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- PDB-8oix: CryoEM structure of 20S Trichomonas vaginalis proteasome in compl... -

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Basic information

Entry
Database: PDB / ID: 8oix
TitleCryoEM structure of 20S Trichomonas vaginalis proteasome in complex with proteasome inhibitor Salinosporamid A
Components
  • (Family T1, proteasome alpha subunit, threonine ...) x 4
  • (Proteasome subunit ...) x 8
  • Family T1, proteasome beta subunit, threonine peptidase
  • proteasome endopeptidase complex
KeywordsHYDROLASE / Proteasome / 20S / Trichomonas vaginalis / covalently bound Salinosporamide A / Marizomib
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 / endopeptidase activity / proteasome-mediated ubiquitin-dependent protein catabolic process / nucleus / cytoplasm / cytosol
Similarity search - Function
Proteasome subunit beta 4 / Proteasome subunit beta 2 / Proteasome beta 3 subunit / 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 subunit beta 4 / Proteasome subunit beta 2 / Proteasome beta 3 subunit / 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
Similarity search - Domain/homology
Chem-SA1 / Family T1, proteasome alpha subunit, threonine peptidase / Proteasome subunit beta / Proteasome subunit alpha type / Family T1, proteasome alpha subunit, threonine peptidase / Proteasome subunit beta / proteasome endopeptidase complex / Proteasome subunit beta / Family T1, proteasome beta subunit, threonine peptidase / Family T1, proteasome alpha subunit, threonine peptidase ...Chem-SA1 / Family T1, proteasome alpha subunit, threonine peptidase / Proteasome subunit beta / Proteasome subunit alpha type / Family T1, proteasome alpha subunit, threonine peptidase / Proteasome subunit beta / proteasome endopeptidase complex / Proteasome subunit beta / Family T1, proteasome beta subunit, threonine peptidase / Family T1, proteasome alpha subunit, threonine peptidase / Proteasome subunit beta / Proteasome subunit beta / Proteasome subunit alpha type / Family T1, proteasome alpha subunit, threonine peptidase / Proteasome subunit alpha type
Similarity search - Component
Biological speciesTrichomonas vaginalis G3 (eukaryote)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.89 Å
AuthorsSilhan, J. / Fajtova, P. / Boura, E.
Funding supportEuropean Union, 1items
OrganizationGrant numberCountry
H2020 Marie Curie Actions of the European Commission846688European Union
CitationJournal: Nat Commun / Year: 2024
Title: Structural elucidation of recombinant Trichomonas vaginalis 20S proteasome bound to covalent inhibitors.
Authors: Jan Silhan / Pavla Fajtova / Jitka Bartosova / Brianna M Hurysz / Jehad Almaliti / Yukiko Miyamoto / Lars Eckmann / William H Gerwick / Anthony J O'Donoghue / Evzen Boura /
Abstract: The proteasome is a proteolytic enzyme complex essential for protein homeostasis in mammalian cells and protozoan parasites like Trichomonas vaginalis (Tv), the cause of the most common, non-viral ...The proteasome is a proteolytic enzyme complex essential for protein homeostasis in mammalian cells and protozoan parasites like Trichomonas vaginalis (Tv), the cause of the most common, non-viral sexually transmitted disease. Tv and other protozoan 20S proteasomes have been validated as druggable targets for antimicrobials. However, low yields and purity of the native proteasome have hindered studies of the Tv 20S proteasome (Tv20S). We address this challenge by creating a recombinant protozoan proteasome by expressing all seven α and seven β subunits of Tv20S alongside