+Open data
-Basic information
Entry | Database: PDB / ID: 9buz | ||||||
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Title | Thermoplasma acidophilum 20S proteasome - alphaV24Y | ||||||
Components |
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Keywords | HYDROLASE / Protease / threonine protease / endopeptidase activity | ||||||
Function / homology | Function and homology information proteasome endopeptidase complex / proteasome core complex, beta-subunit complex / proteasome core complex, alpha-subunit complex / threonine-type endopeptidase activity / proteasomal protein catabolic process / ubiquitin-dependent protein catabolic process / endopeptidase activity / cytoplasm Similarity search - Function | ||||||
Biological species | Thermoplasma acidophilum (acidophilic) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.38 Å | ||||||
Authors | Chuah, J. / Smith, D. | ||||||
Funding support | United States, 1items
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Citation | Journal: bioRxiv / Year: 2024 Title: Occupancy of the HbYX hydrophobic pocket is sufficient to induce gate opening in the archaeal 20S proteasomes. Authors: Janelle J Y Chuah / Madalena R Daugherty / David M Smith / Abstract: Enhancing proteasome function has been a long-standing but challenging target of interest for the potential treatment of neurodegenerative diseases, emphasizing the importance of understanding ...Enhancing proteasome function has been a long-standing but challenging target of interest for the potential treatment of neurodegenerative diseases, emphasizing the importance of understanding proteasome activation mechanisms. Most proteasome activator complexes use the C-terminal HbYX motif to bind and trigger gate-opening in the 20S proteasome. This study defines a critical molecular interaction in the HbYX mechanism that triggers gate opening. Here, we focus on the Hb site interaction and find it plays a surprisingly central and crucial role in driving the allosteric conformational changes that induce gate opening in the archaeal 20S. We examined the cryo-EM structure of two mutant archaeal proteasomes, αV24Y T20S and αV24F T20S. These two mutants were engineered to place a bulky aromatic residue in the HbYX hydrophobic pocket and both mutants are highly active, though their mechanisms of activation are undefined. Collectively, our findings indicate that the interaction between the Hb group of the HbYX motif and its corresponding hydrophobic pocket is sufficient to induce gate opening in a mechanistically similar way to the HbYX motif. The involved activation mechanism appears to involve expansion of this hydrophobic binding site affecting the state of the IT switch to triggering gate-opening. Furthermore, we show that the canonical αK66 residue, understood to be critical for proteasome activator binding, plays a key role in stabilizing the open gate, irrespective of activator binding. This study differentiates between the residues in the HbYX motif that support binding interactions ("YX") versus those that allosterically contribute to gate opening (Hb). The insights reported here will guide future drug development efforts, particularly in designing small molecule proteasome activators, by targeting the identified hydrophobic pocket. | ||||||
History |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 9buz.cif.gz | 2.1 MB | Display | PDBx/mmCIF format |
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PDB format | pdb9buz.ent.gz | 1.8 MB | Display | PDB format |
PDBx/mmJSON format | 9buz.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 9buz_validation.pdf.gz | 1.6 MB | Display | wwPDB validaton report |
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Full document | 9buz_full_validation.pdf.gz | 1.6 MB | Display | |
Data in XML | 9buz_validation.xml.gz | 156.7 KB | Display | |
Data in CIF | 9buz_validation.cif.gz | 218.2 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/bu/9buz ftp://data.pdbj.org/pub/pdb/validation_reports/bu/9buz | HTTPS FTP |
-Related structure data
Related structure data | 44926MC C: citing same article (ref.) M: map data used to model this data |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
#1: Protein | Mass: 25893.490 Da / Num. of mol.: 14 / Mutation: V24Y Source method: isolated from a genetically manipulated source Source: (gene. exp.) Thermoplasma acidophilum (acidophilic) / Gene: psmA, Ta1288 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P25156 #2: Protein | Mass: 23169.811 Da / Num. of mol.: 14 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Thermoplasma acidophilum (acidophilic) / Gene: psmB, Ta0612 / Production host: Escherichia coli BL21(DE3) (bacteria) References: UniProt: P28061, proteasome endopeptidase complex #3: Water | ChemComp-HOH / | Has protein modification | N | |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: Thermoplasma acidophilum 20S proteasome alphaV24Y / Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT |
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Molecular weight | Value: 700 kDa/nm / Experimental value: NO |
Source (natural) | Organism: Thermoplasma acidophilum (acidophilic) |
Source (recombinant) | Organism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria) |
Buffer solution | pH: 7.4 |
Specimen | Conc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Vitrification | Cryogen name: ETHANE |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: TFS KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD / Nominal defocus max: 2400 nm / Nominal defocus min: 1200 nm |
Image recording | Electron dose: 50 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: FEI FALCON III (4k x 4k) |
-Processing
EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | |||||||||||||||
3D reconstruction | Resolution: 2.38 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 396542 / Symmetry type: POINT | |||||||||||||||
Atomic model building | Protocol: FLEXIBLE FIT |