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 isresponsible for the regulated degradation of most cytosolic andnuclear proteins in eukaryotes. It consists of 28 subunits thatassemble into a tight complex with the general shape of a cylinderof four stacked rings of seven subunits each. The active sites aresequestered in a cavitity in the interior of the proteasome,inaccessible from the outside. Substrates are recognized andpresented to the proteasome by activators that bind to the ends ofthe cylinder. Recently, our lab showed that the activator PA26 opens>a pore into the yeast proteasome by inducing a symmetric openconformation in the N termini of the proteasomal alpha-subunits. Wecould not show was actually determines binding specificity betweenproteasome and activator because of poor data and the lack of symmetry in that region. I have nowcreated archaeal proteasome mutants that bind to the same activatorthat we used in our previous paper. The mutants individually mimickany of the binding pockets of the yeast proteasome. I expect to extend the resolution of the proteasome activator complex and obtain a clear view of the two features in PA26 implicatedin binding, the activation loop and the C-terminal tails, and theirinteraction with residues in the proteasome. I will greatly benefitfrom non-crystallographic symmetry averaging of the perfectlysevenfold symmetric complex.