Nanoenzymology of the 20S proteasome: proteasomal actions are controlled by the allosteric transition.
The proteasome is a major cytosolic proteolytic assembly, essential for the physiology of eukaryotic cells. Both the architecture and enzymatic properties of the 20S proteasome are relatively well understood. However, despite longstanding interest, the integration of structural and functional properties of the proteasome into a coherent model explaining the mechanism of its enzymatic actions has been difficult. Recently, we used tapping mode atomic force microscopy (AFM) in liquid to demonstrate that the alpha-rings of the proteasome imaged in a top-view position repeatedly switched between their open and closed conformations, apparently to control access to the central channel. Here, we show with AFM that the molecules in a side-view position acquired two stable conformations. The overall shapes of the 20S particles were classified as either barrel-like or cylinder-like. The relative abundance of the two conformers depended on the nature of their interactions with ligands. Similarly to the closed molecules in top view, the barrels predominated in control or inhibited molecules. The cylinders and open molecules prevailed when the proteasome was observed in the presence of peptide substrates. Based on these data, we developed the two-state model of allosteric transitions to explain the dynamics of proteasomal structure. This model helps to better understand the observed properties of the 20S molecule, and sets foundations for further studies of the structural dynamics of the proteasome.
Osmulski
Pawel A
PA
Institute of Biotechnology, University of Texas Health Science Center at San Antonio, 15355 Lambda Drive, San Antonio, Texas 78245, USA.
Gaczynska
Maria
M
D000494:Allosteric Regulation / Q000502:physiology; D003546:Cysteine Endopeptidases / Q000378:metabolism / Q000648:ultrastructure*; D003952:Diagnostic Imaging / Q000379:methods; D018625:Microscopy, Atomic Force / Q000379:methods; D009097:Multienzyme Complexes / Q000378:metabolism / Q000648:ultrastructure*; D036103:Nanotechnology; D009842:Oligopeptides / Q000378:metabolism*; D010447:Peptide Hydrolases / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex; D011487:Protein Conformation; D012568:Schizosaccharomyces / Q000201:enzymology* / Q000648:ultrastructure
D009097:Multienzyme Complexes; D009842:Oligopeptides; D010447:Peptide Hydrolases; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex