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- PDB-9f9p: CryoEM structure of recombinant Trypanosoma cruzi apo proteasome ... -

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Basic information

Entry
Database: PDB / ID: 9f9p
TitleCryoEM structure of recombinant Trypanosoma cruzi apo proteasome 20S subunit
Components
  • (Proteasome subunit ...) x 12
  • (Putative proteasome ...) x 2
KeywordsUNKNOWN FUNCTION / Proteasome
Function / homology
Function and homology information


proteasome core complex / proteasome core complex, beta-subunit complex / threonine-type endopeptidase activity / proteasome core complex, alpha-subunit complex / proteasomal protein catabolic process / proteolysis involved in protein catabolic process / ubiquitin-dependent protein catabolic process / proteasome-mediated ubiquitin-dependent protein catabolic process / nucleus / membrane / cytoplasm
Similarity search - Function
Proteasome subunit alpha 1 / Proteasome subunit beta 4 / Proteasome subunit beta 2 / Proteasome beta 3 subunit / Proteasome subunit alpha6 / Proteasome subunit alpha5 / Proteasome beta-type subunits signature. / Peptidase T1A, proteasome beta-subunit / Proteasome beta-type subunit, conserved site / Proteasome subunit A N-terminal signature ...Proteasome subunit alpha 1 / Proteasome subunit beta 4 / Proteasome subunit beta 2 / Proteasome beta 3 subunit / Proteasome subunit alpha6 / Proteasome subunit alpha5 / 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
Proteasome subunit beta / Putative proteasome beta 3 subunit / Proteasome subunit alpha type / Proteasome subunit beta / Proteasome subunit alpha type / Proteasome subunit alpha type / Proteasome subunit beta / Putative proteasome alpha 7 subunit / Proteasome subunit beta / Proteasome subunit beta ...Proteasome subunit beta / Putative proteasome beta 3 subunit / Proteasome subunit alpha type / Proteasome subunit beta / Proteasome subunit alpha type / Proteasome subunit alpha type / Proteasome subunit beta / Putative proteasome alpha 7 subunit / Proteasome subunit beta / Proteasome subunit beta / Proteasome subunit alpha type / Proteasome subunit alpha type / Proteasome subunit alpha type / Proteasome subunit beta
Similarity search - Component
Biological speciesTrypanosoma cruzi (eukaryote)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.25 Å
AuthorsRowland, P.
Funding support1items
OrganizationGrant numberCountry
Not funded
CitationJournal: J Biol Chem / Year: 2025
Title: Pharmacological and structural understanding of the Trypanosoma cruzi proteasome provides key insights for developing site-specific inhibitors.
Authors: Thomas C Eadsforth / Leah S Torrie / Paul Rowland / Emma V Edgar / Lorna M MacLean / Christy Paterson / David A Robinson / Sharon M Shepherd / John Thomas / Michael G Thomas / David W Gray / ...Authors: Thomas C Eadsforth / Leah S Torrie / Paul Rowland / Emma V Edgar / Lorna M MacLean / Christy Paterson / David A Robinson / Sharon M Shepherd / John Thomas / Michael G Thomas / David W Gray / Vincent L G Postis / Manu De Rycker /
Abstract: The proteasome is considered an excellent drug target for many infectious diseases as well as cancer. Challenges with robust and safe supply of proteasomes from infectious agents, lack of structural ...The proteasome is considered an excellent drug target for many infectious diseases as well as cancer. Challenges with robust and safe supply of proteasomes from infectious agents, lack of structural information, and complex pharmacology due to multiple active sites have hampered progress in the infectious disease space. We recombinantly expressed the proteasome of the protozoan parasite Trypanosoma cruzi, the causative agent of Chagas disease, and demonstrate pharmacological equivalence to the native T. cruzi proteasome. Active-site mutant recombinant proteasomes reveal substrate promiscuity for WT proteasomes, with important implications for assessing pharmacological responses of active-site selective inhibitors. Using these mutant proteasomes, we show that some selective parasite proteasome inhibitors only partially inhibit the chymotrypsin-like activity, including a newly developed 5-(phenoxymethyl)furan-2-carboxamide-based proteasome inhibitor. In spite of partial inhibition, these compounds remain potent inhibitors of intracellular T. cruzi growth. Drug-resistant mutants provide further insights in drug mode-of-inhibition. We also present the high-resolution CryoEM structures of both native and recombinantly-expressed T. cruzi proteasomes which reveal pharmacologically relevant differences in the ligand-binding site compared to the related Leishmania proteasome. Furthermore, we show that the trypanosomatid β4/β5 selectivity pocket is not present in the proteasome structures of other protozoan parasites. This work highlights the need, and provides approaches, to precisely assess proteasome substrate selectivity and pharmacology. It enables structure-guided drug discovery for this promising Chagas disease drug target, provides a new chemical starting point for drug discovery, and paves the road for development of robust proteasome drug discovery programmes for other eukaryotic infectious diseases.
History
DepositionMay 8, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 25, 2024Provider: repository / Type: Initial release
Revision 1.1Jan 8, 2025Group: Data collection / Database references / Category: citation / em_admin / Item: _citation.journal_volume / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Proteasome subunit alpha type
B: Proteasome subunit alpha type
C: Proteasome subunit alpha type
D: Proteasome subunit alpha type
E: Proteasome subunit alpha type
F: Proteasome subunit alpha type
G: Putative proteasome alpha 7 subunit
H: Proteasome subunit beta
I: Proteasome subunit beta
J: Putative proteasome beta 3 subunit
K: Proteasome subunit beta
L: Proteasome subunit beta
M: Proteasome subunit beta
N: Proteasome subunit beta
O: Proteasome subunit alpha type
P: Proteasome subunit alpha type
Q: Proteasome subunit alpha type
R: Proteasome subunit alpha type
S: Proteasome subunit alpha type
T: Proteasome subunit alpha type
U: Putative proteasome alpha 7 subunit
V: Proteasome subunit beta
W: Proteasome subunit beta
X: Putative proteasome beta 3 subunit
Y: Proteasome subunit beta
Z: Proteasome subunit beta
a: Proteasome subunit beta
b: Proteasome subunit beta


