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

Domain: earth_science
Published: 2024-10-04
Editorial outcome: major_revision

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.

Introduction
Trichomonas vaginalis (Tv), a pear-shaped protozoan organism, is the etiological agent of trichomoniasis, the most widespread non-viral sexually transmitted disease (STD) worldwide1–3. This parasite possesses a single flagellum, which enables its motility, as well as several hair-like structures called pili, facilitating its attachment to host cells. Tv has a complex cytoskeleton that gives it the ability to alter its shape and traverse host tissues. In women, Tv infection can cause vaginitis, while in men it can cause urethritis and prostatitis. Notably, this infection heightens the risk of transmission of HIV and other STDs in both sexes. Current treatment relies on 5-nitroimidazoles4,5, however, the emergence of resistant strains poses a significant public health threat due to the lack of alternative treatment options6,7. Consequently, effective anti-parasitic compounds are urgently needed. Recently, the critical role of the proteasome in the survival of Tv was demonstrated, validating it as a potential drug target for treating trichomoniasis8–11.

The proteasome is a large protein complex that plays a pivotal role in the degradation and homeostasis of cellular proteins12–15. It consists of two main components: the 20S core particle and the 19S regulatory particle. The 20S core particle forms a cylindrical structure consisting of four stacked rings, comprised of two inner rings of seven different β subunits and two outer rings of seven different α subunits. Importantly, three of the β subunits, β1, β2 and β5, have proteolytic activity and are therefore responsible for protein degradation16,17. Structural studies using yeast and mammalian proteasomes have greatly improved the understanding of the mechanisms of protein degradation by these enzymes, as well as their interactions with inhibitors and other proteins. To perform structural studies, highly pure and homogeneous samples are required. We and others have previously isolated proteasomes from parasites such as Plasmodium falciparum that are sufficiently pure for structural studies18,19 but efforts to isolate highly pure proteasomes from Tv were unsuccessful10,20. One promising strategy to circumvent these technical limitations is to produce recombinant Tv proteasome. Archaeal 20S proteasomes, composed of homo-heptameric rings can be produced by co-expressing the two subunits in E. coli21. However, the tightly regulated biogenesis pathway of eukaryotic 20S proteasome makes expression in bacteria unfeasible22. The assembly of the human 20S proteasome involves the stepwise incorporation of 14 distinct protein subunits, α1–α7 and β1–β7, assisted by five dedicated chaperones. These chaperones consist of Ump-1 and the heterodimers, PAC1-PAC2 and PAC3-PAC423,24. The seven α subunits form the α-ring which then serves as a scaffold for the ordered incorporation of β subunits. After dimerization of two pre-assembled half-proteasomes, the final maturation step involves self-cleavage of the β subunit pro-peptides. These N-terminal extensions, present in immature β subunits, are believed to shield their proteolytic activity until formation of the entire 20S proteasome and may also contribute as scaffolds for proteasome assembly16,25.

Following maturation, the β1, β2, and β5 subunits contain a threonine residue as the N-terminal amino acid. This threonine residue can initiate a catalytic reaction through nucleophilic attack of the carbonyl carbon within the peptide backbone of the substrate. This reaction leads to cleavage of the protein or peptide substrates. The β1, β2, and β5 subunits have distinct substrate specificity preferences and are generally considered to have caspase-like, trypsin-like, and chymotrypsin-like activities, respectively26. These three subunits enable the proteasome to cleave a wide variety of substrates at distinct sites allowing the cell to efficiently degrade proteins and maintain cellular protein homeostasis27,28.

Proteasome inhibitors bind to the proteasome, blocking its proteolytic activity and impeding protein degradation. This results in the accumulation of proteins within cells, ultimately triggering cell death. Proteasome inhibitors have been shown to selectively kill cancer cells and three (bortezomib, carfilzomib, and ixazomib) have been approved for treatment of multiple myeloma and mantle cell lymphoma29. Additionally, proteasome inhibitors have recently been investigated as potential treatments for parasitic diseases, such as malaria19,30,31, Chagas disease and leishmaniasis32–34, with one molecule, LXE408, developed by Novartis Pharmaceuticals entering Phase II clinical trials for leishmaniasis in December 2022. These clinical studies in different parasites support our rationale for developing proteasome inhibitors to treat trichomoniasis.

Carmaphycin B is a marine cyanobacterial metabolite that was isolated from extracts of Symploca sp. collected from Curaçao35. This compound is a potent inhibitor of the 20S proteasome from mammals, yeast, trematodes and protozoa30,35–37 and has potent cytotoxic activity in these cells. We have synthesized more than 100 analogs that exhibit a range of biological activities. Carmaphycin-17 (CP-17) is one analog that has been shown to have reduced cytotoxicity for human cells while exhibiting significant activity against Tv10,35. Additionally, it has shown to be potent against metronidazole-resistant Tv strains10. In topical treatment studies of mice vaginally infected with the related trichomonad, Trichomonas fetus, CP-17 reduced parasite burden without noticeable adverse effects. These studies not only validated Tv proteasome as a therapeutic target for trichomonacidal agents but also highlighted CP-17 as a starting point for further medicinal chemistry studies10.

Marizomib (MZB) is a natural product isolated from the marine bacterium Salinispora tropica38. This non-peptidic proteasome inhibitor is currently in Phase III clinical trials for the treatment of various types of glioblastoma and stands out as the only proteasome inhibitor capable of readily crossing the blood-brain barrier39–41. While MZB primarily targets the β5 subunit of the 20S proteasome, it has been reported to also inhibit the β1 and β2 subunits at higher concentrations42. This ability to target multiple subunits of the proteasome may contribute to its potent anti-cancer activity. Compared to clinically approved proteasome inhibitors such as bortezomib and carfilzomib, MZB offers advantages including enhanced stability, bioavailability, and the potential for a broader range of anti-cancer activity. While MZB is too toxic for use as an anti-microbial drug, analogs of this molecule could be developed that are selective for Tv over mammalian cells.

In this study, we successfully employed the baculovirus expression system to generate recombinant Tv20S proteasome. Biochemical comparison with the native enzyme confirmed that all three catalytic subunits of the recombinant enzyme were functional, as they hydrolyzed three different subunit-specific fluorogenic substrates. A broad-spectrum activity-based probe covalently labeled the catalytic threonine from each subunit. Furthermore, we determined the structure of Tv20S in complex with MZB and CP-17. MZB was found to bind to six sites within Tv20S that corresponded to three subunits in each β-ring, albeit the electron density was insufficient to fully resolve its binding to the β5 subunit. By comparison, CP-17 bound to four of six catalytic subunits, corresponding to β2 and β5 from each β-ring. These findings not only offer valuable insights into the structure and function of Tv20S proteasome but also shed light on the underlying molecular mechanisms of MZB and CP-17 inhibition.

Results
Expression of recombinant Tv20S
The genome of T. vaginalis G3 is available from trichdb.org and all 14 subunits of the Tv20S proteasome were identified by alignment with the human constitutive proteasome10 (Supplementary Fig. 1). From these analyses, we identified the putative β7 subunit as A2F3X4 (Uniprot ID) which in the human and yeast proteasome has been shown to be the last subunit incorporated into the α-ring/β-ring half-proteasome43,44. A sequence encoding a C-terminal twin-strep tag was added to the β7 subunit (Supplementary Fig. 2) as this was expected to have minimal effect on the assembly of the proteasome complex. In addition, the Tv genome was searched for chaperone proteins with homology to PAC1–PAC2, PAC3–PAC4 or Ump-1 that are known to play a role in proteasome assembly in human cells22,45. A homolog of human Ump-1 in the Tv genome (Uniprot A2FJW0) with 21% sequence identity was identified (Supplementary Fig. 1) however, no homologs of PAC1–PAC2, PAC3–PAC4 were found.

Three baculoviruses were prepared, one bearing seven α subunit genes, one with seven β subunit genes, and a third with the Ump-1 chaperone gene (Fig. 1a). Insect cells were simultaneously infected with the three baculoviruses (Fig. 1b). The resulting recombinant protein complex was enriched from cell lysates on a streptavidin column and further purified by size exclusion chromatography. Only fractions containing catalytically active protein were selected as determined by assays with the Suc-LLVY-amc substrate (Fig. 1c). This substrate is cleaved by the β5 subunits of Tv20S that are found on each of the β rings (Fig. 1d). A protein yield of ~1 mg per liter of cell culture was achieved.

Protein quality control
To assess the functional similarity of rTv20S and native Tv20S (nTv20S), both enzymes were incubated with the fluorescent inhibitor probe Me4BodipyFL-Ahx3Leu3VS. This probe irreversibly binds to the active sites of β1, β2, and β5 subunits. We then analyzed the labeled enzymes on denaturing and native gels. Consistent with our previous findings on nTv20S20, the probe strongly labeled the β2 and β5 subunits of both rTv20S and nTv20S, with weaker labeling of β1 subunits (Fig. 1e). On a native gel both enzymes exhibited a similar molecular weight of approximately 690 kDa (Fig. 1f) although rTv20S appeared slightly larger. This could be attributed to the presence of a 28-amino acid Strep-tag on each β7 subunit. This tag alters the overall isoelectric point (pI) of rTv20S and adds approximately 6 kDa in size.

Native Tv20S was isolated by sequential steps of ammonium sulfate precipitation, size exclusion chromatography (SEC), and anion exchange chromatography purification while rTv20S was affinity purified of a streptavidin column and then further refined by SEC. To assess the purity of both enzymes, we compared them using silver staining on native and denaturing gels. As shown in Fig. 1e, f, these analyses revealed the high purity of the rTv20S compared to nTv20S.

To understand the role of Tv Ump-1, we expressed rTv20S with and without this chaperone protein. In the absence of Ump-1, the eluted fraction from the streptavidin column contained a higher ratio of half-proteasomes to full-proteasomes, indicating incomplete assembly. Interestingly, incubating these proteins for 72h at room temperature partially rescued this defect (Supplementary Fig. 3). Additionally, β5 subunit activity, measured using both the fluorogenic probe and substrate, was significantly weaker when Ump-1 was absent (Supplementary Fig. 3). The connection between Ump-1 and β5 activity is supported by previous studies of the yeast proteasome where Ump-1 was shown to directly interact with the propeptide sequence of β5 and properly orientate it between the β6 and β7 subunits46. These findings collectively demonstrate that recombinant Tv Ump-1 functions as a critical chaperone, essential for the complete maturation and full activity of rTv20S.

Since rTv20S was expressed in Spodoptera frugiperda insect cells, we investigated the possibility of contamination with insect proteasome subunits. While the overall size difference allowed clear separation of rTv20S and the host cell proteasome (Sf20S) on a native gel (Supplementary Fig. 3), the potential incorporation of individual insect subunits into rTv20S remained a concern. To address this, the rTv20S band from the native gel was excised and analyzed by proteomics. All 14 Tv subunits were identified and quantified when the mass spectrometry data was searched against the Tv proteome (Fig. 1g). Additionally, the data was searched against the insect proteome. While six insect subunits were detected, their abundance was significantly lower (>100-fold) than the equivalent Tv subunits (details are provided in the Source Data file). These quality control measures demonstrate that rTv20S is a highly pure and enzymatically active enzyme complex, suitable for further biochemical and structural studies.

Biochemical characterization of rTv20S
We next compared the catalytic activity of the rTv20S and nTv20S using a combination of inhibitors and fluorogenic substrates. As revealed in Fig. 1e, the fluorescent probe, Me4BodipyFL-Ahx3Leu3VS, binds to all three catalytic subunits. Preincubation of the proteasome with irreversible-binding inhibitors prevents labeling with the probe. When rTv20S and nTv20S were incubated with 50 μM of CP-17 followed by the probe, the β2 and β5 subunits were no longer labeled (Fig. 2a). When the same proteins were incubated with 50 μM MZB none of the catalytic subunits were labeled with the probe. These data reveal that CP-17 binds to β2 and β5, while MZB binds to all three catalytic subunits (Fig. 2b). In addition, these studies show that rTv20S and nTv20S have the same inhibition profile when using Me4BodipyFL-Ahx3Leu3VS as a reporter of subunit targeting. As a control, human 20S (Hs20S) was preincubated with CP-17 and MZB prior to labeling with the probe. CP-17 completely inhibited β5, partially inhibited β2 and did not inhibit β1, while MZB targeted all three subunits.

