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- PDB-9buz: Thermoplasma acidophilum 20S proteasome - alphaV24Y -

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Basic information

Entry
Database: PDB / ID: 9buz
TitleThermoplasma acidophilum 20S proteasome - alphaV24Y
Components
  • Proteasome subunit alpha
  • Proteasome subunit beta
KeywordsHYDROLASE / 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
Peptidase T1A, proteasome beta-subunit, archaeal / Proteasome alpha subunit, archaeal / Proteasome beta-type subunits signature. / Peptidase T1A, proteasome beta-subunit / Proteasome beta-type subunit, conserved site / Proteasome subunit A N-terminal signature / Proteasome alpha-type subunits signature. / Proteasome alpha-subunit, N-terminal domain / Proteasome subunit A N-terminal signature Add an annotation / : ...Peptidase T1A, proteasome beta-subunit, archaeal / Proteasome alpha subunit, archaeal / Proteasome beta-type subunits signature. / Peptidase T1A, proteasome beta-subunit / Proteasome beta-type subunit, conserved site / Proteasome subunit A N-terminal signature / Proteasome alpha-type subunits signature. / Proteasome alpha-subunit, N-terminal domain / Proteasome subunit A N-terminal signature Add an annotation / : / Proteasome alpha-type subunit / Proteasome alpha-type subunit profile. / Proteasome B-type subunit / Proteasome beta-type subunit profile. / Proteasome subunit / Proteasome, subunit alpha/beta / Nucleophile aminohydrolases, N-terminal
Similarity search - Domain/homology
Proteasome subunit alpha / Proteasome subunit beta
Similarity search - Component
Biological speciesThermoplasma acidophilum (acidophilic)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.38 Å
AuthorsChuah, J. / Smith, D.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01AG064188 United States
CitationJournal: 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
DepositionMay 18, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 30, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Proteasome subunit alpha
B: Proteasome subunit alpha
C: Proteasome subunit alpha
D: Proteasome subunit alpha
E: Proteasome subunit alpha
F: Proteasome subunit alpha
G: Proteasome subunit alpha
H: Proteasome subunit beta
I: Proteasome subunit beta
J: Proteasome subunit beta
K: Proteasome subunit beta
L: Proteasome subunit beta
M: Proteasome subunit beta
N: Proteasome subunit beta
O: Proteasome subunit alpha
P: Proteasome subunit alpha
Q: Proteasome subunit alpha
R: Proteasome subunit alpha
S: Proteasome subunit alpha
T: Proteasome subunit alpha
U: Proteasome subunit alpha
V: Proteasome subunit beta
W: Proteasome subunit beta
X: Proteasome subunit beta
Y: Proteasome subunit beta
Z: Proteasome subunit beta
a: Proteasome subunit beta
b: Proteasome subunit beta


Theoretical massNumber of molelcules
Total (without water)686,88628
Polymers686,88628
Non-polymers00
Water7,134396
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
Proteasome subunit alpha / 20S proteasome alpha subunit / Proteasome core protein PsmA


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
Proteasome subunit beta / 20S proteasome beta subunit / Proteasome core protein PsmB


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 / water


Mass: 18.015 Da / Num. of mol.: 396 / Source method: isolated from a natural source / Formula: H2O
Has protein modificationN

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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: Thermoplasma acidophilum 20S proteasome alphaV24Y / Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightValue: 700 kDa/nm / Experimental value: NO
Source (natural)Organism: Thermoplasma acidophilum (acidophilic)
Source (recombinant)Organism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
Buffer solutionpH: 7.4
SpecimenConc.: 1 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: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2400 nm / Nominal defocus min: 1200 nm
Image recordingElectron dose: 50 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: FEI FALCON III (4k x 4k)

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Processing

EM software
IDNameVersionCategoryDetails
7Coot0.9.6.model fittingWinCoot CCP4
9PHENIX1.21-5207model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.38 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 396542 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT

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