Structural Investigation of Proteasome Inhibition^*

The novel proteolytic mechanism of the 20S proteasome from T.acidophilumhas been investigated by Xray 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 β-subunits. The chromogenic substrate analogue Suc-LLVY-AMC binds in the same pocket of the proteolytically inactive T1A mutant of the β-subunit, but with a significantly altered geometry. The heavy-atom cluster Ta₆Br₁₂²⁺ used in Xray 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.