Structural studies of the proteasome activator Blm10

The 20S proteasome is an intracellular protease found in all kingdoms of life. In eukaryotes, the ubiquitin-proteasome is a major proteolytic pathway involved in the turn-over of cytosolic and nuclear proteins and controls many cellular processes. The 20S recruits one of three classes of proteasome activators that modulate and enforces its function: the PA700, the PA28 and the PA200/Blm10 families. In contrast to PA700 and PA28 that function by binding to the 20S as oligomeric structures, PA200/Blm10 binds to the 20S as a single polypeptide chain of about 250kDa.;To further advance understanding of Blm10 function, we determined the crystal structure of Blm10:20S complex at 3.4A resolution. Blm10 is a HEAT-repeat containing protein that folds into a left-handed solenoid burying a large solvent cavity over the 20S a subunits. Blm10 contains a pore large enough to allow passage of small peptides explaining the biochemical activity of Blm10:20S complex. Binding to the 20S occurs through many loops from different HEAT repeats and also by Blm10's single C-terminal carboxylate that forms a salt bridge with a conserved lysine of 20S. Intriguingly, the penultimate residue of Blm10 is a conserved tyrosine that opens the gate locally at the proline reverse turn of alpha-5. This repositioning at alpha-5 is propagated to the neighboring subunits: alpha-6, 7, and 1 triggering their open conformation while disordering the gate at alpha-2, 3, and 4. Though the partial disordering of the 20S gate seems to be unique to Bim10, the penultimate tyrosine of Blm10 is shared by subunits of the PA700 activator and provides a model for proteasome activation by ATPase activators.;This dissertation focuses on structural studies of the Blm10 proteasome activator from the budding yeast S. cerevisiae. The 18A cryo-EM reconstruction of Blm10:20S complex shows that Blm10 is a dome-shaped protein that binds to the ends of the proteasome and opens its gate. The similarity of the cryo-EM structures of Blm10 and its bovine homologue, PA200, suggested a similar biological function; however, the precise biological role of PA200/Blm10 remains to be determined.