|Entry||Database: PDB / ID: 7mdm|
|Title||Structure of human p97 ATPase L464P mutant|
|Components||Transitional endoplasmic reticulum ATPase|
|Function / homology|
Function and homology information
positive regulation of Lys63-specific deubiquitinase activity / flavin adenine dinucleotide catabolic process / positive regulation of protein K63-linked deubiquitination / protein-DNA covalent cross-linking repair / positive regulation of oxidative phosphorylation / ATPase complex / VCP-NSFL1C complex / deubiquitinase activator activity / endosome to lysosome transport via multivesicular body sorting pathway / cellular response to arsenite ion ...positive regulation of Lys63-specific deubiquitinase activity / flavin adenine dinucleotide catabolic process / positive regulation of protein K63-linked deubiquitination / protein-DNA covalent cross-linking repair / positive regulation of oxidative phosphorylation / ATPase complex / VCP-NSFL1C complex / deubiquitinase activator activity / endosome to lysosome transport via multivesicular body sorting pathway / cellular response to arsenite ion / endoplasmic reticulum stress-induced pre-emptive quality control / Derlin-1 retrotranslocation complex / BAT3 complex binding / mitotic spindle disassembly / ERAD pathway / VCP-NPL4-UFD1 AAA ATPase complex / NADH metabolic process / aggresome assembly / vesicle-fusing ATPase / regulation of protein localization to chromatin / ER-associated misfolded protein catabolic process / K48-linked polyubiquitin modification-dependent protein binding / stress granule disassembly / positive regulation of mitochondrial membrane potential / retrograde protein transport, ER to cytosol / autophagosome maturation / regulation of aerobic respiration / MHC class I protein binding / regulation of synapse organization / positive regulation of ATP biosynthetic process / negative regulation of smoothened signaling pathway / ubiquitin-specific protease binding / ubiquitin-like protein ligase binding / RHOH GTPase cycle / ATP metabolic process / Attachment and Entry / polyubiquitin modification-dependent protein binding / proteasomal protein catabolic process / endoplasmic reticulum to Golgi vesicle-mediated transport / Protein methylation / translesion synthesis / HSF1 activation / proteasome complex / interstrand cross-link repair / endoplasmic reticulum unfolded protein response / lipid droplet / ubiquitin-dependent ERAD pathway / Josephin domain DUBs / N-glycan trimming in the ER and Calnexin/Calreticulin cycle / macroautophagy / ADP binding / Hh mutants are degraded by ERAD / Defective CFTR causes cystic fibrosis / Hedgehog ligand biogenesis / Translesion Synthesis by POLH / ABC-family proteins mediated transport / positive regulation of protein catabolic process / positive regulation of protein-containing complex assembly / double-strand break repair / Aggrephagy / establishment of protein localization / cytoplasmic stress granule / autophagy / positive regulation of canonical Wnt signaling pathway / azurophil granule lumen / activation of cysteine-type endopeptidase activity involved in apoptotic process / positive regulation of proteasomal ubiquitin-dependent protein catabolic process / site of double-strand break / viral genome replication / Attachment and Entry / Ovarian tumor domain proteases / E3 ubiquitin ligases ubiquitinate target proteins / proteasome-mediated ubiquitin-dependent protein catabolic process / cellular response to heat / secretory granule lumen / protein phosphatase binding / regulation of apoptotic process / protein ubiquitination / ficolin-1-rich granule lumen / lipid binding / : / glutamatergic synapse / DNA repair / intracellular membrane-bounded organelle / protein domain specific binding / cellular response to DNA damage stimulus / ubiquitin protein ligase binding / Neutrophil degranulation / endoplasmic reticulum membrane / perinuclear region of cytoplasm / endoplasmic reticulum / protein-containing complex / RNA binding / extracellular exosome / extracellular region / nucleoplasm / ATP binding / identical protein binding / nucleus / cytosol
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 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 species||Homo sapiens (human)|
|Method||ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.86 Å|
|Authors||Zhang, X. / Gui, L. / Li, S. / Nandi, P. / Columbres, R.C. / Wong, D.E. / Moen, D.R. / Lin, H.J. / Chiu, P.-L. / Chou, T.-F.|
|Funding support|| United States, 2items |
|Citation||Journal: Biochem J / Year: 2021|
Title: Conserved L464 in p97 D1-D2 linker is critical for p97 cofactor regulated ATPase activity.
