Structural and Mechanistic Insights into the Yeast Disaggregase Hsp104

Hsp104 is a hexameric, AAA+ disaggregase from yeast, which couples ATP hydrolysis to remodeling diverse substrates ranging from amorphous aggregates to amyloid fibers. A mechanistic understanding of Hsp104\u27s substrate remodeling activities remains poorly defined. The hexamer undergoes large conformational changes upon ATP hydrolysis, but the details of these changes and how they are coupled to substrate remodeling are unresolved. The goals of this thesis were to elucidate low and high-resolution structural information about the Hsp104 hexamer and to discover new details of the mechanism of substrate remodeling. We used the in solution structural techniques small angle x-ray scattering and synchrotron x-ray footprinting, complemented by several biochemical assays, to elucidate novel roles for several Hsp104 domains, and to develop a comprehensive model for how the Hsp104 hexamer engages substrate and unleashes its remodeling capabilities. We discovered that the N-terminal domain (NTD) is involved in substrate binding, productive interactions with Hsp70, and an interface with nucleotide binding domain 1 (NBD1) and the middle domain (MD). We discovered a loop in NBD1 that may engage substrate in the ADP bound state to prevent premature substrate release, identified the region of the MD (helix 2) responsible and the mechanism of signal transmission between NBD1 and NBD2, and confirmed the validity of a hexameric model of the NBD2 domain. Hsp104 engages substrate in the ATP-bound state. We have found that in this state Hsp104 displays an increase in rigidity, which we propose as a pre-payment of the entropic cost of substrate binding. Initial substrate engagement in the NTD and NBD1, as well as Hsp70 interactions at the NTD:NBD1:MD interface, serve to `prime the pump\u27. These interactions result in large conformational changes of the MD, specifically in helix 2, which spans the entirety of the domain. These conformational changes increase MD dynamics, partially releasing MD:NBD2 contacts, and allow signal transmission between NBD1 and NBD2. As NBD2 responds to these signals, a positive feedback loop is created. Further nucleotide binding in NBD2 stimulates ATP hydrolysis in NBD1, and substrate is remodeled by iterative binding events and peristaltic motions of the Hsp104 hexamer channel