The functionality of rTv20S was further compared to nTv20S through enzyme kinetic assays using subunit-specific fluorescent substrates20. Using a concentration range of 2 to 250 μM for the β2 and β5 substrates and 10 to 1250 μM for the β1 substrate, the specific activity was indistinguishable between the two enzymes, and the catalytic rate constant (kcat) and Michaelis constant (KM) were calculated for all three subunits of rTv20A and nTv20S (Fig. 2c) to reveal that these enzymes were equivalent.

Finally, we used fluorogenic assays to show that the relative activity of nTv20S and rTv20S in the presence of inhibitors is similar. Using the β5 and β2 substrates, the activity of both enzymes was reduced to comparable levels following pre-incubation with 50 μM CP-17 or MZB (Fig. 2d). For the β1 substrate, MZB reduced activity for both enzymes to comparable levels. However, CP-17 activated the β1 activity of nTv20S by 200% but activated rTv20S by only 120%. Although activation of β1 in the presence of β5 and β2 inhibitors was observed previously for nTv20S20 it is unclear why this occurs. Our follow-up studies revealed that β1 activation of rTv20S in the presence of CP-17 is independent of the substrate concentration used in the assay with activation ranging from 120% to 160% (Supplementary Fig. 4). In summary, these probe-labeling and enzyme activity assays revealed that rTv20S is enzymatically identical to nTv20S and therefore, validates the use of the recombinant enzyme for biochemical and structural studies.

Overall structural characterization of the Tv20S proteasome
To obtain atomic models of the Tv20S proteasome structure with bound inhibitors, we mixed rTv20S with a 50-fold excess of CP-17 or MZB and subjected it to cryo-EM. Initial data analysis revealed that the recombinant sample with MZB contained a significant amount of unassembled proteasome. During the 2D classification of the collected dataset, only 2% of the particles corresponded to the assembled 20S proteasome, while the remaining ring particles mostly consisted of half-proteasome complexes made up of a single α and β ring. The low abundance of full proteasomes was not due to inhibitor-dependent disassembly of proteasome (Supplementary Fig. 3e) and therefore may have occurred during the grid set-up.

We were able to obtain a 2.86 Å cryo-EM map of the Tv20S proteasome, which was used to build the initial atomic model of the rTv20S with MZB; PDB ID: 8OIX. For rTv20S bound to CP-17, we performed a second round of gel filtration to enrich the larger 20S proteasomes over the half-proteasomes. Each fraction was then analyzed for the presence of fully assembled Tv20S using negative-stain electron microscopy. This procedure improved yields so that approximately 80% of all particles could be assigned to the fully assembled Tv20S proteasomes. The data collected from this sample allowed the cryo-EM reconstruction and determination of the atomic structure of Tv20S with CP-17; PDB ID: 8P0T.

The initial Tv20S atomic model was built by leveraging AlphaFold models, which were superimposed onto the existing 20S proteasome structure of Leishmania tarentolae 20S proteasome (PDB ID: 7ZYJ)47 and fitted to the cryo-EM maps. Subsequently, the model was refined and rebuilt based on these maps. Unsurprisingly, Tv20S exhibits a shape and fold consistent with the characteristic overall structure of 20S proteasomes. Tv20S displays C2 symmetry, with the two sets of 14 subunits arranged in the conventional ring configuration of α1–α7, β1–β7 / β1–β7, α1–α7. Despite the homology among the individual chains between Tv and L. tarentolae 20S proteasome variations in their arrangement were discovered. The overall fold of the Tv20S proteasome somewhat differs from other proteasomes with known structures (e.g., RMSD = 2.729 Å for Cα atoms in structural alignment with the L. tarentolae 20S proteasome). It is worth noting that, in these structures, the Tv20S sequence (3167 residues) was mapped onto the cryo-EM maps, achieving 96.8% coverage for Tv20S/MZB and 96.6% for Tv20S/CP-17 structures. This includes well-defined regions, such as the active site pockets, which allowed for the modeling of inhibitor molecules in both structures.

Structure of active sites of the catalytic subunits β1, β2, and β5 bound to inhibitors
All three active sites in Tv20S are defined by a conserved catalytic triad Thr1, Asp17, and Lys3326. In addition, other well-conserved residues, such as Asp168, Ser131, and Ser170 in β1, are predicted to be required for efficient catalysis and active site structural integrity48. In the Tv20S-MZB dataset, the electron map within the β1 active site exhibited clear density extending beyond Thr1, providing sufficient coverage for the inhibitor MZB. As a result, the atomic model of MZB was successfully built and fit accurately into the cryo-EM map (Supplementary Fig. 5). Conversely, no density was observed within the β1 active site for CP-17 in the other dataset. This is consistent with the biochemical data (Fig. 2d), which demonstrated that MZB inhibits β1 activity while CP-17 does not.

The second active subunit, β2, can accommodate both MZB and CP-17 inhibitors in its active site, as clearly shown by the cryo-EM maps of those regions (Supplementary Figs. 5 and 6). MZB adopts a conformation like that observed in the β1 site. In both cases, the cyclohexenyl moiety of MZB and the phenyl residue of CP-17 occupy a deep pocket that is shared by several residues, including Lys33, and a loop composed of Ala46, Ala49, Glu31, and Asn52 (Fig. 3f). This pocket is commonly referred to as the S1 pocket of the enzyme because it serves as the binding site for the P1 amino acid of the substrate, which is on the N-terminal side of the scissile bond. For CP-17, the α,β-epoxyketone group is covalently bound to Thr-1 forming a morpholino derivative. Beyond the S1 pocket, the P2 tryptophan of CP-17 interacts with the β2 active site through its indole ring that resides on the hydrophilic ridge of the shallow S2 pocket, whereas the indole ring of the P3 tryptophan is buried and fits well within the deep S3 pocket. The S3 site is characterized by the presence of aliphatic chains, including Val28 and six alanine residues (Ala20, Ala22, Ala27, Ala124, Ala130, Ala132). Additionally, Glu31 and Asp126 line the S3 pocket, creating an acidic environment. The N-terminal capping group of CP-17 is a hexyl chain, which is orientated in a shallow S4 pocket.

In the catalytic site of β5, electron density corresponding to MZB was observed in the cryo-EM map. However, unlike the observations made for the β1 and β2 sites, the MZB in β5 was not sufficiently defined to allow for a satisfactory fit. Even though we found that MZB inactivates the β5 catalytic activity, it could not be included in the final model. In contrast, the density of CP-17 in the β5 site was clearly defined. The ligand was positioned in a highly similar conformation to that observed in the β2 site (Fig. 3). The α,β-epoxyketone group was covalently bound to Thr-1 and the P1 phenylalanine is located in the S1 pocket, which is lined with Lys33, Ala46, and Ala69. In contrast to the β1 and β2 sites, the S1 of β5 harbors Val31 and Met35. Moving forward, the P2 indole ring is positioned on the ridge of S2, formed by the loop between Ala46 and Ala49, and is enclosed by Ser96. The P3 indole is anchored in the relatively deep S3 pocket, defined by π–π interaction with the residue Phe27. Finally, the hexyl group of CP-17 sits in a well-defined shallow pocket formed by the hydrophobic parts of four residues belonging to the neighboring β6 chain (Tyr116, Asp136, Pro137, and Val138).

Comparison of the active proteasome sites of human and T. vaginalis for improved design of selective inhibitors
At first glance, the overall fold of the human constitutive proteasome and Tv20S appears very similar. However, upon structural alignment, the overall root mean square deviation (RMSD) is calculated to be 2.638 Å (Fig. 4a), which is higher than anticipated given the conservation of individual subunits. This disparity can be attributed to the slightly different packing of Tv20S, leading to less-than-optimal overall alignment. When aligning the individual subunits, the RMSD values decrease accordingly as the aligned sequences exhibit a range of 25–51% amino acid identity, with conserved regions in the active sites (Supplementary Table 1). Despite this relatively high sequence identity, the fine structural variances within the active site pockets present opportunities for designing selective inhibitors targeting Tv20S over human 20S, as illustrated by CP-17, which inhibits the Tv20S β5 subunit with a 10-fold higher potency than the equivalent subunit of the human constitutive proteasome10. In the human 20S proteasome, several amino acids pose steric hindrances, resulting in clashes when hypothetically positioning the CP-17 inhibitor within the active sites based on the alignment of Tv20S and the human proteasome.

In the β2 active site, many of the residues are conserved but in the S3 pocket where the second indole ring is positioned, the human 20S contains bulkier residues, such as Met131 and Ser122, in contrast to the significantly smaller alanine residues found at these positions in the Tv20S β2 site (Fig. 4b, c). The S3 pocket of the β5 subunit of Tv20S contains Phe27, which forms a favorable π-π interaction with the indole ring and represents the sole significantly different residue in this site. Conversely, the S1 pocket of the human active site contains Met45 and Ile35, which may obstruct the binding of the phenyl ring (Fig. 4d, e). It is plausible that Met45 from Tv20S exhibits greater mobility, moving away from the approaching ligand and not being hindered by Ile35, while the π-π interaction with Phe27 enhances the binding of the ligand in the case of Tv20S proteasome.

Furthermore, upon exploring the surfaces and binding interfaces of all the active sites in Tv20S and human 20S proteasomes, it becomes evident that the β2 and β5 active sites, along with their respective S1 and S3 pockets, exhibit significant differences in shape. These differences offer additional opportunities for further inhibitor design (Fig. 5). Although the distinct amino acid composition and slight differences in the arrangement of the β2 compared to β5 active sites pose challenges for the design of selective β2 inhibitors.

Methods
Cloning, expression, and protein purification
The cDNA sequences of the 14 proteasome subunits and the assembly chaperone (maturation factor) Ump-1 were identified for the T. vaginalis G3 strain on TrichDB by aligning them with human proteins (Supplementary Fig. 1). Required genes were E. coli codon-optimized, synthesized (Azenta) and cloned into three separate pACEBac1 vectors by restriction cloning (Supplementary Fig. 2). The plasmids were transposed to DH10EmBacY cells (Geneva Biotech) and the baculoviruses and protein expression was performed in Sf9 insect cells (Thermo Fisher, catalog number #11496015) according to the manufacturer’s instructions.

The expression of the 20S proteasome was performed in the presence or absence of the Ump-1 chaperone to understand its importance. Sf9 cells were collected after 72h post infection and centrifuged for 15 min at 2,000 x g and the pellet was diluted in Buffer A (50 mM Tris pH 7.5, 150 mM NaCl, 1 mM dithiothreitol, 1 mM EDTA) at a volume corresponding to 4-times the volume of the pellet. Cells were lysed by sonication and the lysate was cleared by centrifugation at 30,000g for 20 min. The cleared lysate was loaded onto tandem Streptactin XP columns (QIAGEN), equilibrated in Buffer A. Upon extensive washing with Buffer A, protein was eluted in the same buffer supplemented with 50 mM biotin. Proteasome-containing fractions were pooled, concentrated using ultrafiltration (Amicon) and loaded onto a Superose 6 Increase 10/300 gel filtration column (GE Healthcare) equilibrated with 50 mM HEPES pH 7.5, 150 mM NaCl, 1 mM EDTA. Fractions of 0.5 ml were obtained and assayed for protease activity. Enzymatically active fractions were pooled and concentrated to 1 mg/ml. The native Tv20S proteasome was purified from frozen pellets of Tv parasites as described previously20.