Theoretical massNumber of molelcules
Total (without water)784,67628
Polymers784,67628
Non-polymers00
Water19,3481074
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-ID
11
22
33
44
55
66
77
88
99
1010
1111
1212
1313
1414
/ NCS ensembles :
ID
1
2
3
4
5
6
7
8
9
10
11
12
13
14

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Components

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Proteasome subunit ... , 12 types, 24 molecules AOBPCQDRESFTHVIWKYLZMaNb

#1: Protein Proteasome subunit alpha type


Mass: 29876.949 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Trypanosoma cruzi (eukaryote) / Gene: C4B63_2g302 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: A0A2V2W7U6
#2: Protein Proteasome subunit alpha type


Mass: 25080.297 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Trypanosoma cruzi (eukaryote) / Gene: C3747_243g34, C4B63_43g135 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: A0A2V2V7B1
#3: Protein Proteasome subunit alpha type


Mass: 31997.125 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Trypanosoma cruzi (eukaryote) / Gene: C4B63_25g312 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: A0A2V2VJR6
#4: Protein Proteasome subunit alpha type


Mass: 28109.943 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Trypanosoma cruzi (eukaryote) / Gene: C3747_43g63, C3747_44g28, ECC02_002871 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: A0A2V2WZ04
#5: Protein Proteasome subunit alpha type


Mass: 27163.762 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Trypanosoma cruzi (eukaryote) / Gene: C4B63_43g129 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: A0A2V2V560
#6: Protein Proteasome subunit alpha type


Mass: 29400.271 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Trypanosoma cruzi (eukaryote) / Gene: pr29A, C4B63_48g135 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q3ZMB6
#8: Protein Proteasome subunit beta


Mass: 30777.965 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Trypanosoma cruzi (eukaryote) / Gene: C4B63_89g44 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: A0A2V2UU31
#9: Protein Proteasome subunit beta


Mass: 32086.119 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Trypanosoma cruzi (eukaryote) / Gene: TCDM_00108 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: V5BCU3
#11: Protein Proteasome subunit beta


Mass: 22745.156 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Trypanosoma cruzi (eukaryote) / Gene: C4B63_13g138 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: A0A2V2VNZ4
#12: Protein Proteasome subunit beta


Mass: 34977.438 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Trypanosoma cruzi (eukaryote) / Gene: C4B63_48g131 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: A0A2V2V5A7
#13: Protein Proteasome subunit beta


Mass: 27832.482 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Trypanosoma cruzi (eukaryote) / Gene: C4B63_6g586 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: A0A2V2VW62
#14: Protein Proteasome subunit beta


Mass: 24222.770 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Trypanosoma cruzi (eukaryote) / Gene: C4B63_7g423 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: A0A2V2VV98

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Putative proteasome ... , 2 types, 4 molecules GUJX

#7: Protein Putative proteasome alpha 7 subunit


Mass: 25643.092 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Trypanosoma cruzi (eukaryote) / Gene: C4B63_12g396 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: A0A2V2VRE2
#10: Protein Putative proteasome beta 3 subunit


