Cryo-Em Structure of the Plasmodium Proteasome in the Search of New Antimalarials

Abstract

The Plasmodium falciparum proteasome has been suggested as possible target for new generation antimalarials. However, the proteasome is essential in all eukaryotes and a proteasome inhibitor with potential to be developed as antimalarial must specifically target the Plasmodium proteasome, while evading cross-reactivity with the human counterpart. A high resolution structure of the Plasmodium proteasome was needed in order to validate it as a drug target, to understand the molecular basis for its specific inhibition and to directly guide candidate drug discovery and development. While X-ray crystallography has been the method of choice for such protein/ligand studies, the small amounts of proteasome that can be realistically purified from cultured parasites impair its crystallisation.Recently cryo-electron microscopy (cryo-EM) has seen an enormous transformation. It is now possible to use cryo-EM and single particle analysis to determine protein structures at resolutions that used to be achievable only by crystallography or NMR methods. Our initial cryo-EM structure of an inhibitor-bound human 20S proteasome, at a resolution of around 3.5A, served as proof of principle that cryo-EM is emerging as a realistic approach for structural studies of protein/ligand interactions, with its own advantages. Cryo-EM requires significantly less amounts of purified protein than other methods and allows structure determination in closer to physiological conditions, where active site specificities can be preserved. Within this context, we used cryo-EM to determine the structure of the Plasmodium falciparum 20S proteasome bound to a newly designed specific inhibitor. This structure, and its comparison with that of the human complex, reveals the molecular basis for the Plasmodium proteasome specific inhibition, therefore validating it as a realistic antimalarial target. Furthermore, the cryo-EM structure of the Plasmodium proteasome can guide the development of the bound prototype inhibitor towards potential next-generation antimalarials.