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- PDB-8f66: Thermoplasma acidophilum 20S proteasome - L81Y mutation in alpha ... -

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Basic information

Entry
Database: PDB / ID: 8f66
TitleThermoplasma acidophilum 20S proteasome - L81Y mutation in alpha subunit
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 / threonine-type endopeptidase activity / proteasome core complex, alpha-subunit complex / 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 / Aminohydrolase, N-terminal nucleophile (Ntn) domain / Glutamine Phosphoribosylpyrophosphate, subunit 1, domain 1 / 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 ...Peptidase T1A, proteasome beta-subunit, archaeal / Proteasome alpha subunit, archaeal / Aminohydrolase, N-terminal nucleophile (Ntn) domain / Glutamine Phosphoribosylpyrophosphate, subunit 1, domain 1 / 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 B-type subunit / Proteasome beta-type subunit profile. / : / Proteasome alpha-type subunit / Proteasome alpha-type subunit profile. / Proteasome subunit / Proteasome, subunit alpha/beta / Nucleophile aminohydrolases, N-terminal / 4-Layer Sandwich / Alpha Beta
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: 2.28 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: Commun Biol / Year: 2023
Title: High resolution structures define divergent and convergent mechanisms of archaeal proteasome activation.
Authors: Janelle J Y Chuah / Matthew S Rexroad / David M Smith /
Abstract: Considering the link between neurodegenerative diseases and impaired proteasome function, and the neuro-protective impact of enhanced proteasome activity in animal models, it's crucial to understand ...Considering the link between neurodegenerative diseases and impaired proteasome function, and the neuro-protective impact of enhanced proteasome activity in animal models, it's crucial to understand proteasome activation mechanisms. A hydrophobic-tyrosine-any residue (HbYX) motif on the C-termini of proteasome-activating complexes independently triggers gate-opening of the 20S core particle for protein degradation; however, the causal allosteric mechanism remains unclear. Our study employs a structurally irreducible dipeptide HbYX mimetic to investigate the allosteric mechanism of gate-opening in the archaeal proteasome. High-resolution cryo-EM structures pinpoint vital residues and conformational changes in the proteasome α-subunit implicated in HbYX-dependent activation. Using point mutations, we simulated the HbYX-bound state, providing support for our mechanistic model. We discerned four main mechanistic elements triggering gate-opening: 1) back-loop rearrangement adjacent to K66, 2) intra- and inter- α subunit conformational changes, 3) occupancy of the hydrophobic pocket, and 4) a highly conserved isoleucine-threonine pair in the 20S channel stabilizing the open and closed states, termed the "IT switch." Comparison of different complexes unveiled convergent and divergent mechanism of 20S gate-opening among HbYX-dependent and independent activators. This study delivers a detailed molecular model for HbYX-dependent 20S gate-opening, enabling the development of small molecule proteasome activators that hold promise to treat neurodegenerative diseases.
History
DepositionNov 16, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 30, 2023Provider: 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)686,69028
Polymers686,69028
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Proteasome subunit alpha / 20S proteasome alpha subunit / Proteasome core protein PsmA


Mass: 25879.465 Da / Num. of mol.: 14 / Mutation: L81Y
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, proteasome endopeptidase complex
#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

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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 - alphaL81Y mutant
Type: COMPLEX / Entity ID: #2, #1 / Source: RECOMBINANT
Molecular weightValue: 0.7 MDa / 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

SoftwareName: PHENIX / Version: 1.20rc1_4392: / Classification: refinement
EM software
IDNameVersionCategoryDetails
7Coot0.9.6.model fittingWinCoot CCP4
9PHENIX1.20.1-4487model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.28 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 131453 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.01547236
ELECTRON MICROSCOPYf_angle_d2.163756
ELECTRON MICROSCOPYf_dihedral_angle_d20.6217794
ELECTRON MICROSCOPYf_chiral_restr0.1237434
ELECTRON MICROSCOPYf_plane_restr0.0098176

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