Design principles to tailor Hsp104 therapeutics

The hexameric AAA+ disaggregase, Hsp104, collaborates with Hsp70 and Hsp40 via its autoregulatory middle domain (MD) to solubilize aggregated proteins. However, how ATP- or ADP-specific MD configurations regulate Hsp104 hexamers remains poorly understood. Here, we define an ATP-specific network of interprotomer contacts between nucleotide-binding domain 1 (NBD1) and MD helix L1, which tunes Hsp70 collaboration. Manipulating this network can (1) reduce Hsp70 collaboration without enhancing activity, (2) generate Hsp104 hypomorphs that collaborate selectively with class B Hsp40s, (3) produce Hsp70-independent potentiated variants, or (4) create species barriers between Hsp104 and Hsp70. Conversely, ADP-specific intraprotomer contacts between MD helix L2 and NBD1 restrict activity, and their perturbation frequently potentiates Hsp104. Importantly, adjusting an NBD1:MD helix L1 rheostat via rational design enables finely tuned collaboration with Hsp70 to safely potentiate Hsp104, minimize off-target toxicity, and counteract FUS and TDP-43 proteinopathies in human cells. Thus, we establish design principles to tailor Hsp104 therapeutics. [Display omitted] •ATP-specific NBD1-MD contacts in Hsp104 regulate collaboration with Hsp70•ADP-specific NBD1-MD contacts in Hsp104 restrict activity•Rational design to tune Hsp70 collaboration and safely enhance Hsp104 activity•Safely enhanced Hsp104 variants reduce FUS and TDP-43 proteinopathies in human cells In this paper, Lin et al. establish design principles for therapeutic Hsp104 variants. By leveraging high-resolution structures of the Hsp104 disaggregase, they rationally engineer enhanced disaggregases with retuned Hsp70 collaboration and minimal off-target unfoldase activity. These safely enhanced Hsp104 variants effectively mitigate FUS and TDP-43 proteinopathies in human cells.