Structural elucidation of recombinant Trichomonas vaginalis 20S proteasome bound to covalent inhibitors

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 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.

Fig. 1. Cloning, Recombinant Expression, and Purification of Tv20S Proteasome.

a Cloning of three baculovirus vectors containing either 7 α-subunits, 7 β-subunits or Ump-1 from T. vaginalis. b Co-infection of Sf9 insect cells with the three baculoviruses for proteasome expression results in assembly of half proteasomes initially and then 20S proteasomes. The twin-strep tag is indicated by ~. c Final purification step of recombinant Tv20S using Superose 6 chromatography. Absorbance at 280 nm (blue line) and proteasome activity assessed using a fluorogenic substrate, Suc-LLVY-amc (grey bars). d Side and planar views of a 20S proteasome complex showing two β rings sandwiched between two α rings. Within the β ring there are three catalytic subunits (β1, β2, β5) located within the central core. e Denaturing gel comparing Me4BodipyFL-Ahx3Leu3VS-labeled nTv20S and rTv20S. The gel was imaged by silver staining and fluorescent scan. f Native-PAGE gel of the same proteins from panel e. The protein band in the red box was excised for proteomic studies. Gels were repeated independently three times with similar results. g Proteomics analysis of the excised band and searched against T. vaginalis and S. frugiperda proteomes. The blue shading correlates with peak intensity. Source data are provided in the Source Data file.

Fig. 2. Biochemical and enzymatic characterization of recombinant Tv20S.

a Schematic representation of active sites during inhibition by MZB or CP-17, followed by visualization through fluorescent Me4BodipyFL-Ahx3Leu3VS probing. The inhibitor is depicted binding to specific active sites, thereby blocking enzymatic activity. Subsequent visualization techniques provide insights into the binding interaction and potential conformational changes induced by the inhibitor. b rTv20S, nTv20S and Hs20S were first incubated for 1 h with 50 µM CP-17 or 50 µM inhibitor or the solvent control, DMSO, and then incubated with 2 µM Me4BodipyFL-Ahx3Leu3VS for 16 h, followed by fractionation on a denaturing gel and fluorescence imaging at 470 nm excitation and 530 nm emission. The experiment was repeated independently three times with similar results. ‘ST’ stands for the molecular weight ladder. c Comparison of dose response curves for nTv20S and rTv20S with K_M and k_cat values indicated. Assays were performed in triplicate, with each dot representing the mean of 3 technical replicates. d Comparison and validation of inhibitor specificity using the indicated subunit-specific Tv20S proteasome substrates. Assays were performed in technical replicates (n = 3).

Fig. 3. Cryo-EM structures of Tv20S bound to inhibitors.

Processing of the cryo-EM datasets for a Tv20S bound to MZB and f CP17. The electron maps highlight the inhibitors in the active sites (b), g, shown as green mesh), while the catalytic subunits are depicted in different colors (β1 yellow, β2 cyan, and β5 magenta). MZB was observed only in the catalytic sites of β1 and β2 (b–e), while CP-17 was observed only in the catalytic sites of β2 and β5 (g–j). Both inhibitors were covalently bound to Thr1 as detailed for MZB in panels b–e) and for CP-17 in panels g–j. Amino acid residues within a reach of non-covalent interactions (up to 4 Å) are shown as sticks. More detailed interactions between inhibitors and active site pockets are shown in Supplementary Figs. 12–14.

Fig. 4. Structural comparison of Tv20S and human 20S.

The panel a comparison of human (orange) and T. vaginalis (light blue) 20S proteasomes. The further panels (b–d) show structural overlay of human 20S and covalent inhibitor CP-17 (blue sticks) in the active site pockets of β2 (panels b, c) and β5 (panels d, e) of Tv20S. The residues responsible for major structural differences in these active sites are shown as sticks (human-orange, Tv20S β2 cyan, and β5 magenta, and other subunits shown in white). The predicted clashes between CP-17 and amino acids from human 20S are encircled with dashed lines. In panels b, c, Met131 of human β2 is in clash with one of the indole rings of CP-17, whilst in Tv20S this moiety is accommodated in a deep hydrophobic pocket formed by Ala132 and Ala130. Similarly, in panels d, e, the pocket formed by Met45 of Tv20S β5 allows sufficient space for the phenyl ring of CP-17. In this location, human β5 Met45 is pushed by Ile35 towards the active site, forming a potential barrier for CP-17. The further detailed views highlighting the differences between human 20S and Tv20S-CP-17 in particular are shown in Supplementary Fig. 15.

Fig. 5. Surface detail of comparison of Tv20S and human 20S active sites.

Tv20S surface is in light blue, panels a–f, and its Thr1 residue is shown as blue sticks. Hs20S proteasomes is in light yellow, panels g–i, and its Thr1 is shown as orange sticks. MZB and CP-17, shown as blue sticks, are displayed in the human active site pockets using a structural overlay from Fig. 4. The clashes are encircled with a red dotted line. In panel h, Met131 of human β2 forms shallower binding pocket in this region whilst a similar elevation of active site pocket is observed formed by Met45 from human β5. In this location, human Met45 (β5) in this ligand-free structure is pushed by Ile35 towards the active site. Please refer to Supplementary Fig. 15 for more structural detail highlighting these differences including the architecture of human Glu22 (β2), and Glu106 (β3).