Crystal Structures Illuminate Proteasome Inhibitor Mechanisms

Targeting the proteasome, a key regulator of cellular protein homeostasis, is a strategy to restrict cancer growth. Several proteasome inhibitors are approved or in clinical trials for cancer treatment, but the dearth of structural information required to identify ligand binding sites and understand the mechanisms of inhibition has limited the development of new inhibitors. Most of the structural information comes from studies of the yeast proteasome, or from relatively low-resolution X-ray crystallography and electron cryomicroscopy studies of the human proteasome. To get higher-resolution structural information, Schrader, Henneberg, and colleagues developed an optimized pipeline for production, purification, and crystallization of human 20S proteasomes, allowing production of large quantities of protein and hundreds of crystals that routinely diffract to high resolution. The structure of the 20S proteasome was solved at a 1.8 Å resolution and in complex with 6 different proteasome inhibitors at a 1.9 to 2.1 Å resolution. These structures revealed functionally relevant differences compared with previously reported structures, including a chloride ion in all active sites previously assigned as catalytic water, and 3 water molecules in the active site that had not been previously observed. Although all of the inhibitors bound to the same subunit as in earlier studies, there were some structural differences at the active site affecting the mode of inhibition; for example, oprozomib formed a seven-membered 1,4-oxazepane ring structure with the proteasome active site, contrary to what had previously been observed in lower-resolution structures, whereas so-called ketoaldehydes formed six-membered 1,4-morpholine rings. In addition to providing structural insights to refine our understanding of the mechanisms of action of proteasome inhibitors in clinical development and guide the development of next-generation proteasome inhibitors, these findings provide a potential framework for screening structures of potential cancer therapeutics in large numbers at high resolution.

Schrader J, Henneberg F, Mata RA, Tittmann K, Schneider TR, Stark H, et al. The inhibition mechanism of human 20S proteasomes enables next-generation inhibitor design. Science 2016;353:594–8.