The proteasome activator PA200 binds to the catalytic core of both standard proteasome (s20S) and the immunoproteasome (i20S); however, whether PA200 uses the same mechanisms to activate i20S remains unknown. In this work, the cryo-EM structures of singly- and doubly-capped i20S-PA200 complexes, combined with complementary in vitro biochemical assays, show that binding of the first PA200 induces allosteric bending of the i20S and widens the opposite alpha-ring, promoting higher PA200 occupancy and stronger activation compared to the s20S. PA200 also selectively modulates i20S proteolytic activity by enhancing peptide production and shifting cleavage specificity toward caspase-like activity. In cells and tissues co-expressing PA200, s20S, and i20S, PA200 preferentially associates with the i20S. Moreover, PA200 and i20S catalytic subunits are differentially regulated, with PA200 playing a potential role in regulating the i20S subunits' expression. Overall, these findings suggest that PA200 contributes to the regulation of i20S function and dynamics.
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