the Ump-1 chaperone in insect cells. The recombinant Tv20S displays biochemical equivalence to its native counterpart, confirmed by various assays. Notably, the marizomib (MZB) inhibits all catalytic subunits of Tv20S, while the peptide inhibitor carmaphycin-17 (CP-17) specifically targets β2 and β5. Cryo-electron microscopy (cryo-EM) unveils the structures of Tv20S bound to MZB and CP-17 at 2.8 Å. These findings explain MZB's low specificity for Tv20S compared to the human proteasome and demonstrate CP-17's higher specificity. Overall, these data provide a structure-based strategy for the development of specific Tv20S inhibitors to treat trichomoniasis.
History
DepositionMar 23, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Apr 10, 2024Provider: repository / Type: Initial release
Revision 2.0Apr 17, 2024Group: Advisory / Atomic model ...Advisory / Atomic model / Database references / Derived calculations / Polymer sequence / Source and taxonomy / Structure summary
Category: atom_site / entity ...atom_site / entity / entity_poly / entity_poly_seq / entity_src_gen / pdbx_poly_seq_scheme / pdbx_struct_sheet_hbond / pdbx_unobs_or_zero_occ_residues / struct_conf / struct_ref / struct_ref_seq / struct_sheet_range
Item: _atom_site.label_seq_id / _entity.formula_weight ..._atom_site.label_seq_id / _entity.formula_weight / _entity_poly.pdbx_seq_one_letter_code / _entity_poly.pdbx_seq_one_letter_code_can / _entity_src_gen.pdbx_end_seq_num / _pdbx_struct_sheet_hbond.range_1_label_seq_id / _pdbx_struct_sheet_hbond.range_2_label_seq_id / _struct_conf.beg_label_seq_id / _struct_conf.end_label_seq_id / _struct_ref.pdbx_align_begin / _struct_ref.pdbx_seq_one_letter_code / _struct_ref_seq.db_align_beg / _struct_ref_seq.pdbx_auth_seq_align_beg / _struct_ref_seq.seq_align_end / _struct_sheet_range.beg_label_seq_id / _struct_sheet_range.end_label_seq_id
Revision 2.1Oct 9, 2024Group: Data collection / Structure summary
Category: em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update / _pdbx_entry_details.has_protein_modification
Revision 2.2Oct 23, 2024Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Family T1, proteasome alpha subunit, threonine peptidase
B: Family T1, proteasome alpha subunit, threonine peptidase
C: Proteasome subunit alpha type
D: Proteasome subunit alpha type
E: Proteasome subunit alpha type
F: Family T1, proteasome alpha subunit, threonine peptidase
G: Family T1, proteasome alpha subunit, threonine peptidase
H: Proteasome subunit beta
I: proteasome endopeptidase complex
J: Proteasome subunit beta
K: Proteasome subunit beta
L: Proteasome subunit beta
M: Proteasome subunit beta
N: Family T1, proteasome beta subunit, threonine peptidase
O: Family T1, proteasome alpha subunit, threonine peptidase
P: Family T1, proteasome alpha subunit, threonine peptidase
Q: Proteasome subunit alpha type
R: Proteasome subunit alpha type
S: Proteasome subunit alpha type
T: Family T1, proteasome alpha subunit, threonine peptidase
U: Family T1, proteasome alpha subunit, threonine peptidase
V: Proteasome subunit beta
W: proteasome endopeptidase complex
X: Proteasome subunit beta
Y: Proteasome subunit beta
Z: Proteasome subunit beta
a: Proteasome subunit beta
b: Family T1, proteasome beta subunit, threonine peptidase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)708,40632
Polymers707,28828
Non-polymers1,1174
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, Negative Stain with Uranyl Acetate SEC
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area117030 Å2
ΔGint-412 kcal/mol
Surface area177780 Å2
MethodPISA

