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.