Lars Ditzel

Crystal structure of the thermosome, the archaeal chaperonin and homolog of CCT.

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.

Bifunctional inhibitors of the trypsin-like activity of eukaryotic proteasomes

BACKGROUND The 20S proteasome is a multicatalytic protease complex that exhibits trypsin-like, chymotrypsin-like and post-glutamyl-peptide hydrolytic activities associated with the active sites of the beta2, beta5 and beta1 subunits, respectively. Modulation of these activities using inhibitors is essential for a better understanding of the proteasomes mechanism of action. Although there are highly selective inhibitors of the proteasomes chymotryptic activity, inhibitors of similar specificity have not yet been identified for the other activities. RESULTS The X-ray structure of the yeast proteasome reveals that the sidechain of Cys118 of the beta3 subunit protrudes into the S3 subsite of the beta2 active site. The location of this residue was exploited for the rational design of bidentated inhibitors containing a maleinimide moiety at the P3 position for covalent linkage to the thiol group and a carboxy-terminal aldehyde group for hemiacetal formation with the Thr1 hydroxyl group of the active site. Structure-based modelling was used to determine the optimal spacing of the maleinimide group from the P2-P1 dipeptide aldehydes and the specificity of the S1 subsite was exploited to limit the inhibitory activity to the beta2 active site. X-ray crystallographic analysis of a yeast proteasome-inhibitor adduct confirmed the expected irreversible binding of the inhibitor to the P3 subsite. CONCLUSIONS 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.

Structural Investigation of Proteasome Inhibition

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.

Synthesis, Kinetic Characterization and X-Ray Analysis of Peptide Aldehydes as Inhibitors of the 20S Proteasomes from Thermoplasma acidophilum and Saccharomyces cerevisiae

A comparative kinetic characterization of the peptide aldehydes Ac-Leu-Leu-X-H [X = Trp, Tyr and Tyr(tBu)] and Z-Gly-Pro-Gly-Gly-Leu-Leu-Nle-H as inhibitors of the chymotryptic activity of 20S proteasomes from the archaebacterium T. acidophilum and yeast S. cerevisiae revealed significantly differentiated inhibitory potencies that can be rationalized on the basis of X-ray crystallographic data.