Conformational changes in the 20S proteasome upon macromolecular ligand binding analyzed with monoclonal antibodies

Proteasomes interact with a variety of macromolecular ligands that modulate their ability to degrade peptide and protein substrates. The effector PA28 increases the peptidase activities of proteasomes whereas HSP90 and alpha-crystallin inhibit a peptide-hydrolyzing activity. Four monoclonal antibodies were used as probes to detect conformational changes of proteasome subunits. Conformational changes in alpha- or beta-subunits were found upon binding PA28, HSP90, alpha-crystallin, and the substrate casein but not with the peptide substrate analogs calpain inhibitor 1 (Ac-Leu-Leu-norleucinal), calpain inhibitor 2 (Ac-Leu-Leu-methioninal), or MG 132 (N-Cbz-Leu-Leu-leucinal).