Proteasome activity and inhibition assays
The β5 proteolytic activity of the 20S proteasome was measured in insect cell extracts and in fractions isolated from chromatography columns using the fluorogenic substrate, Suc-LLVY-amc (Cayman Chemical). For enzyme characterization studies, substrates specific for Tv20S β1, β2, and β5 subunits, namely Ac-RYFD-amc, Ac-FRSR-amc, and Ac-GWYL-amc, were used as described previously20. These substrates were custom-synthesized by GenScript, NJ. For inhibition studies, 1 μM recombinant Tv20S was incubated with 50 μM MZB, 50 μM CP-17 inhibitor, or 0.5% DMSO (vehicle control) for 1.5h at room temperature. The inhibition of individual Tv20S subunits was confirmed using the subunit specific fluorogenic substrates. Kinetic assays in 384-well plates were performed using 4.4 nM recombinant Tv20S (rTv20) or enriched native Tv20S (nTv20S) in 50 mM HEPES pH 7.5 with 80 μM of Ac-RYFD-amc, 25 μM of Ac-FRSR-amc, and 25 μM of Ac-GWYL-amc, in a final volume of 8 μL per well. All assays were performed in triplicates in 384-well black plates (Nunc) at 37°C using a Synergy HTX Multi-Mode Microplate Reader (BioTek, Winooski, VT) with excitation and emission wavelengths of 360 and 460 nm, respectively.

Gel-based active site probing
Protein lysates, nTv20S, rTv20S, Hs20S, and 20S-inhibitor complex were diluted with 50 mM HEPES pH 7.5 then mixed with 2 μM Me4BodipyFL-Ahx3Leu3VS (R&D Systems #I-190). After probe addition, samples were incubated at room temperature for 16h. For denaturing gels, samples were mixed with 4X Bolt LDS sample buffer (Thermo Fisher Scientific) containing 250 μM dithiothreitol, heated at 100°C for 5 min, and loaded onto a NuPAGE 12% Bis-Tris gel (Thermo Fisher Scientific, P/N). PageRuler Plus pre-stained protein ladder (Thermo) was included on each gel. Gels were run with 1X MOPS SDS buffers (Invitrogen) at 130 V. For native gels, samples were mixed with 2X Novex Tris-glycine native sample buffer and loaded onto NuPAGE 3-8% Tris-glycine gels (Invitrogen) with NativeMark unstained protein standard (Thermo Fisher Scientific, P/N 57030). Gels were run at 100 V with Novex Tris-glycine running buffer (Invitrogen). All gels were imaged on Bio-Rad ChemiDoc XRS+ at 470 nm excitation and 530 nm emission for Me4BodipyFL-Ahx3Leu3VS probe visualization and silver stained.

Native gel digestion and desalting
After the native gel was silver stained, the band with recombinant proteasome (n=1) was excised, sliced into smaller fragments, and washed three times with 25 mM NH4HCO3, 50% acetonitrile for 10 min each time. Next, the band pieces were dried completely in a Savant SpeedVac Plus AR (Thermo Fisher Scientific). Reduction and alkylation were performed by adding a mixture of 10 mM TCEP and 25 mM iodoacetamide in 25 mM NH4HCO3 to the gel pieces and this reaction proceeded in the dark for 1h, followed by a 25 mM NH4HCO3 wash and subsequent 25 mM NH4HCO3, 50% acetonitrile wash to dehydrate the gel. The sample was then dried in a Savant SpeedVac Plus AR and 12.5 ng/μL trypsin in 25 mM NH4HCO3 was added. The sample was incubated at 4°C before being covered in 25 mM NH4HCO3 where the digestion proceeded at 37°C for 20h. The supernatant was transferred to a clean tube and the remaining peptides were extracted from the gel by the addition of 50% acetonitrile, 5% formic acid. The extracted digest was dried and resuspended in 0.1% formic acid before C18 ZipTip desalting. A C18 column was washed with methanol and spun for 45 s at 3500 x g. The column was equilibrated with 0.1% formic acid in 50% acetonitrile followed by 0.1% formic acid in water. The sample was loaded onto the column and spun for 2 min at 2000 x g. It was then washed with 0.1% formic acid then eluted from C18 with 50% acetonitrile, 0.1% formic acid by spinning at 3500 x g for 45 s. The sample was dried in a Savant SpeedVac Plus AR and stored at −80°C for 1 week prior to preparing it for mass spectrometry.

LC-MS/MS
The sample was redissolved in 0.1% formic acid prior to LC-MS/MS injection. Chromatography was performed on an Easy-nLC 1200 (Thermo Fisher Scientific), with a 75 μm i.d. PicoFrit (New Objective, Woburn, MA) column packed with 1.9 μm AQ-C18 material (Dr. Maisch, Germany) to 50 cm in length. Peptides were separated at 50°C over 46 min run which consisted of 6% solvent B at 1 min to 30% B in 21 mins, 72% B by 30 min, 90% by 31 min, and finally 60% B until 46 min. Mass spectrometry was performed on a Orbitrap Eclipse with ETD and PTCR (Thermo Fisher Scientific). The scan range was 350–1800 m/z, resolution of 60,000, and a 50 ms maximum injection time. The top 8 scans were selected for MS2. MS/MS spectra were analyzed in PEAKS Studio (v8.5) software (Bioinformatics Solutions Inc.). MS2 data were searched against the T. vaginalis and S. frugiperda proteome (UniProt taxon ID: 412133, ID: UP000829999). A precursor tolerance of 20 ppm and 0.01 Da was defined. Trypsin digestion was specified. The data can be found at ftp://massive.ucsd.edu/v07/MSV000094569/ or at proteomeXchange: PXD051584.

Preparation of cryo-EM grids and data acquisition
Recombinant purified Tv20S (1.4 μM) was mixed with 1.25 mM of either MZB or CP-17 to achieve a final concentration of 50 μM inhibitor. The mixture was prepared in a solution of 50 mM HEPES pH 7.5 and incubated at room temperature for 1h. After the incubation period, the sample was cooled on ice. Simultaneously, the cryo-EM Quantifoil R2/1 300-mesh copper grids (EM Sciences, Prod. No. Q350CR1) were glow discharged at 15 mA for 30 seconds to enhance their hydrophilicity. The proteasomes-inhibitor complex (4 μL) at a concentration of 1 mg/ml was transferred to the grid. The grids were blotted at −5 power for 5 s in FEI Vitrobot Mark IV (Thermo Fisher Scientific) at 4°C and 100% humidity and immediately frozen in liquid ethane. Immersion freezing and screening of cryo-EM data and all data acquisition were performed at the Umeå Core Facility for Electron Microscopy, Sweden. Screening was performed using a 200 kV Glacios system (Thermo Fisher Scientific) equipped with a Falcon 4i direct electron detector. 7983 images of Tv20S with MZB and 10,037 images of Tv20S with CP-17 were acquired with a pixel size of 0.7 Å with an exposure of 40 electrons per Å2 on Titan Krios (Thermo Fisher Scientific) at 300 kV using a Falcon 4i direct electron detector. Data collection was performed using the EPU v3.0.0 data collection software (Thermo Fisher).

Cryo-EM imaging
The data for Tv20S complexed with MZB was processed using cryoSPARC (v4.0.1). Images were imported and corrected using default settings for patch motion and patch contrast transfer function (CTF). Particles were selected using a blob picker (with a particle search size of 190 Å), extracted with a box size of 440 pixels, and classified using a 2D classification job. For Tv20S-MZB initial 100 2D classes were sorted using 2D classification. Particles and exposures were reduced to 6,135 micrographs by manual curation. Subsequent 2D classification revealed that of the 916,422 good particles, only 2% of the 2D classes contained the entire proteasome. After several rounds of 2D classification to remove unwanted particles, the subset of 13,933 particles (side views only) was used for an ab initio reconstruction and further homogeneous refinement. The resolution changed from 2.79 Å to 2.86 Å during 3D classification and removal of poor 3D classes and further reiterations including 2D classifications. We have observed rather a large gap between the unmasked-calculated resolution (3.73 Å with the 0.143 threshold and 6.91 Å with the 0.5 threshold). The C2 symmetry was applied in 3D homologous refinement steps for the generation of the final model. The cryo-EM map (GSFSC) achieved a final resolution of 2.86 Å, utilizing 14,257 particles.

The cryo-EM 3D map for Tv20S complexed with CP-17 was generated in a similar manner. Images were imported in small batches and then pooled, motion-corrected, and CTF-corrected in four batches, each containing approximately 2000 images. Particle picking was performed using well-defined Tv20S-MZB particles as templates. 2D classifications were also conducted in four batches, and subsequently, visually well-defined particles were selected (528,679), reclassified, and filtered using both 2D and 3D classification methods. After several rounds of further selection processes, 80,145 particles were retained for the final 3D map generation. The resulting cryo-EM map of Tv20S complexed with CP-17 exhibited a final resolution estimate of 2.60 Å, as determined by GSFSC analysis. An overview of cryo-EM data processing workflow is shown in Supplementary Fig. 10.

Model building and refinement
A high-resolution proteasome structure of Leishmania tarentolae (PDB ID: 7ZYJ) was used as an initial template to build the model47. The initial model was built by alignment of Tv20S the structures predicted by AlphaFold onto the 7ZYJ template. The Tv20S-MZB model was fitted to density using ChimeraX51 and refined by rigid body refinement and real space refinement in Coot 0.9.8.5 EL52 and structure optimization using ISOLDE package53 in ChimeraX54. Representative images of the structures and maps were generated using ChimeraX and Pymol. The Tv20S-CP-17 structure was refined in a similar manner as the Tv20S-MZB structure using the Tv20S-MZB complex as a starting model. Local resolution maps for both cryoEM maps were estimated using individual half maps and conducting calculations in Phenix v1.20.1-448755. The data were implemented into Tv20S-MZB and Tv20S-CP-17 maps and colored according to the local resolution estimates in ChimeraX54 (Supplementary Fig. 11). The reported maximum resolution (Supplementary Table 2) for deposited Cryo-EM maps was determined through FSC analysis by applying a tight mask to selected Cryo-EM map, with a threshold of 0.143. The Tv20S-MZB structure was deposited to the world protein databank under the ID number PDB ID: 8OIX and the Tv20S-CP-17 structure under PDB ID: 8P0T (Supplementary Table 2).

