The crystal structure of the 20S proteasome from the yeast Saccharomyces cerevisiae shows that its 28 protein subunits are arranged as an (alpha1...alpha7, beta1...beta7)2 complex in four stacked rings and occupy unique locations. The interior of the particle, which harbours the active sites, is only accessible by some very narrow side entrances. The beta-type subunits are synthesized as proproteins before being proteolytically processed for assembly into the particle. The proforms of three of the seven different beta-type subunits, beta1/PRE3, beta2/PUP1 and beta5/PRE2, are cleaved between the threonine at position 1 and the last glycine of the pro-sequence, with release of the active-site residue Thr 1. These three beta-type subunits have inhibitor-binding sites, indicating that PRE2 has a chymotrypsin-like and a trypsin-like activity and that PRE3 has peptidylglutamyl peptide hydrolytic specificity. Other beta-type subunits are processed to an intermediate form, indicating that an additional nonspecific endopeptidase activity may exist which is important for peptide hydrolysis and for the generation of ligands for class I molecules of the major histocompatibility complex.

We have determined to 2.6 A resolution the crystal structure of the thermosome, the archaeal group II chaperonin from T. acidophilum. The hexadecameric homolog of the eukaryotic chaperonin CCT/TRiC shows an (alphabeta)4(alphabeta)4 subunit assembly. Domain folds are homologous to GroEL but form a novel type of inter-ring contact. The domain arrangement resembles the GroEL-GroES cis-ring. Parts of the apical domains form a lid creating a closed conformation. The lid substitutes for a GroES-like cochaperonin that is absent in the CCT/TRiC system. The central cavity has a polar surface implicated in protein folding. Binding of the transition state analog Mg-ADP-AIF3 suggests that the closed conformation corresponds to the ATP form.

Proteasomes are large multisubunit proteases that are found in the cytosol, both free and attached to the endoplasmic reticulum, and in the nucleus of eukaryotic cells. Their ubiquitous presence and high abundance in these compartments reflects their central role in cellular protein turnover. Proteasomes recognize, unfold, and digest protein substrates that have been marked for degradation by the attachment of a ubiquitin moiety. Individual subcomplexes of the complete 26S proteasome are involved in these different tasks: The ATP-dependent 19S caps are believed to unfold substrates and feed them to the actual protease, the 20S proteasome. This core particle appears to be more ancient than the ubiquitin system. Both prokaryotic and archaebacterial ancestors have been identified. Crystal structures are now available for the E. coli proteasome homologue and the T. acidophilum and S. cerevisiae 20S proteasomes. All three enzymes are cylindrical particles that have their active sites on the inner walls of a large central cavity. They share the fold and a novel catalytic mechanism with an N-terminal nucleophilic threonine, which places them in the family of Ntn (N terminal nucleophile) hydrolases. Evolution has added complexity to the comparatively simple prokaryotic prototype. This minimal proteasome is a homododecamer made from two hexameric rings stacked head to head. Its heptameric version is the catalytic core of archaebacterial proteasomes, where it is sandwiched between two inactive antichambers that are made up from a different subunit. In eukaryotes, both subunits have diverged into seven different subunits each, which are present in the particle in unique locations such that a complex dimer is formed that has six active sites with three major specificities that can be attributed to individual subunits. Genetic, biochemical, and high-resolution electron microscopy data, but no crystal structures, are available for the 19S caps. A first step toward a mechanistic understanding of proteasome activation and regulation has been made with the elucidation of the X-ray structure of the alternative, mammalian proteasome activator PA28.

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Heat shock locus V (HslV; also called ClpQ) is the proteolytic core of the ATP-dependent protease HslVU in Escherichia coli. It has sequence similarity with the ␤-type subunits of the eukaryotic and archaebacterial proteasomes. Unlike these particles, which display 72-point symmetry, it is a dimer of hexamers with 62-point symmetry. The crystal structure of HslV at 3.8-Å resolution, determined by isomorphous replacement and symmetry averaging, shows that in spite of the different symmetry of the particle, the fold and the contacts between subunits are conserved. A tripeptide aldehyde inhibitor, acetyl-Leu-Leu-norleucinal, binds to the Nterminal threonine residue of HslV, probably as a hemiacetal, relating HslV also functionally to the proteasomes of archaea and eukaryotes.Heat shock leads to increased levels of misfolded proteins. In response, Escherichia coli expresses ATP-dependent chaperones and complexes that appear to combine chaperone-like activity with proteolysis. The discovery of the operon hslVU (heat shock locus VU) (1) in E. coli under transcriptional control of a 32 -dependent heat shock promoter added protease HslVU to this class that already contained the cytosolic proteases Lon (La) and Clp (Ti) (2). Protease HslVU (3) is a hybrid of Clp and the proteasome. The subunits of HslV and the ␤-subunits of the 20S proteasome of the archaeon Thermoplasma acidophilum are 18% identical in amino acid sequence, and, after processing, they both have a threonine at the N terminus (Fig. 1). The regulatory caps HslU that contain the Walker consensus ATP-binding motif are highly homologous to ClpX from E. coli (4). They seem to play the role of both the ␣-subunits of the 20S particle and the 19S caps. Unlike the situation in archaea, where the presence of the ␣-subunits is essential for assembly of the ␤-subunits (5), HslV assembles in the absence of HslU, and unlike the ␤-subunits in T. acidophilum, it forms a dimer of hexamers rather than heptamers as first shown by electron microscopy (6, 7). In vitro, it has been shown that HslU stimulates proteolytic activity of HslV against casein (6) and small chromogenic peptides in the presence of ATP (8). The range of small peptide substrates for HslVU appears to be rather limited. Z-Gly-Gly-Leu-AMC (7-amido-4 methylcoumarin) and some, but not all, hydrophobic substrates are degraded (3). Weak peptidase activity has been reported for HslV in the absence of HslU (9). To our knowledge, tryptic and peptidyl-glutamyl-like activities, both present in eukaryotic proteasomes, have not been found in HslV.

Maleoyl-beta-alanyl-valyl-arginal is a new type of inhibitor that is highly selective for the trypsin-like activity of eukaryotic proteasomes. Despite the reactivity of the maleinimide group towards thiols, and therefore the limited use of this inhibitor for in vitro studies, it might represent an interesting new biochemical tool.

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The novel proteolytic mechanism of the 20S proteasome from T. acidophilum has been investigated by X-ray crystallography using small-molecule inhibitors and substrate analogues. The 20S proteasome degrades unfolded substrates into small peptides of a defined length. Calpain inhibitor II, chymostatin and lactacystin all bind in the previously identified active site pocket near Thr1 of all fourteen beta-subunits. The chromogenic substrate analogue Suc-LLVY-AMC binds in the same pocket of the proteolytically inactive T1A mutant of the beta-subunit, but with a significantly altered geometry. The heavy-atom cluster Ta6Br12(2+) used in X-ray structure determination occupies seven sites in the inner compartment of the proteasome and exhibits inhibition of the chymotrypsin-like activity. Other effectors of proteasome activity showed no significant difference in electron density.