The Plasmodium falciparum 20S proteasome (Pf20S) is a promising antimalarial target. Therapeutic development has previously relied on native purifications of Pf20S, which is challenging and has limited the scope of previous efforts. Here, we report an effective recombinant Pf20S platform to facilitate drug discovery. Complex assembly is carried out in insect cells by co-expressing all fourteen subunits along with the essential Pf chaperone homolog, Ump1. Unexpectedly, the isolated proteins consisted of both a mature and an immature complex. Cryo-EM analysis of the immature complexes revealed structural insights detailing how Ump1 and β2/β5 pro-peptides coordinate β-ring assembly, which differ from human and yeast homologs. Biochemical validation confirmed that β1, β2, and β5 subunits of the mature proteasome were catalytically active. Clinical proteasome inhibitors, bortezomib, carfilzomib and marizomib were potent but lacked Pf20S selectivity. However, the tripeptide-epoxyketone J-80 potently and selectively inhibited Pf20S β5 (IC50= 22.4 (1.3) nM, 90-fold over human β5), with cryo-EM elucidating the structural basis for its specific binding. Further evaluation of novel Pf20S-selective inhibitors such as the reversible TDI-8304 and irreversible analogs 8304-vinyl sulfone and 8304-epoxyketone confirmed their potency and selectivity over the human constitutive proteasome. This recombinant Pf20S platform facilitates detailed biochemical and structural studies, accelerating the development of selective antimalarial therapeutics.

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