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The authors report the first recombinant overexpression of the Trichomonas vaginalis 20S proteasome (Tv20S) in the insect cells, which will facilitate many studies in the field. They also report cryo-EM structures of Tv20S bound to two inhibitors, revealing potential strategy for the development of species selective inhibitors in the future. Overall, this is a potentially useful study. However, there are significant concerns about the recombinant system they have developed, as well as major concerns on the data quality based on the PDB validation report and listed statistics in the SI Table.\nMajor concerns:\n1. The authors added a C-terminal twin-strep tag to β7 and expected this to have minimal effect on the assembly of the proteasome complex. However, previous studies have shown that β7 is the last subunit incorporated into the half proteasome and the C-terminal tail is important for the holo- proteasome assembly and β1 propeptide processing (Marques AJ, Glanemann C, Ramos PC, Dohmen RJ. The C-terminal extension of the beta7 subunit and activator complexes stabilize nascent 20 S proteasomes and promote their maturation. J. Biol. Chem. 2007; 282:34869–34876). I don’t know about the Tv20S, but in other proteasomes, the β7 C-terminal tail inserts into the space between β1 and β2 of the opposing half proteasome, and this interaction is considered the first event when two half-proteasomes come together. If this holds true in the Tv20S assembly, the C- terminal twin-strep tag may have hindered the assembly of the full complex. This would explain why there were so many immature half proteasome particles in their cryo-EM images, and the use of very low percentage of raw particles in 3D reconstruction.\n2. Fig. 1b is confusing. Because Trichomonas doesn’t contain the PAC1–PAC2 and PAC3–PAC4 chaperones, Tv likely uses PAC1-2 and PAC3-4 of the insect cells to help assemble the rTv20S, and this should be clearly stated. If so, Tv may also use the insect Ump-1, rendering the coexpression of Tv Ump-1 redundant? This can be easily figured out by comparing the Tv20S expression with or without the Ump-1 co-infection. A related question is the author’s use of Ump-1 condon- optimization for E. coli in the SF9 cells. Are they sure the introduced Ump-1 is functional? In the introduction, the authors state the rTv20S is functional by biochemical comparison with native Tv20S. However, we didn’t find any activity comparison between the native and rTv20S. It’s important to perform a detailed comparison of the activity and stability between native and rTv20S.\n3. In Fig. 1e, the native gel shows that the rTv20S runs higher than the native one. But it is hard for imagine a minor difference of a twin Strep tag (6kDa) could cause the observed shift in the native gel. Is it possible that the recombinant proteins have acquired additional modifications in the SF9 expression system? The authors should run an SDS-PAGE gel and perform mass spectrometry to find out.\n4. it seems that the authors used merely 2% of total particles for final reconstruction. This raises the serious concern that vast majority of their rTv20S particle may not have matured or fully assembled. Could this be due to the Ump-1 codon not being optimized for SF9 expression, or the system has overwhelmed the limited supply of the native insect PAC1/2 and PAC3/4 chaperones? Or the purification tag may have interfered with the assembly? This should be investigated.\n5. As 98% of the rTv20s become half proteasome after adding inhibitors (Fig. S2a), the stability of rTv20S is a concern. Will these inhibitors also destroy the wild type Ts20S? If the answer is yes, the measured inhibitory activity may not reflect the real inhibitions of all sites because the inhibitors may get into the active site through the open ring of half proteasome. Furthermore, if the inhibitors can break up most of the proteasome particles, the inhibition activity may be irrelevant? If the inhibitors only break up the rTv20s but not the native 20S, the result in Fig. 2c does not represent the real inhibition of the native Tv20s. Will the inhibitor also break up the human proteasome core particle?\n6. Fig. 2d shows CP-17 may enhance the beta-1 activity. The author reasoned that the beta-1 substrate being directed to the beta-1 site when both beta-2 and beta-5 are blocked by CP-17. But this is confusing: if the substrate is only specific for beta-1 site, it won’t go to the beta-2 and beta-5 sites. And blocking beta-2 and -5 should not affect the beta-1 activity. The author should use other beta-1 specific substrates to test the hypothesis.\n7. Both structures describe only the residues of binding pockets. No potential interactions were proposed. Is there any polar interaction between inhibitors and the binding pockets? The author didn’t mention any. Both inhibitors contain peptide bond structure which should contribute several polar interactions. Thr1 covalent bond and the π–π interaction may not be all interactions. The authors should do more detailed analysis.\n8. The authors didn’t provide the human PDB code(s) used in the selectivity analysis (Figs. 4-5), making it hard to trace the structural differences. We downloaded two human constitutive proteasome PDBs with or without ligand (5LF3and 4R3O) and one human immunoproteasome with a ligand (7AWE). In both constitutive and immune-proteasome structures, we were unable to identify the described residues Ser122, Cys128, and Met130 in beta-2, nor Ser129 in beta-5. In fact, Ser123, Cys129, and Met131 of beta-3 and Ser129 of b6 might correspond to the residues the authors referred to. This makes us wonder if the structural analysis was done carefully, and whether chain ID and residue number were assigned incorrectly.\n9. Using ligand free human 20S to compare with ligand-bound rTv20S is not convincing. The author stated the Met130 of beta-2 (assuming the residue # is correct) and Met45 of beta-5 will clash with CP-17. In fact, comparison of the ligand-bound with ligand-free human 20S (4R3O) show that Met45 of ligand-bound 20S (5LF3 and 7AWE) adopts a new rotamer to accommodate the ligand. Met has 13 rotamers. The author should consider solving the structure of human 20S with CP-17.\n10. In Section 6 of validation report of 8OIX, the calculated raw map from half maps is barely visible in all projections. The resolution of calculated map at FSC=0.5 is 6.91 which is much worse than the stated 3.31 Å. This may indicate inconsistence of the two half maps. Either the map quality should be improved, or the noted issue resolved.\n11. From Fig. S8, MZB is most potent to b5. However, no inhibitor density was found in b5. Is this due to map quality or sample quality? The author should resolve this issue.\nMinor concerns:\n1. The submitted manuscript may not be the final version, because several communications between co-authors were not removed.\n2. Insect expression of rTv20S is an important part of the manuscript. The description in Method is too brief, expression conditions e.g., expression time should be stated. In Fig. S1, the structures of the plasmids should be depicted clearly since seven genes are in one plasmid. Only sequences in plain text without any labeling, the construct structure is not clear. The polyhedrin promoter, Kozak sequence, start and stop sites of a gene, accession code of the gene, SV40 polyA tail should all be clearly labeled.\n3. Production of the rTv20s in SF9 cells used a similar approach as for the human 20S. The authors should cite the recombinant human 20S paper.\n4. MV151 should be noted the Proteasome Activity Probe (it is named as Me4BodipyFL-Ahx3Leu3VS in R&D Systems, not MV151, are they the same thing?).\n5. Fig. S1 is inconsistent. The left panels indicate a contiguous fragment of alpha-1 to alpha-7 (or beta-1 to beta-7) was inserted into the pACEBac1 vector, but the right panel clearly shows that each inserted subunit contains its own promoter region and termination region.\n6. In the “Cryo-EM imaging” section, Fig. S2C says 13,933 particles were used to for ab initio reconstruction, but the final model has 14,257 particles. It appears the 13,933 particles are side views, and they added 4,381 particles in top views to obtain the final 3D map. But it is not clear where and how they obtained these extra top views.\n7. From the validation report (8OIX), it looks like the tag was removed and the beta5 propeptide was not processed. Is this true?\n8. Fig 2c should include a Coomassie staining of the same gel to serve as a loading control (without fluorescence).\n9. “CP-17, which inhibits the Tv20S β5 subunit with a 10-fold higher potency than the equivalent subunit of the human constitutive proteasome”: it is only 5-fold difference in ref 8?\n10. What is homologous refinement in “The C2 symmetry was applied in 3D “homologous refinement” steps for generation of final model”. Is this a typo or using a homologous map as reference? Please clarify.\n11. In the Fig. S2c flow chart, the resolution of rTv20S-mzb was reduced from 2.70 Å to 2.86 Å. Similarly, the resolution of Tv20S-CP17 was reduce from 2.48 Å to 2.60 Å. Is there any reason to reduce the resolution? Also, the particle number was reduced. Did the authors use any procedures that were not included in the flow chart to reduce the particle number and the resolution?\n12. The symmetry used should be included in the table. The clash scores (24.83, 28.72) and rotamer (2.41, 3.84, reported, 5.1 and 4.0- validation reports) are way too high for a 2.6 Å structure.\n13. The RMS deviations of bond lengths and angles, b-factor of model and ligand, model composition, model resolution are all missing in the data statistics.\n14. Local resolution map and model-map correlation are both missing.\n15. Distance of clash should be labeled.", "reviewer_2": "This manuscript describes structures of Trichomonas vuginalis (Tv) 20S proteasome covalently bound to two small molecule proteasome inhibitors. Proteasome has been indicated as a therapeutic target for STDs caused by this protozoan parasite. Therefore, these results have value in the development of drugs to treat this widespread disease.\nA significant technical advance reported in the paper is the production of recombinant Tv 20S proteasome. This technically challenging achievement overcomes previous problems in producing endogenous Tv proteasome in sufficient quantity and quality for biochemical and structural studies. The results show that the purified recombinant proteasome has features that are indistinguishable from endogenous Tv and therefore make the current work possible. In addition to the production and functional characterization of Tv 20S, this manuscript describes high resolution cryo electron microscopic structures of Tv20S with bound to previously identified Tv 20 inhibitors MZB and CP-17. The results show that whereas MZB binds to each of three different proteasome catalytic sites, CP-17 binds more selectively to only two: the beta 2 and beta 5 subunits. Overall, these results provide a platform for the possible future development of more specific Tv20S proteasome inhibitors as useful drugs.\nCritique:\nThe manuscript is clearly written, and the results are largely convincing. I am not competent to critically judge all technical aspects of the structural determination and model building. Nevertheless, the overall description of the models seems biochemically reasonable and generally conform to the functional data. Likewise differences of inhibitors between Tv an human proteasomes can be explained by the structure reported here and elsewhere (e.g. the greater specificity of CP-17 for Tv versus human 20S). This work builds on previous work on this topic this this and other groups. The extent to which this represents a large breakthrough in the stated goal of developing new drugs is less certain. This is a step forward (aided by the technical advance) but not yet delivered and might be viewed differently by different people.\nThe data in Figure 2d should be expanded, in my opinion. In addition to a representative time course for a single inhibitor concentration, as shown, it would be useful to determine relative IC50s for these inhibitors with each of the subunit-specific substrates for both the recombinant and native enzymes. This would validate the broader claims about both relative specificity of the inhibitors (including differential sites) and the quality of the recombinant protein. This is an issue since the data show that only a small fraction of expressed protein fully assembles into intact 20S. This raises questions about interpretations of minor differences in structure. Finally, activities should be reported in more quantitative fashion as nmol AMC/min/ug. This will allow direct quantitative comparisons that are central to the conclusions.