Structural elucidation of recombinantTrichomonas vaginalis20S proteasome bound to covalent inhibitors

Abstract

ABSTRACTProteasomes are essential for protein homeostasis in mammalian cells1-4and in protozoan parasites such asTrichomonas vaginalis (Tv).5Tvand other protozoan 20S proteasomes have been validated as druggable targets.6-8However, in the case ofTv20S proteasome (Tv20S), biochemical and structural studies were impeded by low yields and purity of the native proteasome. We successfully made recombinantTv20S by expressing all seven α and seven β 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 recombinantTv20S is inhibited by the natural product marizomib (MZB)9and the recently developed peptide inhibitor carmaphycin-17 (CP-17)8,10. Specifically, MZB binds to the β1, β2 and β5 subunits, while CP-17 binds the β2 and β5 subunits. Next, we obtained cryo-EM structures ofTv20S in complex with these covalent inhibitors at 2.8Å resolution. The structures revealed the overall fold of theTv20S and the binding mode of MZB and CP-17. Our work explains the low specificity of MZB and higher specificity of CP-17 towardsTv20S as compared to human proteasome and provides the platform for the development ofTv20S inhibitors for treatment of trichomoniasis.