Cell survival under severe thermal stress requires the activity of the ClpB (Hsp104) AAA+ chaperone that solubilizes and reactivates aggregated proteins in concert with the DnaK (Hsp70) chaperone system. How protein disaggregation is achieved and whether survival is solely dependent on ClpB-mediated elimination of aggregates or also on reactivation of aggregated proteins has been unclear. We engineered a ClpB variant, BAP, which associates with the ClpP peptidase and thereby is converted into a degrading disaggregase. BAP translocates substrates through its central pore directly into ClpP for degradation. ClpB-dependent translocation is demonstrated to be an integral part of the disaggregation mechanism. Protein disaggregation by the BAP/ClpP complex remains dependent on DnaK, defining a role for DnaK at early stages of the disaggregation reaction. The activity switch of BAP to a degrading disaggregase does not support thermotolerance development, demonstrating that cell survival during severe thermal stress requires reactivation of aggregated proteins.

ClpB of Escherichia coli is an ATP-dependent ringforming chaperone that mediates the resolubilization of aggregated proteins in cooperation with the DnaK chaperone system. ClpB belongs to the Hsp100/Clp subfamily of AAA؉ proteins and is composed of an N-terminal domain and two AAA-domains that are separated by a "linker" region. Here we present a detailed structurefunction analysis of ClpB, dissecting the individual roles of ClpB domains and conserved motifs in oligomerization, ATP hydrolysis, and chaperone activity. Our results show that ClpB oligomerization is strictly dependent on the presence of the C-terminal domain of the second AAA-domain, while ATP binding to the first AAAdomains stabilized the ClpB oligomer. Analysis of mutants of conserved residues in Walker A and B and sensor 2 motifs revealed that both AAA-domains contribute to the basal ATPase activity of ClpB and communicate in a complex manner. Chaperone activity strictly depends on ClpB oligomerization and the presence of a residual ATPase activity. The N-domain is dispensable for oligomerization and for the disaggregating activity in vitro and in vivo. In contrast the presence of the linker region, although not involved in oligomerization, is essential for ClpB chaperone activity.

The AAA+ protein ClpB cooperates with the DnaK chaperone system to solubilize and refold proteins from an aggregated state. The substrate-binding site of ClpB and the mechanism of ClpB-dependent protein disaggregation are largely unknown. Here we identified a substrate-binding site of ClpB that is located at the central pore of the first AAA domain. The conserved Tyr251 residue that lines the central pore contributes to substrate binding and its crucial role was confirmed by mutational analysis and direct crosslinking to substrates. Because the positioning of an aromatic residue at the central pore is conserved in many AAA+ proteins, a central substrate-binding site involving this residue may be a common feature of this protein family. The location of the identified binding site also suggests a possible translocation mechanism as an integral part of the ClpB-dependent disaggregation reaction.

TorsinA is a membrane-associated AAA+ (ATPases associated with a variety of cellular activities) ATPase implicated in primary dystonia, an autosomal-dominant movement disorder. We reconstituted TorsinA and its cofactors in vitro and show that TorsinA does not display ATPase activity in isolation; ATP hydrolysis is induced upon association with LAP1 and LULL1, type II transmembrane proteins residing in the nuclear envelope and endoplasmic reticulum. This interaction requires TorsinA to be in the ATP-bound state, and can be attributed to the luminal domains of LAP1 and LULL1. This ATPase activator function controls the activities of other members of the Torsin family in distinct fashion, leading to an acceleration of the hydrolysis step by up to two orders of magnitude. The dystonia-causing mutant of TorsinA is defective in this activation mechanism, suggesting a loss-of-function mechanism for this congenital disorder.DYT1 dystonia | LINC complex | nuclear egress