\nMinor:\nPg 10, paragraph 2, line 1: “catalytical” should be “catalytic”.", "reviewer_3": "The manuscript from Boura and coworkers reports the structure of a fully recombinant 20S proteasome from the human pathogen Trichomonas vaginalis that is responsible for urinary tract infections. The manuscript reports structures of Tv20S bound to two different inhibitors at 2.8 and 2.6 Angstrom resolutions. While the technological advance of producing the Tv20S proteasome by separately expressing the alpha and beta subunits in Sf9 insect cells is noteworthy, overall the manuscript fails to significantly advance our understanding of TV20S function or its inhibition. For example, high resolution structures already exist for several bacterial proteasomes, including Leishmania tarentolae and Mycobacterium tuberculosis proteasomes and cryo-EM proteasome structures are no longer considered novel per se. Meanwhile, the inhibitor MZB was not observed in the beta5 subunit structure, despite its known ability to inhibit the proteolytic activity of this subunit, indicating incomplete applicability of the current technique toward visualizing covalent adducts of key beta subunit residues. Finally, while the authors hypothesize several opportunities to improve selective inhibitor design based on their structural conclusions regarding differences between human and Tv proteasomes; no evidence is provided that this can actually be accomplished with molecules based on the authors' comments of significant differences in shape of the active sites. Indeed, this exact challenge of selectivity has been explored extensively for Tuberculosis proteasome inhibitors and not led to any known TB therapeutics. Thus, the manuscript falls well short of providing any new leads based on the reported structure. Along these lines, it is already known that CP-17 inhibits Tv20S with 10-fold higher potencyas the authors mention, which would have been a great starting point for optimization of the lead based on their structure. Finally, the conclusion that beta2 and beta5 inhibition by the inhibitor CP17 led to increased activity at beta1 due to increased substrate concentration is obvious as the substrate is not divided among subunit \"enzymes\". However, it is unclear what the ramifications of this observation are, or indeed if the activity is the same at 1/3 total substrate with inhibited beta2 and beta5, as it would be for total substrate with no inhibition. The authors also state they performed enzyme kinetics, however, no kcat and Km values are reported and only gain in AMC fluorescence as a function of time is presented in Figure 2d. As an additional minor note, the manuscript appears hastily written because several personal comments from the authors have been left behind in the manuscript. With the overall scientific limitations in mind, this reviewer does not feel that the manuscript rises to the level of publication in Nature Communications. In its current form it is likely more appropriate for a biological structure-focused journal where the protein expression technique would be better appreciated."}, "author_response": "Responses to Reviewers' comments:\nReviewer #1 (Remarks to the Author):\nThe authors report the first recombinant overexpression of the Trichomonas vaginalis 20S proteasome (Tv20S) in the\ninsect cells, which will facilitate many studies in the field. They also report cryo-EM structures of Tv20S bound to\ntwo inhibitors, revealing potential strategy for the development of species selective inhibitors in the future. Overall,\nthis is a potentially useful study. However, there are significant concerns about the recombinant system they have\ndeveloped, as well as major concerns on the data quality based on the PDB validation report and listed statistics in the\nSI Table.\nWe appreciate that Reviewer #1 considers our study potentially useful and believes it will facilitate many studies in\nthe field. We are grateful for all the suggestions by Reviewer #1 that we have now addressed and believe that these\nchanges have improved our manuscript.\nMajor concerns:\n1. The authors added a C-terminal twin- pected this to have minimal effect on the assembly of\nproteasome and the C-terminal tail is important for the holo- 1 propeptide processing\n(Marques AJ, Glanemann C, Ramos PC, Dohmen RJ. The C-terminal extension of the beta7 subunit and activator\ncomplexes stabilize nascent 20 S proteasomes and promote their maturation. J. Biol. Chem. 2007; 282:34869 34876).\n-\n-\n-proteasomes come\ntogether. If this holds true in the Tv20S assembly, the C-terminal twin-strep tag may have hindered the assembly of\nthe full complex. This would explain why there were so many immature half proteasome particles in their cryo-EM\nimages, and the use of very low percentage of raw particles in 3D reconstruction.\nResponse: There are numerous reasons why there is an abundance of immature half proteasomes. One hypothesis for\nthis may be due to the absence of the T. vaginalis PAC1-PAC2 and PAC3-PAC4 assembly chaperones. It is likely\nthat insect chaperones may be able to assist with the full proteasome assembly but are likely to be sub-optimal. It is\ndifficult to test this hypothesis since we cannot find PAC1-PAC2 and PAC3-PAC4 equivalents in the T. vaginalis\ngenome.\nAn alternative hypothesis is that the C-\nTo test this hypothesis, the recombinant Tv proteasome was enriched on the Streptactin XP column, and the eluted\nprotein was incubated for 24 and 72 h at room temperature and compared to a sample that was stored at 4°C. All\nsamples were run on a native gel after 72 h incubation, and we show that the amount of half-proteasome decreases\nwith time following incubation at room temperature (SI Fig. 3c). These data reveal that a higher percentage of mature\n20S proteasome can be achieved but requires additional time for the half-proteasomes to interact.\nIn the structures of the 20S proteasome our study revealed that the C-\n. 7). In the resubmitted manuscript, we now discuss\nthe C-\nSpecifically, we now state: \"...by the observation of fully assembled Tv20S with the C-\nis known to stabilize nascent 20S (Marques et al.),\nhalf proteasome (SI Fig. S7).\"\nPAC2 and PAC3 PAC4 chaperones, Tv\nlikely uses PAC1-2 and PAC3-4 of the insect cells to help assemble the rTv20S, and this should be clearly stated. If\nso, Tv may also use the insect Ump-1, rendering the coexpression of Tv Ump-1 redundant? This can be easily figured\nout by comparing the Tv20S expression with or without the Ump-1 co-\nuse of Ump-1 codon-optimization for E. coli in the SF9 cells. Are they sure the introduced Ump-1 is functional?\nResponse: We used E. coli codon optimized genes because E. coli prefers to use codons that have moderate GC content\n(high GC content codons are rare in the E. coli genome) and therefore these genes express well in every other common\nexpression system including insect cells.\nWe have performed the suggested experiment where Tv20S was expressed with and without Tv Ump-1 and can\nconclusively show that the recombinant Tv Ump-1 is functional. When using the activity-based probe, Me4BodipyFL-\nAhx3Leu3VS, Sf9 cell extracts contain a mixture of two functional proteasomes on a native gel, the Sf9 insect\nproteasome with a mw of ~720 kDa and the recombinant Tv20S proteasome with a mw of ~700 kDa. When the same\nprobe-labelled samples are evaluated on a denaturing gel, there is very little labelling of the Tv\nabsence of TvUmp-1 (compare Lane 2 vs Lane 3 in SI Fig. S3a). Therefore, Tv Ump-1 promotes full maturation of\nthe recombinant Tv20S thereby revealing that it is functional.\nTv Ump-1, we\nexpressed rTv20S with and without this chaperone protein. In the absence of Ump-1, the eluted fraction from the\nstreptavidin column contained more half-proteasomes, indicating incomplete assembly. Interestingly, incubating these\nproteins for 72 hours at room temperature partially rescued this defect (SI Fig. S3c).\nmeasured using both the fluorogenic probe and substrate, was significantly weaker when Ump-1 was absent (SI Fig.\nS3d). These findings collectively demonstrate that recombinant TvUmp-1 functions as a critical chaperone, essential\nfor the complete maturation and full activity of rTv\nIn the introduction, the authors state the rTv20S is functional by biochemical comparison with native Tv20S. However,\nperform a detailed comparison\nof the activity and stability between native and rTv20S.\nResponse: We have now performed an in-depth comparison of the native and recombinant enzymes and generated\nMichaelis Menton plots for both enzymes using the three subunit-selective substrates. A new Fig. 2c shows that the\nenzymes cleave the substrates at the same rate across a wide range of substrate concentrations. A table containing the\nK values was also generated and included as Table S1.\nM\nIn addition, we pre-incubated nTv20S and rTv20S with 50 of MZB or CP-17 for 1 h and then quantified the\nremaining activity with the fluorogenic substrates. MZB and CP-17 had similar inhibitory effects on proteasome\nactivity (Fig. 2d). nTv20S and rTv20S to the same level, however CP-17 activated\nnTv20S by 200% while the same compound activated rTv20S by only 120%. It is unclear why the native\nactivates more than the recombinant enzyme. As outlined in the manuscript, we do not see any structural differences\nin in the presence of either MZB and CP-17 and therefore the general mechanism of subunit activation\ncannot be explained based on structure.\none or more subunits is inhibited.\n3. In Fig. 1e, the native gel shows that the rTv20S runs higher than the native one. But it is hard for imagine a minor\ndifference of a twin Strep tag (6kDa) could cause the observed shift in the native gel. Is it possible that the recombinant\nproteins have acquired additional modifications in the SF9 expression system? The authors should run an SDS-PAGE\ngel and perform mass spectrometry to find out.\nResponse: We have performed an in-depth comparison of the two protein complexes and have been unable to find\nmodifications to the recombinant enzyme that would cause the observed mass difference seen on the native gels. The\nfollowing studies were performed.\n1. rTv20S and nTv20S were incubated with the Me4BodipyFL-Ahx3Leu3VS probe and run\non a denaturing gel. 5 (lower) bands on\nWhen comparing the first and forth lanes,\nthe banding pattern for rTv20S and nTv20S are identical. This gel was then silver stained\nand all of the subunits bands can now been seen. While the rTv20S preparation is much\ncleaner than nTv20S the banding pattern in the region of 25 kDa to 30 kDa is the same.\nTherefore, from these studies, there is no evidence that the rTv20S have higher molecular\nmass than the nTv20S subunits.\n2. The intact insect proteasome is clearly higher in molecular mass than nTv20S and therefore,\nwe hypothesized that some of the insect proteasome subunits may get incorporated into\nrTv20S during the assembly. This would then increase the overall mass of rTv20S. We\ntherefore performed a proteomic analysis on the recombinant enzyme and searched the\nresulting data against the T. vaginalis and Spodoptera frugiperda proteomes. We found\nthat the 14 most abundant subunits match to the T. vaginalis proteome (new Fig. 1g).\nSeveral insect proteasome subunits were found but their abundance was significantly lower\n(>100-fold). These studies indicate that population of rTv20S expressed in insect cell lines\nmay contain a low percentage of host subunits mass but there was no dominant\nTv20S/Sf20S chimeric proteasome present that could explain the slightly different masses\nbetween rTv20S and nTv20S.\n4. it seems that the authors used merely 2% of total particles for final reconstruction. This raises the serious concern\nthat vast majority of their rTv20S particle may not have matured or fully assembled. Could this be due to the Ump-1\ncodon not being optimized for SF9 expression, or the system has overwhelmed the limited supply of the native insect\nPAC1/2 and PAC3/4 chaperones? Or the purification tag may have interfered with the assembly? This should be\ninvestigated.\nResponse: We have already addressed concerns about the function of Ump-1, the chaperones and the potential\ninterference by the purification tag. Our use of only ~2% of all the particles has no impact on the quality of the model\nof the fully assembled proteasome as only these particles were selected for reconstruction (Fig. S10).\n5. As 98% of the rTv20s become half proteasome after adding inhibitors (Fig. S2a), the stability of rTv20S is a\nconcern. Will these inhibitors also destroy the wild type Tv20S? If the answer is yes, the measured inhibitory activity\nmay not reflect the real inhibitions of all sites because the inhibitors may get into the active site through the open ring\nof half proteasome. Furthermore, if the inhibitors can break up most of the proteasome particles, the inhibition activity\nmay be irrelevant? If the inhibitors only break up the rTv20s but not the native 20S, the result in Fig. 2c does not\nrepresent the real inhibition of the native Tv20s. Will the inhibitor also break up the human proteasome core particle?\nResponse: The presence of half proteasomes is not a result of addition of inhibitor, as we observed half\nproteasomes during the purification process (in the absence of MZB).\n6. Fig. 2d shows CP-17 may enhance the beta-1 activity. The author reasoned that the beta-1 substrate being directed\nto the beta-1 site when both beta-2 and beta-5 are blocked by CP-17. But this is confusing: if the substrate is only\nspecific for beta- -2 and beta-5 sites. And blocking beta-2 and -5 should not affect the\nbeta-1 activity. The author should use other beta-1 specific substrates to test the hypothesis.\nResponse: For substrates to be cleaved by a proteasome, they will ultimately sample all binding sites within the central\nwith them, albeit for a\nsubstrate will spend less time sampling this site and therefore the apparent concentration within the core of the\nproteasome would increase. In our Michaelis Menton plot (Fig 2c), we show that activity of Ac-RYFD-amc increases\n-\n17) is also due to an increase in substrate concentration.\nTo examine this, rTv20S was pre- of CP-\n-17, there is between 120% to 160%\nTherefore, for example, when rTv20S is pre-treated with CP- -RYFD, the\nvelocity of the reaction is 1.4 RFU/sec (SI Fig. S4). To achieve this velocity in the non-\nof substrate is needed. Therefore, in the presence of CP- -RYFD-amc, the apparent concentration\nTv -inhibited enzyme.\nIn a separate study, we have shown that inhibition of nTv\nsubstrates for Trichomonas vaginalis either\ncarfilzomib or CP-\ncleavage occurs when the substrate cannot sample other subunits within the core of the proteasome.\nIn the discussion section\n-\ninteraction, leading to a higher observed activi\n7. Both structures describe only the residues of binding pockets. No potential interactions were proposed. Is there any\npeptide bond structu\nmay not be all interactions. The authors should do more detailed analysis.\nResponse: We acknowledge that our manuscript did not thoroughly discuss certain interactions. Our revised\nmanuscript now contains three new figures (Fig. S12 - S14) that present an extensive and more detailed analysis,\nemphasizing the contributions of all relevant interactions between the inhibitors and the binding pockets.