Crowding Modulates the Conformation, Affinity, and Activity of the Components of the Bacterial Disaggregase Machinery

Chaperone-mediated protein aggregate reactivation is a complex reaction that depends on the sequential association of molecular chaperones on their interaction with protein aggregates and on substrate refolding. This process could be modulated by the highly crowded intracellular environment, which is known to affect protein conformational change, enzymatic activity, and protein–protein interactions. Here, we report that molecular crowding shapes the chaperone activity of bacterial disaggregase composed of the DnaK system (DnaK, DnaJ, and GrpE) and the molecular motor ClpB. A combination of biophysical and biochemical methods shows that the excluded volume conditions modify the conformation of DnaK and DnaJ without affecting that of GrpE. These crowding-induced conformational rearrangements activate DnaK, enhance the affinity of DnaK for DnaJ, but not for GrpE, and increase the sensitivity of the chaperone activity to cochaperone concentration, explaining the tight control of their relative intracellular amounts. Furthermore, crowding-mediated disordering of the G/F domain of DnaJ facilitates the reversible formation of intermolecular DnaJ conglomerates. These assemblies could drive the formation of Hsp70 clusters at the aggregate surface with the consequent enhancement of the disaggregation efficiency through their coordinated action via entropic pulling. Finally, crowding helps ClpB to outcompete GrpE for DnaK binding, a key aspect of DnaK/ClpB cooperation given the low affinity of the disaggregase for DnaK. Excluded volume conditions promote the formation of the bichaperone complex that disentangles aggregates, enhancing the efficiency of the disaggregation reaction. [Display omitted] •Protein aggregate reactivation involved the sequential association of different proteins.•Crowding selectively modifies the conformation and affinity of these proteins.•Crowding promotes the reversible formation of DnaJ conglomerates.•Crowding helps ClpB to outcompete GrpE for DnaK binding.•Excluded volume conditions shapes bacterial disaggregase activity.