Over the last few years 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 human 20S proteasome core.
The proteasome is a highly regulated protease complex fundamental for cell homeostasis and controlled cell cycle progression. The proteolytic active sites of the proteasome are enclosed within its 20S core. In eukaryotes, the 20S core 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 explored for an increasing range of further therapeutic usages. 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. This has the potential benefits of extending such studies to complexes unsuitable for other methods of structure determination and allowing closer to physiological conditions to be used. 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 therefore a major threat to human health and to the current programs aiming at controlling and eventually eradicating malaria. We determined the structure of the Plasmodium falciparum 20S proteasome core bond to a new specific inhibitor, at a resolution of around 3.6Å. This inhibitor was developed by our collaborator Matt Bogyo, Stanford University, based on the profiling of substrate cleavage sites specific to the parasite proteasome. 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. The structure obtained has further guided the improvement of this ligand into a more effective anti-malaria drug prototype, with demonstrated low toxicity to in vivo model hosts.
