The Yeast 20S Proteasome

As discussed above, the eubacterial HslVU is distantly related in structure to the proteasome found in archaea and eukaryotes. Surprisingly, however, the structural relationship is not reflected in the regulatory properties as will be described in Section 10.3, which focuses on structural studies of the yeast 20S proteasome and its activation, activity, and inhibition. 

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This notion was further confirmed by the crystal structure of a 20S/11S heterologous complex between the yeast 20S-proteasome and the Trypanosoma IIS-regulator (Whitby et al. 2000). This approach was justified by the ability of IIS reg-... 

Fig. 10.7. Inh ibitor binding to individual active sites of the yeast 20S proteasome. The inhibitors lactacystin (A), epoxomicin (B) and TMC95A (C) are colored green and are shown in stereo mode together with their unbiased electron densities. The active-site Thrl is highlighted in black. (A) Covalent binding of the Streptomyces metabolite lactacystin to the active site of 5. The SI pockets of the active subunits and differ from that of 5 and are not suitably constructed to bind the inhibitor. As discussed in the text, Met45 (black), which is located at the bottom of the 5-Sl pocket, makes the difference for inhibitor...

Nussbaum, a. K., Dick, T. P., Keilholz, W., Schiele, M., Stevanovic, S., Dietz, K., Heinemeyee, W., Geoll, M., Wole, D. H., Hubee, R., et al. Cleavage motifs of the yeast 20S proteasome beta subunits deduced from digests of enolase 1. Proc. Natl. Acad. Sci. USA 1998, 95, 12504-12509. 

T. acidcphilum, yeast and mammals. The arrangement of subunits in the bovine 20S proteasome is identical with that in the yeast 20S proteasome. 

The a ring of the bovine proteasome was superimposed onto the P ring of the yeast proteasome by a least-squares fitting of main chain atoms. R.m.s. deviations between all atoms of the functional amino acids in the bovine yffl, pi and pS subunits and the corresponding subunits in the yeast 20S proteasome were 0.3 A,... 

TMC-95A, a cydic peptide metabolite from Apiospra montagnei, is a potent competitive inhibitor of all active sites and forms characteristic hydrogen bonds with the protein backbone. The crystal structure of the yeast 20S proteasome in complex with TMC-95A indicates a non-covalent linkage to the active y -subunits the N-terminal threonine residues are not modified. The TMC-95A backbone adopts a -conformation and extends the -strand SI by the generation of an antiparallel P-sheet. This stmcture is similar to that seen with the aldehyde and epoxyketone inhibitors. An interactions of TMC-95A are formed with main-chain atoms and strictly conserved residues of the 20S proteasome. 

To allow for an access of two anchor groups to two identical or different active sites from the non-primed S subsites, the crystal structure of Ac-Leu-Leu-Nle-H bound to pS and pS of the yeast 20S proteasome was used as a template [34]. The entry of substrates into the proteolytic chamber is restricted by the bottle-neck of the a ring, which recruits from outside only fully unfolded linear polypeptides for digestion. This fact significantly restricts the choice of spacers for bivalent inhibitor constructs. Such a spacer should mimic as much as possible the unstructured polypeptide chain of an unfolded protein, and reach a length of about 50 A. Peptides of appropriate size are known to be rapidly degraded by the yeast proteasome, and thus linear polyoxyethylene (PEG) chains were selected as mimic of random-coiled polypeptide chains [37, 64], since this polymer is known to be highly solvated and... 

Homo- and Heterobivalent Inhibitors of the Yeast 20S Proteasome 401 Table 2.2 Inhibition of 20S proteasome by mono- and bivalent inhibitors (/C50, tM)... 

Fig. 2.5 Upper panel Stereoview of a section of the X-ray structure of the yeast 20S proteasome/compound (5) adduct. Lower panel Schematic representation of the inhibitor linked via hemiacetal bond to two active-site Thrl residues.

It has been known that the treatment of mammalian [67, 68] or yeast proteasome [69] with larger excesses of the thiol-reagent N-ethylmaleinimide (NEM) leads to selective inhibition of the trypsin-like activity. In the crystal structure of the yeast 20S proteasome the conserved CysllS residue of the P3 subunit protrudes into the S3 sub site of the p2 active site [34], a fact that could explain the inactivation of the trypsin-like activity of proteasomes by its chemical modification with NEM. The par-... 

Fig. 2.6 Schematic representation of the S subsites of the p2 active site of the yeast 20S proteasome as structural model for the design of maleoyl-y -alanyl-dipeptide aldehydes as a new type selective heterobifunctional inhibitors.

Proteasomes - Both 20S and 26S proteasomes are known. Figure 28.44 shows the structure of the yeast 20S proteasome. Structurally, the proteasome shows remarkable similarity to the GroEL chaperonin (see here). Both are multitiered cylinders with 7-fold symmetry. Both can accept an unfolded polypeptide chain in their hollow interior. But whereas GroEL protects the polypeptide chain, the proteasome degrades it. 

Figure 28.44 Three-dimensional strueture of the yeast 20S proteasome. 

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