\n8. The -5), making it hard to\ntrace the structural differences. We downloaded two human constitutive proteasome PDBs with or without ligand\n(5LF3 and 4R3O) and one human immunoproteasome with a ligand (7AWE). In both constitutive and immune-\nproteasome structures, we were unable to identify the described residues Ser122, Cys128, and Met130 in beta-2, nor\nSer129 in beta-5. In fact, Ser123, Cys129, and Met131 of beta-3 and Ser129 of b6 might correspond to the residues\nthe authors referred to. This makes us wonder if the structural analysis was done carefully, and whether chain ID and\nresidue number were assigned incorrectly.\nResponse: We apologize that PDB code for human 20S were omitted and the chain IDs for individual residues were\nnot included. In the revised manuscript Fig. 4 and 5 have been corrected. We used one of the suggested human\nstructures (PDB ID = 4R3O) and also included a new SI figure (Fig. S15) that illustrates the major structural\ndifferences between h20S vs Tv20S in the vicinity of the CP-17 inhibitor binding sites (beta-2 and beta-5).\n9. Using ligand free human 20S to compare with ligand-bound rTv20S is not convincing. The author stated the Met130\nof beta-2 (assuming the residue # is correct) and Met45 of beta-5 will clash with CP-17. In fact, comparison of the\nligand-bound with ligand-free human 20S (4R3O) show that Met45 of ligand-bound 20S (5LF3 and 7AWE) adopts a\nnew rotamer to accommodate the ligand. Met has 13 rotamers. The author should consider solving the structure of\nhuman 20S with CP-17.\nResponse: Thank you for this suggestion. The structural superposition has now been done as suggested by Reviewer\n#1. Specifically, we have carefully re-examined the structural analysis/alignments with all three suggested structures\n(4R3O, 5LF3 and 7AWE) in addition to 7PG9, which we have used previously. We have rephrased our statements\nabout differences between human 20S proteasome and Tv20S. In the discussion we now more carefully state the\npossibilities of exploiting these differences in the future research and drug development.\nThank you for the suggestion to solve the structure of the human proteasome in complex with CP-17. While\nthis is beyond the scope of the current study, the recombinant expression of the human proteasome and\nstructural studies with peptide epoxyketone inhibitors are a long-term goal of this project.\n10. In Section 6 of validation report of 8OIX, the calculated raw map from half maps is barely visible in all projections.\nThe resolution of calculated map at FSC=0.5 is 6.91 which is much worse than the stated 3.31 Å. This may indicate\ninconsistence of the two half maps. Either the map quality should be improved, or the noted issue resolved.\nResponse: We apologize for a lack of clarity in the validation report, but we do not have control over the quality of\nthe maps' contours in the PDB report. Here, we present a figure of raw maps with different contours solely for the\nreviewer's revision (Figure for Reviewer 1).\nWe would like to clarify the other concern. The aforementioned resolution is an estimate of Fourier shell correlation\ncurve at 50% FSC(0.5) = 6.9. This corresponds to a resolution estimate calculated for the RAW map at FSC 0.5. The\nauthor-provided FSC(0.5)=3.31 which corresponds to resolution of FCS curve calculated for a tight mask applied on\nthe map. Moreover, the maximum resolution reported in the manuscript (e.g., [8OIX] = 2.86 Å) is determined through\nFSC analysis, employing a specific threshold of 0.143.\nNow we state in the manuscript (methods section): -\nEM maps was determined through FSC analysis by applying a tight mask to selected Cryo-EM map, with a threshold\n11. From Fig. S8, MZB is most potent to b5. However, no inhibitor density was found in b5. Is this due to map quality\nor sample quality? The author should resolve this issue.\nactive site does not allow to model a bound MZB molecule (Fig. S6c). It is\nhighly improbable that the absence of a clear electron density is attributable to poor sample quality, as all\n. Our biochemical data (Fig. 2) confirms that the inhibitor is\nbound. The lack of density, therefore, is probably caused by the flexibility of the inhibitor in the .\nWe now state in the manuscript: esponded to three subunits\n-\nMinor concerns:\n1. The submitted manuscript may not be the final version, because several communications between co-authors were\nnot removed.\nResponse: We apologize for this oversight. It has been corrected.\n2. Insect expression of rTv20S is an important part of the manuscript. The description in Method is too brief,\nexpression conditions e.g., expression time should be stated. In Fig. S1, the structures of the plasmids should be\ndepicted clearly since seven genes are in one plasmid. Only sequences in plain text without any labeling, the construct\nstructure is not clear. The polyhedrin promoter, Kozak sequence, start and stop sites of a gene, accession code of the\ngene, SV40 polyA tail should all be clearly labeled.\nResponse: Thank you for the suggestion. The SI Figure (currently Fig. S2) has been modified as suggested,\nand indeed, it is now clearer.\n3. Production of the rTv20s in SF9 cells used a similar approach as for the human 20S. The authors should cite the\nrecombinant human 20S paper.\nResponse: We do cite the study by Toste Rego, A. & da Fonseca (Characterization of Fully Recombinant Human 20S\nand 20S-PA200 Proteasome Complexes). We are not aware of other studies that have expressed recombinant human\n20S that should be cited.\n4. MV151 should be noted the Proteasome Activity Probe (it is named as Me4BodipyFL-Ahx3Leu3VS in R&D\nSystems, not MV151, are they the same thing?).\nResponse: Thank you for asking us this question. We assumed that MV151 and Me4BodipyFL-Ahx3Leu3VS were\nthe same compound, but they are actually slightly different. MV151 is BodipyTMR-Ahx3Leu3VS and therefore has\na tetramethylrhodamine fluorescent label while Me4BodipyFL-Ahx3Leu3VS contains a fluorescein dye. We have\nupdated the manuscript and used Me4BodipyFL-Ahx3Leu3VS instead of MV151.\n5. Fig. S1 is inconsistent. The left panels indicate a contiguous fragment of alpha-1 to alpha-7 (or beta-1 to beta-7)\nwas inserted into the pACEBac1 vector, but the right panel clearly shows that each inserted subunit contains its own\npromoter region and termination region.\nResponse: Each subunit has its own promoter region and termination region. These were also highlighted in the\nupdated Fig. S2.\n- . S2C says 13,933 particles were used to for ab initio reconstruction, but the\nfinal model has 14,257 particles. It appears the 13,933 particles are side views, and they added 4,381 particles in top\nviews to obtain the final 3D map. But it is not clear where and how they obtained these extra top views.\nResponse: We apologize for the confusion; 13,724 particles were used for the ab initio model. In the next step, 13,933\nside view particles were used to obtain an improved model using homogeneous reconstruction. Then 4381 top view\nparticles were added in next round. For the final model, more particles were removed during the several rounds of\nhomogeneous reconstruction and 3D classifications. Please note: clear top views of full proteasome were assumed to\nbe the particles with highest contrast. Figure S10 and its legend was modified to improve the clarity of the processing.\nWe now state in the Figure legend:\nFig. S10. Cryo-EM workflow of data processing. This image processing workflow was employed to reconstruct the\nTv20S structures in cryo-EM. Both datasets were acquired at Titan Krios with the Falcon 4i detector under identical\nconditions using the same setup (refer to Supplementary Table 1 for details). The ab initio model served as the starting\npoint for 3D homogeneous refinement (Homogeneous Refinement) to enhance the quality of the maps. Multiple\niterations, including 3D classification and 2D classification, were carried out in several rounds to eliminate unwanted\nparticles, refine the resolution, and improve the maps. The unsharpened maps of the final reconstruction and the gold-\nstandard Fourier Shell Correlation (FSC) curve using different masks are shown. The figures of the maps were\ngenerated by ChimeraX2. The resolution in italics corresponds to a particular Fourier Shell Correlation Cryosparc and\nwas used to navigate the process of data analysis. The final resolution was estimated by a Fourier Shell Correlation\njob in Cryosparc when a tight mask was applied and was estimated to be 2.86 Å for 8IOX and 2.60 Å for 8P0T. The\nfinal resolution was estimated by Fourier Shell Correlation job in Cryosparc3 when a tight mask was applied and was\nestimated to be 2.86 Å for 8IOX and 2.60 Å for 8P0T. An example of the Tv20S-MZB route of processing is as\nfollows: 1,436,978 particles were extracted from 6135 processed images. After several rounds of 2D classification to\nsort out unwanted classes, only 40,916 particles remained. These were then reclassified with independent 2D\nclassifications for the top views and the side views. Separately, classes with 13,724 particles of both views were used\nfor an ab initio model. This initial cryo-EM map was used as a starting model for homogeneous reconstruction, where\n13,933 side view particles were used. Next, 4381 particles corresponding to the top views were added in the next\nround. For the final model, additional particles were removed during several rounds of homogeneous reconstruction\nand 3D classifications. Note: clear top views of the full proteasome were assumed to be the particles with the highest\ncontrast.\n7. From the validation report (8OIX), it looks like the tag was removed and the beta5 propeptide was not processed.\nIs this true?\nResponse: We apologize for this error. The entire sequence of the propeptide was included in the deposition. The\ndeposition was updated not to contain the sequences of the propeptides.\n8. Fig 2c should include a Coomassie staining of the same gel to serve as a loading control (without fluorescence).\nResponse: We added the same silver-stained gel after fluorescence visualization to Fig. 2b.\n- -fold higher potency than the equivalent subunit of the human\n-fold difference in ref 8?\nResponse: Thank you for catching this. We have updated the manuscript now to indicate 5-fold.\nWe apologize for misunderstanding; this was a typo. The corrected term is now the 3D homogeneous refinement.\n11. In the Fig. S2c flow chart, the resolution of rTv20S-mzb was reduced from 2.70 Å to 2.86 Å. Similarly, the\nresolution of Tv20S-CP17 was reduced from 2.48 Å to 2.60 Å. Is there any reason to reduce the resolution? Also, the\nparticle number was reduced. Did the authors use any procedures that were not included in the flow chart to reduce\nthe particle number and the resolution?\nResponse: We apologize for a lack of clarity in this respect. We have now corrected the resolution. The resolutions\nthat are only produced by a particular Fourier Shell Correlation Cryosparc run were used to navigate the process of\n3D refinement. Final resolution is estimated by Fourier Shell Correlation job in Cryosparc when a tight mask was\napplied (detailed in Fig. S10). Several iterations of individual processesing steps were often carried out. The final\nlocal resolution maps are now shown in new Fig. S11.\n12. The symmetry used should be included in the table. The clash scores (24.83, 28.72) and\nrotamer (2.41, 3.84, reported, 5.1 and 4.0- validation reports) are way too high for a 2.6 Å structure.\nResponse: We have significantly improved our models clash score (11.17 & 11.9) and rotamers geometry (0.93 &\n1.21), and redeposited the models into the PDB database. The new validation reports are attached. We have also\nincluded symmetry of the model (C2) in Table S3.\n13. The RMS deviations of bond lengths and angles, b-factor of model and ligand, model composition, model\nresolution are all missing in the data statistics.\nResponse: The RMSDs of angles and lengths along with other details are now included in updated SI Table S3.\n14. Local resolution map and model-map correlation are both missing.\nResponse: Both local resolution map and model-map correlation are now included as new Fig. S11 and Table S3.\n15. Distance of clash should be labeled.\nResponse: It is unclear to us what exactly should be labeled. However, with some additional explanation for\nReviewer #1, we will implement all the requested changes.\nReviewer #2 (Remarks to the Author):\nThis manuscript describes structures of Trichomonas vaginalis (Tv) 20S proteasome covalently bound to two small\nmolecule proteasome inhibitors. Proteasome has been indicated as a therapeutic target for STDs caused by this\nprotozoan parasite. Therefore, these results have value in the development of drugs to treat this widespread disease.\nA significant technical advance reported in the paper is the production of recombinant Tv 20S proteasome. This\ntechnically challenging achievement overcomes previous problems in producing endogenous Tv proteasome in\nsufficient quantity and quality for biochemical and structural studies. The results show that the purified recombinant\nproteasome has features that are indistinguishable from endogenous Tv and therefore make the current work possible.\nIn addition to the production and functional characterization of Tv 20S, this manuscript describes high resolution cryo\nelectron microscopic structures of Tv20S with bound to previously identified Tv 20 inhibitors MZB and CP-17. The\nresults show that whereas MZB binds to each of three different proteasome catalytic sites, CP-17 binds more\nselectively to only two: the beta 2 and beta 5 subunits. Overall, these results provide a platform for the possible future\ndevelopment of more specific Tv20S proteasome inhibitors as useful drugs.\nWe appreciate that Reviewer #2 believes that our results have value in the development of drugs to treat this\nwidespread disease and provide a platform for the possible future development of more specific Tv20S proteasome\ninhibitors as useful drugs.