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Components

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Family T1, proteasome alpha subunit, threonine ... , 4 types, 8 molecules AOBPFTGU

#1: Protein Family T1, proteasome alpha subunit, threonine peptidase


Mass: 26511.184 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Trichomonas vaginalis G3 (eukaryote) / Gene: TVAG_267300 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: A2F568
#2: Protein Family T1, proteasome alpha subunit, threonine peptidase


Mass: 25444.994 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Trichomonas vaginalis G3 (eukaryote) / Gene: TVAG_103780 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: A2FJV7
#6: Protein Family T1, proteasome alpha subunit, threonine peptidase


Mass: 25682.287 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Trichomonas vaginalis G3 (eukaryote) / Gene: TVAG_206040 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: A2E1I9
#7: Protein Family T1, proteasome alpha subunit, threonine peptidase


Mass: 27067.422 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Trichomonas vaginalis G3 (eukaryote) / Gene: TVAG_185200 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: A2D8G5

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Proteasome subunit ... , 8 types, 16 molecules CQDRESHVJXKYLZMa

#3: Protein Proteasome subunit alpha type


Mass: 27989.707 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Trichomonas vaginalis G3 (eukaryote) / Gene: TVAG_058050 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: A2FT79
#4: Protein Proteasome subunit alpha type


Mass: 26306.916 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Trichomonas vaginalis G3 (eukaryote) / Gene: TVAG_340740 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: A2DTN3
#5: Protein Proteasome subunit alpha type


Mass: 27330.986 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Trichomonas vaginalis G3 (eukaryote) / Gene: TVAG_293540 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: A2FCM7
#8: Protein Proteasome subunit beta


Mass: 21930.805 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Trichomonas vaginalis G3 (eukaryote) / Gene: TVAG_127250 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: A2E7Z2
#10: Protein Proteasome subunit beta


Mass: 23028.574 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Trichomonas vaginalis G3 (eukaryote) / Gene: TVAG_286960 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: A2F3H9
#11: Protein Proteasome subunit beta


Mass: 21421.600 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Trichomonas vaginalis G3 (eukaryote) / Gene: TVAG_403280 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: A2F8W4
#12: Protein Proteasome subunit beta


Mass: 22572.598 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Trichomonas vaginalis G3 (eukaryote) / Gene: TVAG_013010 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: A2DD57
#13: Protein Proteasome subunit beta


Mass: 25001.025 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Trichomonas vaginalis G3 (eukaryote) / Gene: TVAG_152400 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: A2F716

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Protein , 2 types, 4 molecules IWNb

#9: Protein proteasome endopeptidase complex


Mass: 26869.426 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Trichomonas vaginalis G3 (eukaryote) / Gene: TVAG_231360 / Production host: Spodoptera frugiperda (fall armyworm)
References: UniProt: A2F2T6, proteasome endopeptidase complex
#14: Protein Family T1, proteasome beta subunit, threonine peptidase


Mass: 26486.643 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Trichomonas vaginalis G3 (eukaryote) / Gene: TVAG_290960 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: A2F3X4

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Non-polymers , 1 types, 4 molecules

#15: Chemical
ChemComp-SA1 / (3AR,6R,6AS)-6-((S)-((S)-CYCLOHEX-2-ENYL)(HYDROXY)METHYL)-6A-METHYL-4-OXO-HEXAHYDRO-2H-FURO[3,2-C]PYRROLE-6-CARBALDEHYDE / Salinosporamide A, bound form


Mass: 279.332 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C15H21NO4 / Feature type: SUBJECT OF INVESTIGATION

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Details

Has ligand of interestY
Has protein modificationY

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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: Ternary complex of 20S proteasome from Trichomonas vaginalis
Type: COMPLEX
Details: Subunits beta 1 and beta 2 a covalently attached to protease inhibitor marizomib (Salinosporamid A)
Entity ID: #1-#14 / Source: RECOMBINANT
Molecular weightValue: 0.704 MDa / Experimental value: NO
Source (natural)Organism: Trichomonas vaginalis G3 (eukaryote)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMHEPESHEPES1
2150 mMsodium chlorideNaCl1
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 15 mA / Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 3600 nm / Nominal defocus min: 800 nm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 50 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 7983
EM imaging opticsEnergyfilter name: TFS Selectris
Image scansWidth: 4096 / Height: 4096

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Processing

EM softwareName: PHENIX / Version: 1.20.1_4487: / Category: model refinement
CTF correctionType: NONE
Particle selectionNum. of particles selected: 2303719
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 2.89 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 14257 / Details: Standard Homo Refine job in Cryosparc v4 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00448680
ELECTRON MICROSCOPYf_angle_d0.60865880
ELECTRON MICROSCOPYf_dihedral_angle_d13.03717850
ELECTRON MICROSCOPYf_chiral_restr0.0457434
ELECTRON MICROSCOPYf_plane_restr0.0088518

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