Structural elucidation of recombinantTrichomonas vaginalis20S proteasome bound to covalent inhibitors

Abstract

Proteasomes are essential for protein homeostasis in mammalian cells and in protozoan parasites such as Trichomonas vaginalis (Tv). Tv and other protozoan 20S proteasomes have been validated as druggable targets. However, in the case of Tv 20S proteasome (Tv20S), biochemical and structural studies were impeded by low yields and purity of the native proteasome. We successfully made recombinant Tv20S by expressing all seven alpha and seven beta subunits together with the Ump-1 chaperone in insect cells. We isolated recombinant proteasome and showed that it was biochemically indistinguishable from the native enzyme. We confirmed that the recombinant Tv20S is inhibited by the natural product marizomib (MZB) and the recently developed peptide inhibitor carmaphycin-17 (CP-17). Specifically, MZB binds to the beta1, beta2 and beta5 subunits, while CP-17 binds the beta2 and beta5 subunits. Next, we obtained cryo-EM structures of Tv20S in complex with these covalent inhibitors at 2.8A resolution. The structures revealed the overall fold of the Tv20S and the binding mode of MZB and CP-17. Our work explains the low specificity of MZB and higher specificity of CP-17 towards Tv20S as compared to human proteasome and provides the platform for the development of Tv20S inhibitors for treatment of trichomoniasis.

Visualizing chaperone-mediated multistep assembly of the human 20S proteasome

“…Recombinant systems have proven useful for studying mature proteasome complexes 16,[50][51][52] . To express the human CP in insect cells, we utilized a combination of the biGBac 53 and MultiBac 54 systems to generate baculovirus transfer vectors: one with all seven α-subunits, another with all seven β-subunits, and the third with the five CP assembly chaperones.…”