Science 353, 594–598 (2016)

High-resolution structures of the human 20S proteasome reveal four coaxially stacked heteroheptameric rings, with the two inner rings containing the proteolytic active sites. Proteasome structures in complex with small-molecule inhibitors have yet to enable definition of the inhibition details at an atomic level. Using a newly developed workflow for purification and crystallization of human 20S proteasomes, Schrader et al. solved X-ray structures of the proteasome in complex with six inhibitors, including bortezomib, a drug used to treat multiple myeloma. The structures with two epoxyketones, oprozomib and dihydroeponemycin, revealed a 1,4-oxazepane (seven-membered) ring structure formed by nucleophilic attack of the active site threonine amine moiety to the compound epoxide β-carbon. This is notably different from the 1,4-morpholine ring structure and attack by the epoxide α-carbon that had been proposed in previous structural studies. The structures with these epoxyketones, the ketoaldehyde Z-LLY-ketoaldehyde, and three boronic inhibitors also define a proton-shuttling role for a water molecule both in catalysis of peptide cleavage and in the cyclization step of the inhibition reaction, findings that were corroborated by enzyme kinetics and cluster quantum chemical calculations. These findings refine the proteasome active site mechanism and suggest that the new workflow could allow for the development of novel active site inhibitors.