Constitutive proteasomes and immunoproteasomes shape the peptide repertoire presented by major histocompatibility complex class I (MHC-I) molecules by harboring different sets of catalytically active subunits. Here, we present the crystal structures of constitutive proteasomes and immunoproteasomes from mouse in the presence and absence of the epoxyketone inhibitor PR-957 (ONX 0914) at 2.9 Å resolution. Based on our X-ray data, we propose a unique catalytic feature for the immunoproteasome subunit b5i/LMP7. Comparison of ligand-free and ligand-bound proteasomes reveals conformational changes in the S1 pocket of b5c/X but not b5i, thereby explaining the selectivity of PR-957 for b5i. Time-resolved structures of yeast proteasome:PR-957 complexes indicate that ligand docking to the active site occurs only via the reactive head group and the P1 side chain. Together, our results support structure-guided design of inhibitory lead structures selective for immunoproteasomes that are linked to cytokine production and diseases like cancer and autoimmune disorders.

The 26 S proteasome is the central protease involved in ubiquitin-mediated protein degradation and fulfills vital regulatory functions in eukaryotes. The proteolytic core of the complex is the 20 S proteasome, a cylindrical particle with two outer rings each made of 7 different ␣-type subunits and two inner rings made of 7 different ␤-type subunits. In the archaebacterial 20 S proteasome ancestor proteolytically active sites reside in the 14 uniform ␤-subunits. Their N-terminal threonine residues, released by precursor processing, perform the nucleophilic attack for peptide bond hydrolysis. By directed mutational analysis of 20 S proteasomal ␤-type proteins of Saccharomyces cerevisiae, we identified three active site-carrying subunits responsible for different peptidolytic activities as follows: Pre3 for post-glutamyl hydrolyzing, Pup1 for trypsin-like, and Pre2 for chymotrypsin-like activity. Double mutants harboring only trypsin-like or chymotrypsin-like activity were viable. Mutation of two potentially active site threonine residues in the Pre4 subunit excluded its catalytic involvement in any of the three peptidase activities. The generation of different, incompletely processed forms of the Pre4 precursor in active site mutants suggested that maturation of non-active proteasomal ␤-type subunits is exerted by active subunits and occurs in the fully assembled particle. This trans-acting proteolytic activity might also account for processing intermediates of the active site mutated Pre2 subunit, which was unable to undergo autocatalytic maturation.The proteasome is a large multi-subunit proteinase complex found in the cytoplasm and nucleus of all eukaryotic cells examined so far. This "proteolytic organelle" fulfills vital cellular functions. As part of the ubiquitin-mediated protein degradation machinery, it is responsible not only for the elimination of misfolded proteins, including those derived from the lumen of the endoplasmic reticulum (1), it also controls a multitude of regulatory processes by removing unnecessary or even harmful metabolic enzymes and by balancing the levels of many regulatory proteins (for reviews see Refs. 2-4). Proteasomes exist as particles of 20 S and of 26 S. The 26 S complex of Ϸ2000 kDa is composed of the 20 S particle of Ϸ700 kDa as a proteolytic core unit and two regulatory 19 S caps that dock onto each side of the 20 S cylinder and confer ATP and ubiquitin dependence onto proteasomal protein degradation (2, 3, 5-7).A 20 S proteasome ancestor was isolated from the archaebacterium Thermoplasma acidophilum which exhibits an electron microscopic structure like the eukaryotic proteasome core but a much simpler subunit complexity. Extensive structural studies on this complex (8 -11) were completed by its x-ray crystallographic resolution (12). Two related subunits, ␣ and ␤, form a stack of four heptameric rings, whereby the two outer rings are composed of ␣-subunits and the two inner rings of ␤-subunits. Four narrow gates arranged along the cylinder axis give rise to three cavities...

Proteinase yscE is the yeast equivalent of the proteasome, a multicatalytic‐multifunctional proteinase found in higher eukaryotic cells. We have isolated three mutants affecting the proteolytic activity of proteinase yscE. The mutants show a specific reduction in the activity of the complex against peptide substrates with hydrophobic amino acids at the cleavage site and define two complementation groups, PRE1 and PRE2. The PRE1 gene was cloned and shown to be essential. The deduced amino acid sequence encoded by the PRE1 gene reveals weak, but significant similarities to proteasome subunits of other organisms. Two‐dimensional gel electrophoresis identified the yeast proteasome to be composed of 14 different subunits. Comparison of these 14 subunits with the translation product obtained from PRE1 mRNA synthesized in vitro demonstrated that PRE1 encodes the 22.6 kd subunit (numbered 11) of the yeast proteasome. Diploids homozygous for pre1–1 are defective in sporulation. Strains carrying the pre1–1 mutation show enhanced sensitivity to stresses such as incorporation of the amino acid analogue canavanine into proteins or a combination of poor growth medium and elevated temperature. Under these stress conditions pre1–1 mutant cells exhibit decreased protein degradation and accumulate ubiquitin‐protein conjugates.

We present a biochemical and crystallographic characterization of active site mutants of the yeast 20S proteasome with the aim to characterize substrate cleavage specificity, subunit intermediate processing, and maturation. ␤1(Pre3), ␤2(Pup1), and ␤5(Pre2) are responsible for the postacidic, tryptic, and chymotryptic activity, respectively. The maturation of active subunits is independent of the presence of other active subunits and occurs by intrasubunit autolysis. The propeptides of ␤6(Pre7) and ␤7(Pre4) are intermediately processed to their final forms by ␤2(Pup1) in the wild-type enzyme and by ␤5(Pre2) and ␤1(Pre3) in the ␤2(Pup1) inactive mutants. A role of the propeptide of ␤1(Pre3) is to prevent acetylation and thereby inactivation. A gallery of proteasome mutants that contain active site residues in the context of the inactive subunits ␤3(Pup3), ␤6(Pre7), and ␤7(Pre4) show that the presence of Gly-1, Thr1, Asp17, Lys33, Ser129, Asp166, and Ser169 is not sufficient to generate activity.