Mass: 22424.807 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Trypanosoma cruzi (eukaryote) / Gene: C4B63_112g19 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: A0A2V2UWV1

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Non-polymers , 1 types, 1074 molecules

#15: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 1074 / Source method: isolated from a natural source / Formula: H2O

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Details

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: Proteasome 20S / Type: COMPLEX / Entity ID: #1-#14 / Source: RECOMBINANT
Source (natural)Organism: Trypanosoma cruzi (eukaryote)
Source (recombinant)Organism: Trichoplusia ni (cabbage looper)
Buffer solutionpH: 7.5
SpecimenEmbedding 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: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: OTHER / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 1.05 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM softwareName: REFMAC / Version: 5.8.0405 / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.25 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 92013 / Symmetry type: POINT
RefinementResolution: 2.25→196.68 Å / Cor.coef. Fo:Fc: 0.896 / SU B: 4.508 / SU ML: 0.099 / ESU R: 0.177
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflection
Rwork0.2613 --
obs0.2613 746601 100 %
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso mean: 27.973 Å2
Baniso -1Baniso -2Baniso -3
1-0.12 Å2-0.06 Å20 Å2
2--0.24 Å2-0 Å2
3----0.37 Å2
Refinement stepCycle: 1 / Total: 49596
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.010.01249428
ELECTRON MICROSCOPYr_bond_other_d0.0010.01646680
ELECTRON MICROSCOPYr_angle_refined_deg1.8351.64866880
ELECTRON MICROSCOPYr_angle_other_deg0.5441.57107374
ELECTRON MICROSCOPYr_dihedral_angle_1_deg6.9356236
ELECTRON MICROSCOPYr_dihedral_angle_2_deg6.9735338
ELECTRON MICROSCOPYr_dihedral_angle_3_deg17.418108408
ELECTRON MICROSCOPYr_dihedral_angle_4_deg
ELECTRON MICROSCOPYr_chiral_restr0.0810.27512
ELECTRON MICROSCOPYr_gen_planes_refined0.0090.0258266
ELECTRON MICROSCOPYr_gen_planes_other0.0010.0211374
ELECTRON MICROSCOPYr_nbd_refined
ELECTRON MICROSCOPYr_nbd_other
ELECTRON MICROSCOPYr_nbtor_refined
ELECTRON MICROSCOPYr_nbtor_other
ELECTRON MICROSCOPYr_xyhbond_nbd_refined
ELECTRON MICROSCOPYr_xyhbond_nbd_other
ELECTRON MICROSCOPYr_metal_ion_refined
ELECTRON MICROSCOPYr_metal_ion_other
ELECTRON MICROSCOPYr_symmetry_vdw_refined
ELECTRON MICROSCOPYr_symmetry_vdw_other
ELECTRON MICROSCOPYr_symmetry_hbond_refined
ELECTRON MICROSCOPYr_symmetry_hbond_other
ELECTRON MICROSCOPYr_symmetry_metal_ion_refined
ELECTRON MICROSCOPYr_symmetry_metal_ion_other
ELECTRON MICROSCOPYr_mcbond_it3.4122.26425052
ELECTRON MICROSCOPYr_mcbond_other3.4122.26325052
ELECTRON MICROSCOPYr_mcangle_it5.6034.0631252
ELECTRON MICROSCOPYr_mcangle_other5.6044.06131253
ELECTRON MICROSCOPYr_scbond_it5.6693.2724376
ELECTRON MICROSCOPYr_scbond_other5.6693.2724377
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other9.5555.53335629
ELECTRON MICROSCOPYr_long_range_B_refined12.08821.6150871
ELECTRON MICROSCOPYr_long_range_B_other12.09721.6450739
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded
Refine LS restraints NCS

Refine-ID: ELECTRON MICROSCOPY

Ens-IDDom-IDAuth asym-IDNumberTypeRms dev position (Å)Weight position
1010J1555tight positional00.05
1111K1586tight positional00.05
1212L1578tight positional00.05
1313M1647tight positional00.05
1414N1695tight positional00.05
11A1884tight thermal0.080.5
22B1737tight thermal0.080.5
33C2126tight thermal0.140.5
44D1864tight thermal0.160.5
55E1757tight thermal0.130.5
66F1722tight thermal0.080.5
77G1724tight thermal0.10.5
88H1723tight thermal0.060.5
99I1663tight thermal0.10.5
1010J1555tight thermal0.050.5
1111K1586tight thermal0.070.5
1212L1578tight thermal0.050.5
1313M1647tight thermal0.070.5
1414N1695tight thermal0.050.5
LS refinement shellResolution: 2.247→2.305 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0 0 -
Rwork0.506 55202 -
obs--100 %

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