ARCHAEAL 20S MUTANTS COMPLEXED TO PA26

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The proteasome is a large multi-subunit protease that is responsible for the regulated degradation of most cytosolic and nuclear proteins in eukaryotes. It consists of 28 subunits that assemble into a tight complex with the general shape of a cylinder of four stacked rings of seven subunits each. The active sites are sequestered in a cavitity in the interior of the proteasome, inaccessible from the outside. Substrates are recognized and presented to the proteasome by activators that bind to the ends of the cylinder. Recently, our lab showed that the activator PA26 opens>a pore into the yeast proteasome by inducing a symmetric open conformation in the N termini of the proteasomal alpha-subunits. We could not show was actually determines binding specificity between proteasome and activator because of poor data and the lack of symmetry in that region. I have now created archaeal proteasome mutants that bind to the same activator that we used in our previous paper. The mutants individually mimick any of the binding pockets of the yeast proteasome. Current work is to extend the resolution of the proteasome activator complex and obtain a clear view of the two features in PA26 implicated in binding, the activation loop and the C-terminal tails, and their interaction with residues in the proteasome. I will greatly benefit from non-crystallographic symmetry averaging of the perfectly sevenfold symmetric complex.