Minimal mechanistic component of HbYX-dependent proteasome activation.

The implication of reduced proteasomal function in neurodegenerative diseases combined with numerous studies showing the protective effects of increasing proteasome activity in animal models justify the need to understand how the proteasome is activated for protein degradation. The C-terminal HbYX motif is present on many proteasome binding proteins and functions to tether activators to the 20S core particle. Peptides with a HbYX motif can also autonomously activate 20S gate-opening to allow protein degradation, but the underlying allosteric molecular mechanism is not clear. We designed a HbYX-like dipeptide mimetic that represents only the fundamental components of the HbYX motif to allow rigorous elucidation of the underlying molecular mechanisms of HbYX induced 20S gate-opening in the archaeal and mamalian proteasome. By generating several high-resolution cryo-EM structures (e.g. 1.9Å) we identified multiple proteasome α subunit residues involved in HbYX-dependent activation and the conformational changes involved in gate-opening. In addition, we generated mutants probing these structural findings and identified specific point mutations that strongly activate the proteasome by partially mimicking a HbYX-bound state. These structures resolve 3 novel mechanistic features that are critical for allosteric α subunit conformational changes that ultimately trigger gate-opening: 1) rearrangement of the loop adjacent to K66, 2) inter- and intra- α subunit conformational changes and 3) a pair of IT residues on the α N-terminus in the 20S channel that alternate binding sites to stabilize the open and closed states. All gate-opening mechanisms appear to converge on this "IT switch". When stimulated by the mimetic, the human 20S can degrade unfolded proteins such as tau, and prevent proteasomal inhibition by toxic soluble oligomers. Collectively, the results presented here provide a mechanistic model of HbYX-dependent 20S gate-opening and offer proof of concept for the robust potential of HbYX-like small molecules to stimulate proteasome function, which could be useful to treat neurodegenerative diseases.