Thermotolerance Requires Refolding of Aggregated Proteins by Substrate Translocation through the Central Pore of ClpB

TLDR

Cell survival under severe thermal stress requires the ClpB (Hsp104) AAA+ chaperone, which solubilizes and reactivates aggregated proteins together with the DnaK (Hsp70) system. The study aims to determine whether ClpB-mediated disaggregation alone ensures survival or whether reactivation of aggregated proteins is also required, by engineering a BAP variant that converts ClpB into a degrading disaggregase. BAP is a ClpB variant that binds ClpP and translocates substrates through its central pore directly into ClpP for degradation. The study shows that ClpB-dependent substrate translocation into ClpP is essential for disaggregation, that this process still requires DnaK at early stages, and that converting ClpB into a degrading disaggregase fails to confer thermotolerance, indicating that reactivation of aggregated proteins is necessary for survival.

Abstract

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.