STRUCTURAL BASIS FOR ACTIVATION OF PROTEASOMES BY REG

University Of Utah, Salt Lake City UT

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Abstract

Intracellular proteolysis by the 20S proteasome is central to numerous aspects of cellular metabolism, including regulatory processes such as cell cycle control and the production of antigenic peptides. The 20S proteasome is relatively inactive by itself, since the proteolytic active sites are sequestered in a central chamber that is not accessible in the structure of unliganded proteasome. Proteasomes are activated by complexes that bind to one or both ends of the cylindrical structure, including the 11S regulator (REG), which activates proteasome for the production of antigenic peptides suitable for display on class I MHC molecules. We have already made considerable progress in the study of REG, including crystal structure determination of the 200 kDa recombinant human REGalpha homolog. This is the only proteasome activator whose structure is known. Currently, no crystal structures have been reported for a proteasome/activator complex. This proposal aims to understand the biochemical basis of proteasome activation by REG, including the structure determination of a 1.1 MDa proteasome/REG complex. Crystals have been grown and data collected to 4.4 Angstrom units resolution. A heavy atom derivative has been obtained and the structure has been solved at low resolution. These crystals show good prospects for heavy atom derivatization and density modification, suggesting that the electron density map will reveal important details of the REG/proteasome interaction, even at relatively low resolution. The preliminary low resolution structure determination is already being used in the design of mutagenesis experiments to define the role of individual residues in binding, activation, and homolog-specific effects. Finally, we will solve the structures of inhibitor bound complexes in an effort to determine how proteasome binds substrates and how REG activates proteasome to produce peptides suitable for display by class I MHC molecules.