\nCritique:\nThe manuscript is clearly written, and the results are largely convincing. I am not competent to critically judge all\ntechnical aspects of the structural determination and model building. Nevertheless, the overall description of the\nmodels seems biochemically reasonable and generally conform to the functional data. Likewise differences of\ninhibitors between Tv and human proteasomes can be explained by the structure reported here and elsewhere (e.g. the\ngreater specificity of CP-17 for Tv versus human 20S). This work builds on previous work on this topic this this and\nother groups. The extent to which this represents a large breakthrough in the stated goal of developing new drugs is\nless certain. This is a step forward (aided by the technical advance) but not yet delivered and might be viewed\ndifferently by different people.\nResponse: For all successful protease inhibitor drug development programs, access to the recombinant enzyme is key\nfor hit-to-lead optimization. Examples include the recombinant proteases from SARS-CoV-2, Hepatitis C Virus and\nHIV for antiviral drug development, recombinant renin and angiotensin-converting enzyme for anti-hypertensive drug\ndevelopment and recombinant dipeptidyl peptidase IV for diabetes drug development. One of the major bottlenecks\nfor development of proteasome inhibitors for treatment of malaria (Plasmodium falciparum), leishmaniasis\n(Leishmania donovani), Chagas disease (Trypanosoma cruzi) and African sleeping sickness (Trypanosoma brucei)\nhas been the availability of a consistent source of enzyme for compound screening, hit-to-lead studies and structural\nstudies. Hence, although the parasite proteasomes have been validated as a target for many years, there are only two\nproteasome inhibitors in late-stage clinical trials. Our lab aims to make recombinant proteasome for each of the above\nlisted parasites however, our initial focus has been on T. vaginalis. We believe that the successful expression of a fully\nfunctional 28-subunit proteasome is a breakthrough moment for the field and know that other researchers believe so\ntoo. For example, when this manuscript was posted on bioRxiv, we were contacted by Novartis and have now\nsuccessfully helped them to express a target proteasome for one of their internal anti-parasitic programs.\nThe data in Figure 2d should be expanded, in my opinion. In addition to a representative time course for a single\ninhibitor concentration, as shown, it would be useful to determine relative IC50s for these inhibitors with each of the\nsubunit-specific substrates for both the recombinant and native enzymes. This would validate the broader claims about\nboth relative specificity of the inhibitors (including differential sites) and the quality of the recombinant protein. This\nis an issue since the data show that only a small fraction of expressed protein fully assembles into intact 20S. This\nraises questions about interpretations of minor differences in structure. Finally, activities should be reported in more\nquantitative fashion as nmol AMC/min/ug. This will allow direct quantitative comparisons that are central to the\nconclusions.\nResponse: Thank you for this suggestion. We have now performed an in-depth comparison of the native and\nrecombinant enzymes and generated Michaelis Menton plots for both enzymes using the three subunit-selective\nsubstrates. These data show that the two enzymes cleave the substrates at the same rate across a wide range of substrate\nconcentrations as shown in the figure below. This figure is now included in the modified manuscript as Fig. 2c and\nand K values was also\ncat M\ngenerated and included as Table S1.\nWe pre-incubated nTv20S and rTv of MZB or CP-17 for 1 hour and quantified the remaining activity\nusing fluorogenic substrates. MZB and CP-17 showed similar inhibitory effects on proteasome activity, as shown in\nnTv20S and rTv20S, while CP-17\nactivated nTv20S by 200% and rTv\nunclear, as no structural differences were observed in the presence of MZB or CP-17. We propose that this increase\nis due to a higher substrate concentration within the proteasome core (see in-depth explanation above).\nMinor:\nResponse: Thank you for catching this typo. It has been changed.\nReviewer #3 (Remarks to the Author):\nThe manuscript from Boura and coworkers reports the structure of a fully recombinant 20S proteasome from the\nhuman pathogen Trichomonas vaginalis that is responsible for urinary tract infections. The manuscript reports\nstructures of Tv20S bound to two different inhibitors at 2.8 and 2.6 Angstrom resolutions. While the technological\nadvance of producing the Tv20S proteasome by separately expressing the alpha and beta subunits in Sf9 insect cells\nis noteworthy, overall the manuscript fails to significantly advance our understanding of TV20S function or its\ninhibition. For example, high resolution structures already exist for several bacterial proteasomes, including\nLeishmania tarentolae and Mycobacterium tuberculosis proteasomes and cryo-EM proteasome structures are no\nlonger considered novel per se.\nResponse: In this study, the value of the cryo-EM structure was to understand the binding mode of two active site\ninhibitors. This would not have been possible with the native enzyme as sufficiently high yields and purity could not\nbe achieved. As outlined above, access to a recombinant proteasome is essential for hit-to-lead development of other\nprotease inhibitors that were subsequently approved as drugs. Having a structure will allow us to do structure-based\ndrug design for the next generation of compounds. In addition, if resistant mutants arise, then understanding the\nstructural changes of these mutations will be essential for hit-to-lead optimization. Therefore, we believe that the\ncombination of the expression studies and structural studies add extensive novelty to this project. In the two examples\nprovided by Reviewer #3, it should be noted that the Mycobacterium tuberculosis -\n-subunit that each form into rings of seven. Therefore, this enzyme is simpler that the Tv20S\n- -subunits. Also, Leishmania\ntarentolae protozoa is used as an expression system for making recombinant proteasomes. While the cryo-EM\nstructure of this compound has been published, it is a non-pathogenic organism and therefore important pathogenic\nproteasomes need to be modeled off of this structure. Our study is the first to solve the structure is a recombinant\neukaryotic proteosome from a pathogenic organism that can then be directly used for structure-based drug design.\nMeanwhile, the inhibitor MZB was not observed in the beta5 subunit structure, despite its known ability to inhibit the\nproteolytic activity of this subunit, indicating incomplete applicability of the current technique toward visualizing\ncovalent adducts of key beta subunit residues.\nResponse: As stated above (Response to Referee #1), the map\nmodel a bound MZB molecule (Fig. S6c). It is highly improbable that the absence of a clear electron density\n. Our\nbiochemical data (Fig. 2) confirms that the inhibitor is bound. The lack of density, therefore, is probably caused by\nthe flexibility of the inhibitor in the .\nFinally, while the authors hypothesize several opportunities to improve selective inhibitor design based on their\nstructural conclusions regarding differences between human and Tv proteasomes; no evidence is provided that this\ncan actually be accomplished with molecules based on the authors' comments of significant differences in shape of\nthe active sites. Indeed, this exact challenge of selectivity has been explored extensively for Tuberculosis proteasome\ninhibitors and not led to any known TB therapeutics. Thus, the manuscript falls well short of providing any new leads\nbased on the reported structure. Along these lines, it is already known that CP-17 inhibits Tv20S with 10-fold higher\npotency as the authors mention, which would have been a great starting point for optimization of the lead based on\ntheir structure.\nResponse: This manuscript describes the recombinant expression and biochemical validation of Tv20S and the\nstructure of the enzyme complex bound to two inhibitors. To perform structure-based drug design using CP-17 as a\nstarting point, would require a chemistry team. These libraries will need to be iteratively tested and the cryo-EM\nstructures solved. While the long-term goal of the project is to develop new leads, it is beyond the scope of this\nmanuscript that new lead compounds are identified here. We have updated the discussion to suggest a key Cys residue\n-17 analog.\nFinally, the conclusion that beta2 and beta5 inhibition by the inhibitor CP17 led to increased activity at beta1 due to\nincreased substrate concentration is obvious as the substrate is not divided among subunit \"enzymes\". However, it is\nunclear what the ramifications of this observation are, or indeed if the activity is the same at 1/3 total substrate with\ninhibited beta2 and beta5, as it would be for total substrate with no inhibition.\nResponse: Thank you for your question. We have addressed a similar question from Reviewers 1 and 2. Please see\nour answers above.\nThe authors also state they performed enzyme kinetics, however, no kcat and Km values are reported and only gain\nin AMC fluorescence as a function of time is presented in Figure 2d.\nResponse: Thank you for this suggestion. We have determined the K and k values for each of the substrates using\nM cat\nboth nTv20S and rTv20S. The K plots are now included as Fig. 2c and the K and K values are included in Table\nM cat M\nS1.\nAs an additional minor note, the manuscript appears hastily written because several personal comments from the\nauthors have been left behind in the manuscript.\nResponse: We apologize for this oversight. It has been corrected.\nWith the overall scientific limitations in mind, this reviewer does not feel that the manuscript rises to the level of\npublication in Nature Communications. In its current form it is likely more appropriate for a biological structure-\nfocused journal where the protein expression technique would be better appreciated.\nResponse: Through the valuable feedback provided by all Reviewers, we have substantially enhanced the\nquality of our manuscript. We are now confident that it meets the high standards required for publication in\nNature Communications.\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nThe authors have addressed many of our concerns. The revised manuscript now provides a better\ndescription and characterization of their expression system. A few addition comments are listed\nbelow.\n1. In the Tv20S-MZB dataset (Fig. S10), only 2% of particles were fully assembled proteasomes\nwhile the remaining were half proteasomes. Please provide a full atlas of 2D classes in the first\nround which will better reflect the particle distribution of half and full proteasomes.\n2. In the Tv20S-MZB dataset (Fig. S10), the particle number was reduced from 18,314 to 14,257 but\nthe resolution was decreased from 2.79 to 2.86. But the authors state (inconsistently) in Method\n“cryo-EM imaging” section: The resolution was improved by 3D classification and removal of poor\n3D classes and further reiterations including 2D classifications”.\n3. In Method “cryo-EM imaging” section, particles and exposures were reduced to 6,135 particles\nby manual curation” – did the author mean micrographs rather than particles??\n4. In the validation report of PDB ID 8P0T, the particle number was 197,322, the electron dose was\n50 e/Å2, and the defocus was 800-3600 nm. However, the particle number in Fig. S10 is different.\nThe dose was 40 e/Å2 and the defocus was 800-2400 nm in Table S3.\n5. In the validation report of PDB ID 8OIX, the electron dose was 50 e/Å2, and the defocus was 800-\n3600 nm. However, the dose was 40 e/Å2 and the defocus was 800-2400 nm in Table S3.\n6. In the validation report of PDB ID 8OIX, the Unmasked-calculated resolution is 3.73 Å with 0.143\nthreshold and 6.91Å with the 0.5 threshold. This is an unusually large gap, indicative of low signal-\nto-noise ratio, likely due to limited number of particle (14,257) in the final reconstruction. The\nauthor should either note this shortcoming or collect more data to address the problem.\n7. In Fig. S3c, Tv20S is more abundant in the -Ump1 samples, does not appear to agree with the\ndescription that “in the absence of Ump-1, the eluted fraction from the streptavidin column\ncontained more half-proteasomes, indicating incomplete assembly”.\n8. In Fig. S3b,d, why did the -Ump1 sample lose b5 binding to the fluorogenic probe? Does Ump-1\naffect the b5 maturation?\n9. Fig. 1e and Fig S3b: There is no corresponding band in the silver stain gel compared with the\nfluorogenic probe binding gel. Are they from the same gel?\n10. Fig. 2c: the curves for the b1 of nTv20S and rTv20S never reached Vmax, indicating low affinity or\ninaccessible to the b1 site. Consider another substrate?\n11. From Fig 1c,f, the purified rTv20S was intact, so the proteasome was apparently destroyed by\nMZB, raising the question of the inhibitor entry into the active site. Several scenarios can be\npictured, and different scenario may result in different structure and kinetics. The author should\naddress if half proteasome binds the inhibitor.\n12. Fig. 4e-f and in page 9: there is no fig 4f. Ile35 mentioned in the text but not labeled in the figure.\nReviewer #2 (Remarks to the Author):\nThis revised manuscript about structural relationships between two extablished proteasome\ninhibitors and the Trichomonas vaginalis 20S proteasome addresses many of the concerns raised\nby me and the other reviewers. In my view, a main advance in this manuscript is the description of a\nrecombinant method of producing Tv proteasome for development of therapeutically useful\ninhibitors. That said, despite its usefulness for Tv, analogous methodology for other versions of\nproteasomes is now becoming more common and is therefore less novel. One recent paper not\ncited here (in their response the authors state that they are not aware………….) includes Adolf et al\nNat Struc Mol Biol (https://doi.org/101038/s41594-024-01268-9); maybe this paper crossed during\npreparation of the current manuscript. The promise that the differences between the current 20S Tv\nstructure and human 20S will lead to Tv-specific inhibitors remains to be determined. The\ndescription of structural features of Tv 20S with known inhibitors is useful new information.