Authors: Xiaoyi Zhang / Lin Gui / Shan Li / Purbasha Nandi / Rod Carlo Columbres / Daniel E Wong / Derek R Moen / Henry J Lin / Po-Lin Chiu / Tsui-Fen Chou /
Abstract: p97 protein is a highly conserved, abundant, functionally diverse, structurally dynamic homohexameric AAA enzyme-containing N, D1, and D2 domains. A truncated p97 protein containing the N and D1 ...p97 protein is a highly conserved, abundant, functionally diverse, structurally dynamic homohexameric AAA enzyme-containing N, D1, and D2 domains. A truncated p97 protein containing the N and D1 domains and the D1-D2 linker (ND1L) exhibits 79% of wild-type (WT) ATPase activity whereas the ND1 domain alone without the linker only has 2% of WT activity. To investigate the relationship between the D1-D2 linker and the D1 domain, we produced p97 ND1L mutants and demonstrated that this 22-residue linker region is essential for D1 ATPase activity. The conserved amino acid leucine 464 (L464) is critical for regulating D1 and D2 ATPase activity by p97 cofactors p37, p47, and Npl4-Ufd1 (NU). Changing leucine to alanine, proline, or glutamate increased the maximum rate of ATP turnover (kcat) of p47-regulated ATPase activities for these mutants, but not for WT. p37 and p47 increased the kcat of the proline substituted linker, suggesting that they induced linker conformations facilitating ATP hydrolysis. NU inhibited D1 ATPase activities of WT and mutant ND1L proteins, but activated D2 ATPase activity of full-length p97. To further understand the mutant mechanism, we used single-particle cryo-EM to visualize the full-length p97L464P and revealed the conformational change of the D1-D2 linker, resulting in a movement of the helix-turn-helix motif (543-569). Taken together with the biochemical and structural results we conclude that the linker helps maintain D1 in a competent conformation and relays the communication to/from the N-domain to the D1 and D2 ATPase domains, which are ∼50 Å away.
|Structure viewer||Molecule: |
Downloads & links
B: Transitional endoplasmic reticulum ATPase
C: Transitional endoplasmic reticulum ATPase
D: Transitional endoplasmic reticulum ATPase
E: Transitional endoplasmic reticulum ATPase
A: Transitional endoplasmic reticulum ATPase
F: Transitional endoplasmic reticulum ATPase
Mass: 89420.773 Da / Num. of mol.: 6
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
Mass: 427.201 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Comment: ADP, energy-carrying molecule*YM
|Has ligand of interest||N|
|Experiment||Method: ELECTRON MICROSCOPY|
|EM experiment||Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction|
|Component||Name: Human p97 L464P mutant / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT|
|Molecular weight||Value: 0.536 MDa / Experimental value: NO|
|Source (natural)||Organism: Homo sapiens (human)|
|Source (recombinant)||Organism: Escherichia coli (E. coli)|
|Buffer solution||pH: 7.4|
|Specimen||Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES|
|Specimen support||Grid type: C-flat-2/1|
|Vitrification||Cryogen name: ETHANE|
-Electron microscopy imaging
Model: Titan Krios / Image courtesy: FEI Company
|Microscopy||Model: FEI TITAN KRIOS|
|Electron gun||Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM|
|Electron lens||Mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / C2 aperture diameter: 70 µm|
|Specimen holder||Cryogen: NITROGEN / Model: FEI TITAN KRIOS AUTOGRID HOLDER|
|Image recording||Electron dose: 64.8 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)|
|Software||Name: PHENIX / Version: 1.18.2_3874: / Classification: refinement|
|CTF correction||Type: PHASE FLIPPING AND AMPLITUDE CORRECTION|
|3D reconstruction||Resolution: 4.86 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 81865 / Algorithm: BACK PROJECTION / Symmetry type: POINT|
|Atomic model building||Protocol: RIGID BODY FIT|
|Atomic model building||PDB-ID: 5FTK|
|Refine LS restraints|
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