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R318 --> 496.2 --> Impairment of proteasomal function has been implicated in neurodegenerative diseases, justifying the need to understand how the proteasome is activated for protein degradation. Here, using biochemical and structural (cryo‐EM) strategies in both archaeal and mammalian proteasomes, we further determine the HbYX(‐motif)‐dependent mechanism of proteasomal activation used by multiple proteasome‐activating complexes including the 19S Particle. We identify multiple proteasome α subunit residues involved in HbYX‐dependent activation, a point mutation that activates the proteasome by partially mimicking a HbYX‐bound state, and conformational changes involved in gate‐opening with a 2.1A structure. Through an iterative process of peptide synthesis, we successfully design a HbYX‐like dipeptide mimetic as a robust tool to elucidate how the motif autonomously activates the proteasome. The mimetic induces near complete gate‐opening at saturating concentration, activating mammalian proteasomal degradation of peptides and proteins. Findings using our peptide mimetic suggest the HbYX‐dependent mechanism requires cooperative binding in at least two intersubunit pockets of the α ring. Collectively, the results presented here unambiguously demonstrate the lone role of the HbYX tyrosine in the allosteric mechanism of proteasome activation and offer proof of concept for the robust potential of HbYX‐like small molecules to activate the proteasome. [ABSTRACT FROM AUTHOR]