Mechanistic and Structural Insights into the Prion-Disaggregase Activity of Hsp104.

Hsp104 is a dynamic ring translocase and hexameric AAA+ protein found in yeast, which couples ATP hydrolysis to disassembly and reactivation of proteins trapped in soluble preamyloid oligomers, disordered protein aggregates, and stable amyloid or prion conformers. Here, we highlight advances in our structural understanding of Hsp104 and how Hsp104 deconstructs Sup35 prions. Although the atomic structure of Hsp104 hexamers remains uncertain, volumetric reconstruction of Hsp104 hexamers in ATPγS, ADP-AlFx (ATP hydrolysis transition-state mimic), and ADP via small-angle x-ray scattering has revealed a peristaltic pumping motion upon ATP hydrolysis. This pumping motion likely drives directional substrate translocation across the central Hsp104 channel. Hsp104 initially engages Sup35 prions immediately C-terminal to their cross-β structure. Directional pulling by Hsp104 then resolves N-terminal cross-β structure in a stepwise manner. First, Hsp104 fragments the prion. Second, Hsp104 unfolds cross-β structure. Third, Hsp104 releases soluble Sup35. Deletion of the Hsp104 N-terminal domain yields a hypomorphic disaggregase, Hsp104(∆N), with an altered pumping mechanism. Hsp104(∆N) fragments Sup35 prions without unfolding cross-β structure or releasing soluble Sup35. Moreover, Hsp104(∆N) activity cannot be enhanced by mutations in the middle domain that potentiate disaggregase activity. Thus, the N-terminal domain is critical for the full repertoire of Hsp104 activities.