ION / Number of Copies: 12 / Recombinant expression: No|
|Mass||Theoretical: 2.430505 MDa|
|Specimen||Specimen State: 2D array / Method: cryo EM|
|Sample solution||Specimen conc.: 2 mg/mL / Buffer solution: Protein Storage Buffer with ATPgS / pH: 8|
|Vitrification||Cryogen Name: ETHANE|
-Electron microscopy imaging
Model: Titan Krios / Image courtesy: FEI Company
|Imaging||Microscope: FEI TITAN KRIOS|
|Electron gun||Electron Source: FIELD EMISSION GUN / Accelerating Voltage: 300 kV / Electron Dose: 50 e/Å2 / Illumination Mode: FLOOD BEAM|
|Lens||Imaging Mode: BRIGHT FIELD|
|Specimen Holder||Model: OTHER|
|Image acquisition||Number of Digital Images: 4797|
|Processing||Method: single particle reconstruction / Number of Projections: 4316075|
|3D reconstruction||Resolution: 3.1 Å / Resolution Method: FSC 0.143 CUT-OFF|
|FSC plot (resolution estimation)|
-Atomic model buiding
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