\nOne issue that I continue to find confusing (also raised by Reviewer 1) is the low percentage of fully\nassembled recombinant protein in the presence of inhibitor used for determination of the structure.\nThat seems at odds with the apparent homogeneous preparations of purified protein on native gels.\nIs the inhibitor promoting dissociation? Why not compare Tv 20S +/- inhibitors on native gels? Did I\nmiss something? On a related issue, the data in Figure S3c/d seem odd. The vast majority of the\nsamples appears to be mature 20S.\nReviewer #3 (Remarks to the Author):\nThe authors have nicely rewritten the manuscript to highlight the new elements of their work and\nperformed additional experimentation in response to reviewer comments. Prior to publication, the\nKm and kcat data should be reported with values and units, not just raw traces. While the overall\nconclusions are still somewhat modest, other than the demonstration that the proteosome can be\npurified from Sf9 cells, the rewritten manuscript does make a strong point of indicating that\ndifferent inhibitors target different active sites in the proteasome. Therefore, I recommend\npublication of a suitably revised manuscript with Km and kcat values in tabulated format with error\nbars and number of repeats indicated.\nREVIEWERS' COMMENTS\nReviewer #1 (Remarks to the Author):\nThe authors have addressed many of our concerns. The revised manuscript now provides a better\ndescription and characterization of their expression system. A few addition comments are listed below.\nWe are happy that the Expert Reviewer #1 believes that our paper was significantly improved during\nrevisions.\n1. In the Tv20S-MZB dataset (Fig. S10), only 2% of particles were fully assembled proteasomes while the\nremaining were half proteasomes. Please provide a full atlas of 2D classes in the first round which will\nbetter reflect the particle distribution of half and full proteasomes.\nPlease, find below a more comprehensive atlas of the original 2D classes from the first round of\nclassification, which more accurately reflects the distribution of half and fully assembled\nproteasomes. The updated\ndataset included approximately 27,000 side-view particles. This additional data supports a clearer\nrepresentation of the particle distribution and confirms the presence of both half and fully assembled\nproteasomes. The new 2D class averages are attached as Figure for Reviewer #1.\n2. In the Tv20S-MZB dataset (Fig. S10), the particle number was reduced from 18,314 to 14,257 but the\nresolution was decreased from 2.79 to 2.86. But the authors state (inconsistently) in Method “cryo-EM\nimaging” section: The resolution was improved by 3D classification and removal of poor 3D classes and\nfurther reiterations including 2D classifications”.\nWe apologize for the confusion regarding the resolution changes and the particle numbers in the Tv20S-\nMZB dataset (Fig. S10). We have included an updated explanation in the figure legend to clarify this issue.\nThe resolution values stated—2.79 Å and 2.86 Å—represent different stages in the analysis process. The\n2.79 Å resolution corresponds to the interim classification obtained from the 3D homogeneous refinement\nsubroutine. The 2.86 Å resolution is the final resolution estimate based on the Fourier Shell Correlation\n(FSC).\nThis number is both affected by the subroutine (program that provides that data) and different number of\nparticles after careful removal of poor-quality particles during the classification process to improve the\ndataset quality, this led to a slight decrease in the overall resolution number. This discrepancy is a result\nof removal of low-quality particles, which, while improving the dataset's integrity, slightly affected the final\nresolution.\nWe now state in the paper (Materials and Methods section): \"The resolution changed from 2.79 Å to 2.86\nÅ during 3D classification.\"\n3. In Method “cryo-EM imaging” section, particles and exposures were reduced to 6,135 particles by\nmanual curation” – did the author mean micrographs rather than particles??\nYes, we apologize, we meant micrographs. This error has been corrected. And we now state in the paper:\n\".. particles and exposures were reduced to 6,135 micrographs by manual curation...\"\n4. In the validation report of PDB ID 8P0T, the particle number was 197,322, the electron dose was 50\ne/Å2, and the defocus was 800-3600 nm. However, the particle number in Fig. S10 is different. The dose\nwas 40 e/Å2 and the defocus was 800-2400 nm in Table S3.\nWe apologize for this inconsistency. We are thankful to Expert Reviewer #1 for spotting this unfortunate\noverlook. The errors originated from inputting setup used during grid screening using the Glacios\nmicroscope and these errors were, unfortunately, not corrected during the finalization. Both 8OIX and\n8P0T final datasets were collected at the same KRIOS instrument with same and identical instrument setup\nwith a was 40 e-/Å2,and using magnification 165000x and defocus range (-2.4)–(-0.9) μm with the\nincrements of 0.3 μm. The table S3 contains correct values including the final number of particles. We have\nreestablished communication with the database and asked for correction of experimental procedures within\neach deposition.\nFigure S10 was corrected, these numbers were deleted.\n5. In the validation report of PDB ID 8OIX, the electron dose was 50 e/Å2, and the defocus was 800-3600\nnm. However, the dose was 40 e/Å2 and the defocus was 800-2400 nm in Table S3.\nSame as above. We apologize for this inconsistency and are thankful to Expert Reviewer #1 for spotting\nthis unfortunate overlook. Both 8OIX and 8P0T datasets were collected at the same KRIOS instrument with\nsame and identical instrument setup with a was 40 e-/Å2, and using magnification 165000x and defocus\nrange (-2.4)–(-0.9) μm with the increments of 0.3 μm. We have reestablished communication with the\ndatabase and asked for correction of experimental procedures within each deposition.\nTable S3 now contains correct values including the final number of particles.\n6. In the validation report of PDB ID 8OIX, the Unmasked-calculated resolution is 3.73 Å with 0.143\nthreshold and 6.91Å with the 0.5 threshold. This is an unusually large gap, indicative of low signal-to-noise\nratio, likely due to limited number of particle (14,257) in the final reconstruction. The author should either\nnote this shortcoming or collect more data to address the problem.\nWe agree with the concern of Expert Reviewer #1 regarding the unusually large gap between the unmasked-\ncalculated resolution (3.73 Å with 0.143 threshold and 6.91 Å with the 0.5 threshold) in the validation\nreport of PDB ID 8OIX. This difference can indeed suggest a lower signal-to-noise ratio, potentially due\nto the limited number of particles (14,257) in the final reconstruction.\nHowever, we have performed extensive iterations from scratch to avoid map bias. The purpose of applying\na mask was to exclude data that are irrelevant to the project (half proteasomes in this case). After applying\nthe mask, the resolution achieved was sufficient, as demonstrated by the quality of the real maps.\nWe believe that attempting to lower the gap between the unmasked and masked data resolutions would not\ncontribute meaningful improvements to our study, as it would involve including redundant data. Moreover,\nour 8P0T map exhibits adequate similarity and accuracy compared to our 8OIX model, confirming the\nvalidity of our results. The 8OIX map, submitted earlier to the RCSB database, supports this consistency.\nWe now state in the manuscript (Materials and Methods section): \" We have observed rather a large gap\nbetween the unmasked-calculated resolution (3.73 Å with 0.143 threshold and 6.91 Å with the 0.5\nthreshold).\n7. In Fig. S3c, Tv20S is more abundant in the -Ump1 samples, does not appear to agree with the description\nthat “in the absence of Ump-1, the eluted fraction from the streptavidin column contained more half-\nproteasomes, indicating incomplete assembly”.\nFigure S3c shows that the ratio of half-proteasome to full-proteasome is higher in the absence of Ump-1.\nThis figure does not compare the abundance of full-proteasome in the presence and absence of Ump-1.\nAlthough the –Ump-1 lanes might seem brighter, this full-proteasome is immature due to non-functional β5\nsubunits (Figures S3b & S3d). Since the native PAGE gel used here cannot differentiate between the\nmature, functional full-proteasome (made with Ump-1) and the immature, non-functional full-proteasome\n(made without Ump-1), then comparing the band intensities is unreliable. Therefore, this figure focuses\nsolely on the ratio of half-proteasome to full-proteasome.\nFor clarity, we now updated the manuscript with the sentence: “In the absence of Ump-1, the eluted fraction\nfrom the streptavidin column contained a higher ratio of more half-proteasomes to full-proteasomes,\nindicating incomplete assembly”.\n8. In Fig. S3b,d, why did the -Ump1 sample lose b5 binding to the fluorogenic probe? Does Ump-1\naffect the b5 maturation?\nFig. S3b and S3d revealed that the full-proteasome generated in the absence of Ump-1 does not have a\nfunctioning β5 subunit. The connection between β5 and Ump-1 has been previously described for the yeast\nproteasome by Velez and colleagues (PMID: 38600323). In that study they show that Ump-1 directly\ninteracts with the propeptide sequence of β5 and properly orientates it between the β6 and β7 subunits.\nTherefore, the interaction of β5 and Ump-1 is key for proteasome maturation in yeast and the same appears\nto be true for Tv20S.\nWe have now added the following sentence to the results section: “The connection between Ump-1 and β5\nactivity are supported by previous studies of the yeast proteasome where Ump-1 was shown to directly\ninteract with the propeptide sequence of β5 and properly orientate it between the β6 and β7 subunits” and\nincluded the reference.\n9. Fig. 1e and Fig S3b: There is no corresponding band in the silver stain gel compared with the\nfluorogenic probe binding gel. Are they from the same gel?\nThese images are from the same gel. Below (Figure for Reviewer 1), we have attached the silver stained\nand fluorescent gel images side-by-side. In addition, we have merged the two gels into one image. The β5\nsubunit is not visible on the silver-stained gel but can be readily detected using the fluorescent probe. Since\nall 14 subunits (7α and 7β) are present at the same ratio on this gel, then the visible bands on the silver\nstained gel are likely to consist of more than one subunit that co-migrate on the gel.\n10. Fig. 2c: the curves for the b1 of nTv20S and rTv20S never reached Vmax, indicating low affinity or\ninaccessible to the b1 site. Consider another substrate?\nThe β1 substrate for Tv20S was developed following extensive substrate profiling studies and it is the most\nefficient substrate available for this enzyme. We have reported those substrate profiling studies in Fajtova\n2024 (PMID: 37066163). The goal of Fig. 2C was to show that the native and recombinant enzymes had\nthe same activity. This comparison can be done with a substrate that has a low Km (such as Ac-GWYL-amc\n(β5) and Ac-FRSR-amc (β2) or with a substrate that has a higher Km such as Ac-RYFD-amc (β1).\n11. From Fig 1c,f, the purified rTv20S was intact, so the proteasome was apparently destroyed by\nMZB, raising the question of the inhibitor entry into the active site. Several scenarios can be\npictured, and different scenario may result in different structure and kinetics. The author should\naddress if half proteasome binds the inhibitor.\nWe did not generate images of the apo-enzyme and therefore we cannot directly compare the ratio of half\nproteasome to full-proteasomes in MZB-treated and untreated images. It is possible the MZB destabilizes\nthe full-proteasome yielding an increase in half-proteasomes. For our structural studies, we used a\npreparation of Tv20S that was co-expressed with Ump-1. However, we later discovered that incubation of\nthis purified preparation at room temperature for 24 to 72 hours decreases the amount of half-proteasome\npresent (see Fig S3c). In our future studies, we will examine the time-dependent assembly of Tv20S.\nBased on published proteasome assembly studies, it is unlikely that MZB or other active site inhibitors bind\nto the half-proteasomes because the propeptides of β1 β2 and β5 are not processed until the full proteasome\nassembly has occurred. Therefore, the catalytic sites of half-proteasomes are unlikely to be accessible by\nan inhibitor.\nEvidence using other protease inhibitors asl suggest that MZB does not bind to half-proteasomes. For\nexample, the activity-based probe, Me4BodipyFL-Ahx3Leu3VS, binds covalently to all three catalytic\nsubunits. However, we do not see labelling of half-proteasomes in insect cell lysates that express Tv20S\nwith a high abundance of half-proteasomes. This is demonstrated by Fig. S3a, Lane 3 where Tv20S is\nexpressed without Ump-1 yet there is no labelling of a half-proteasome bands on this gel.\n12. Fig. 4e-f and in page 9: there is no fig 4f. Ile35 mentioned in the text but not labeled in the figure.\nThank you for pointing out this error. Fig 4e-f was changed to Fig. 4d-e and Ile35 is now labeled.\nReviewer #2 (Remarks to the Author):\nThe authors have responded to my previous concerns satisfactorily. I have no further concerns.\nReviewer #1 (Remarks to the Author)\nOur concerns have been addressed.\nWe are happy to hear that.\nReviewer #2 (Remarks to the Author)\nThe authors have responded to my previous concerns satisfactorily. I have no further concerns.\nWe are happy to hear that."}]

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