High resolution cryo‐EM of eukaryotic proteasomes: an emerging tool for therapeutic drug development

Abstract

Recently the field of biological structural electron microscopy has seen an enormous transformation, primarily triggered by the availability of improved electron microscopes and direct electron detectors. It is now possible to use electron cryo‐microscopy (cryo‐EM) and single particle analysis to determine the structure of proteins to resolutions that used to be achievable only by crystallography or NMR methods. The structural information attainable by such methodologies can in principle be used to infer into the detailed molecular mechanisms of proteins and protein complexes. We explored their application to study protein/ligand interactions using the eukaryotic 20S proteasome core. The proteasome is a highly regulated protease complex essential in all eukaryotes for its role in cell homeostasis and regulation of fundamental mechanisms such as cell cycle progression. The proteasome proteolytic active sites are enclosed within its 20S core. In eukaryotes, the 20S proteasome is a ~750 kDa complex formed by 7 individual α and 7 individual β subunits, arranged in a barrel shaped two‐fold symmetric α7β7β7α7 assembly. While the 20S proteasome core is a well‐established target for cancer therapy, its inhibition is being further explored for an increasing range of varied therapeutic usages, including inflammatory disorders, viral infections and tuberculosis. We used cryo‐EM and single particle analysis to determine the structure of the human 20S proteasome core bound to a substrate analogue inhibitor molecule, at a resolution of around 3.5Å. The resulting map allowed the building of protein coordinates as well as defining the location and conformation of the inhibitor at the different active sites. These results serve as proof of principle that cryo‐EM is emerging as a realistic approach for more general structural studies of protein/ligand interactions, with its own advantages compared with other methods of protein structure determination. Cryo‐EM has the potential benefits of extending such studies to complexes unsuitable for other methods of structure determination, namely by requiring significantly less amounts of sample, and allowing closer to physiological conditions, preserving ligand specificity. Within this context, we extended our studies to assist in the development of new highly specific inhibitors targeting the Plasmodium falciparum proteasome. Plasmodium falciparum is the parasite responsible for the most severe form of malaria, against which artemisinin is currently the forefront medication. The spreading of artemisinin resistant parasites, first identified in the Southeast Asia, represents a major threat to human health and to the current programs aiming at controlling and eventually eradicating malaria and urges the development of new antimalarials. We determined the structure of the Plasmodium falciparum 20S proteasome core bond to a new specific inhibitor, developed by our collaborator Matt Bogyo, Stanford University, at a resolution of around 3.6Å (figure 1). Our structure, and its comparison with that of the human 20S proteasome core, revealed the molecular basis for the inhibitor specificity for the parasite complex and for the improvement of this ligand into a more potent anti‐malaria drug prototype, with demonstrated low toxicity to in vivo model hosts. The cryo‐EM structure of the Plasmodium falciparum 20S proteasome can assist in the development of such inhibitors as ligands with potential as new‐generation antimalarials.