PROJECT SUMMARY/ABSTRACT Protein disaggregation and turnover are essential for protein homeostasis (proteostasis) and cell viability. Malfunction occurs during cell stress and aging, accelerating deleterious protein aggregation and amyloid formation. Improved mechanistic understanding is critical for determining how proteostasis pathways fail and for identifying therapeutic targets in preventing neurodegenerative disease and other protein mis-folding diseases. Heat shock protein (Hsp) 100 members of the conserved AAA+ family serve critical functions in all life as protein unfoldases and disaggregases. They form hexameric, ATP-driven machines that catalyze the translocation of polypeptide substrates through a central channel. The unfolded proteins are then refolded by Hsp molecular chaperones or degraded by an associated protease, such as in the case of the proteasome. Challenges in achieving structures of functional states have led to conflicting mechanistic models across the AAA+ superfamily. Focusing on conserved Hsp100 members, yeast Hsp104 and the bacterial Clp proteins, we have overcome these challenges by using cryo-electron microscopy to determine structures of biochemically defined, functional complexes. We determined the first substrate-bound structures of a AAA+ disaggregase (Hsp104) in distinct translocation states and discovered these machines operate by a rotary mechanism involving precise substrate gripping and release states and a two amino acid translocation step. Since our last submission of this application, we have determined multiple structures of the ClpAP AAA+ protease undergoing active substrate unfolding and proteolysis Together our discoveries reveal a new paradigm for how AAA+s mechanically unfold substrates. The next major question to address is: How is the translocation mechanism (which is now considered highly conserved among AAA+s) coupled to specific cellular functions? Our long-term goal is to determine how translocation and unfolding are precisely tuned for different proteostasis and cell stress response functions. The objective for this application is to identify key allosteric control mechanisms that couple ATP-driven translocation to substrate recognition, unfolding and degradation. Here we will: (SA1) Determine mechanisms of protein unfolding and proteolysis by the ClpAP “bacterial proteasome” complex; (SA2) Determine how Hsp104 interacts with and disaggregates native substrates and amyloids; and (SA3) Determine how the Hsp70 chaperone collaborates with Hsp104 to promote substrate loading. At the completion of this work we will identify conformational networks and protein:protein interactions that define how the core translocation cycle connects allosterically to specify distinct cellular functions of these AAA+ machines.