H2020 Marie Curie Actions of the European Commission
846688
European Union
Citation
Journal: 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.
Model: Quantifoil R2/1 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.015 kPa / Details: 15 mA
Vitrification
Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
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Electron microscopy
Microscope
FEI TITAN KRIOS
Specialist optics
Energy filter - Name: TFS Selectris
Image recording
Film or detector model: FEI FALCON IV (4k x 4k) / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number grids imaged: 1 / Number real images: 7983 / Average electron dose: 50.0 e/Å2
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron optics
Illumination mode: OTHER / Imaging mode: BRIGHT FIELD / Nominal defocus max: 3.6 µm / Nominal defocus min: 0.8 µm
Type of model: INSILICO MODEL In silico model: a startup model was generated using AlphaFold predicted monomers that were superimposed onto 20S PDB:7ZYJ Details: 7ZYJ LEISHMANIA TARENTOLAE PROTEASOME 20S SUBUNIT IN COMPLEX WITH COMPOUND was used as a template for the superimposition of all 14 AlphaFold-generated models.
Final reconstruction
Applied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 2.89 Å / Resolution method: FSC 0.143 CUT-OFF / Details: Standard Homo Refine job in Cryosparc v4 / Number images used: 14257
Initial angle assignment
Type: RANDOM ASSIGNMENT
Final angle assignment
Type: OTHER
FSC plot (resolution estimation)
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Yorodumi
Open data
Basic information
Map data
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Keywords
Function and homology information
Trichomonas vaginalis G3 (eukaryote)
Authors
Citation
Structure visualization
Downloads & links
emd_16901.png
http://ftp.pdbj.org/pub/emdb/structures/EMD-16901
Z (Sec.)
Y (Row.)
X (Col.)
Sample components
Spodoptera frugiperda (fall armyworm)
Processing
Electron microscopy
FIELD EMISSION GUN
