Conformational constraints for protein self-cleavage in the proteasome

Abstract

The proteasome is the central enzyme of protein degradation in the cytosol and the nucleus. It is involved in the removal of abnormal, misfolded or incorrectly assembled proteins, in the processing or degradation of transcriptional regulators in stress response, in degradation of cyclins in cell-cycle control, in the destruction of transcription factors or metabolic enzymes in cell differentiation and metabolic response, and in MHC class I mediated cellular immune response. By the analysis of the crystal and molecular structures of the 20 S proteasomes from the archaeon Thermoplasma acidophilum and from yeast it was shown that the β-type subunits in which the proteolytic activities reside are members of the N-terminal nucleophile (Ntn) protein family. They are synthesized as proproteins and become active by autoprocessing at a Gly - 1-Thr1 bond. The Thr1Ala mutant of subunit β1/Pre3 of the 20 S proteasome from yeast is unable to autolyse. Its crystal and molecular structure at 2.2 Å resolution described here shows that the pro-segment adopts a well-defined γ-turn conformation at Gyl - 1 and provides a first view at an autolysis site in Ntn hydrolases. The Gyl - 1 carbonyl oxygen displays two hydrogen bonds. The modelled Thr1 side-chain is located above the γ-turn bulge such that addition of its nucleophilic hydroxyl group to the electrophilic Gyl - 1 carbonyl carbon atom may proceed by very small motions. The pro-segment binding site and the catalytic site provide a rigid structural framework and appropriate hydrogen bond donors for this reaction. The same structure also supports addition of the Thr1 hydroxyl group to the carbonyl carbon atom of Leu - 2 as a model for the first step in substrate hydrolysis by the proteasome.