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

Proteasomes are essential for protein homeostasis in mammalian cells and in protozoan parasites such as . and other protozoan 20S proteasomes have been validated as druggable targets. However, in the case of 20S proteasome ( 20S), biochemical and structural studies were impeded by low yields and purity of the native proteasome. We successfully made recombinant 20S 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 recombinant 20S is inhibited by the natural product marizomib (MZB) and the recently developed peptide inhibitor carmaphycin-17 (CP-17) . 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 of 20S in complex with these covalent inhibitors at 2.8Å resolution. The structures revealed the overall fold of the 20S and the binding mode of MZB and CP-17. Our work explains the low specificity of MZB and higher specificity of CP-17 towards 20S as compared to human proteasome and provides the platform for the development of 20S inhibitors for treatment of trichomoniasis.