Mechanism of proteasome stimulation by 11S activators

The 20S proteasome is a complex machine that is responsible for the bulk of nonlysosomal intracellular proteolysis in eukaryotes. Its catalytic sites are sequestered in the interior of the cylindrical structure, and activity is tightly regulated at the level of substrate presentation and access. Purified proteasomes are inactive in vitro, but through association with activating complexes they gain the capacity to degrade substrate proteins and peptides. The crystal structure of the yeast proteasome-PA26 complex showed that the activator PA26 functions by opening an axial pore into the proteasome, apparently by introducing disorder in the normally closed pore region. In this dissertation, I address the questions of how the proteasome and activator interact, how 11S activators stimulate proteasomes from multiple diverse species, how the open pore conformation is stabilized, and the extent to which results from one particular type of activator or proteasome can be generalized. To this end, the structure of the yeast proteasome-PA26 structure was reanalyzed, and a cluster of four conserved residues was identified that stabilize an ordered sevenfold symmetric open gate conformation. I performed protein degradation assays with a different activator, PAN, and a number of archaeal Thermoplasma acidophilum proteasome mutants and showed that the four residues that stabilize the open gate in yeast are also important for PAN-dependent proteolysis by the archaeal proteasome. I also demonstrated a direct interaction between PA26 and the T. acidophilum proteasome and determined high resolution crystal structures of three proteasome-activator complexes. The wildtype complex revealed the presence of the open conformation in archaeal proteasomes. In contrast, the absence of the open conformation in two structures of mutant archaeal proteasomes deficient in the stabilizing residues bound to PA26 reinforces the importance of the stabilizing cluster and suggests that all proteasomes are activated through formation of the same open gate conformation. Finally, since PA26 interacts with proteasome through its C termini, I performed protein degradation assays with proteasomes mutated in their binding pockets and with C-terminally truncated PAN. The results of these experiments suggest that PAN, and by homology the eukaryotic activator PA700, might interact with proteasomes similar to PA26.