Crowding Activates ClpB and Enhances Its Association with DnaK for Efficient Protein Aggregate Reactivation

Abstract

Reactivation of intracellular protein aggregates after a severe stress is mandatory for cell survival. In bacteria, this activity depends on the collaboration between the DnaK system and ClpB, which in vivo occurs in a highly crowded environment. The reactivation reaction includes two steps: extraction of unfolded monomers from the aggregate and their subsequent refolding into the native conformation. Both steps might be compromised by excluded volume conditions that would favor aggregation of unstable protein folding intermediates. Here, we have investigated whether ClpB and the DnaK system are able to compensate this unproductive effect and efficiently reactivate aggregates of three different substrate proteins under crowding conditions. To this aim, we have compared the association equilibrium, biochemical properties, stability, and chaperone activity of the disaggregase ClpB in the absence and presence of an inert macromolecular crowding agent. Our data show that crowding i), increases three to four orders of magnitude the association constant of the functional hexamer; ii), shifts the conformational equilibrium of the protein monomer toward a compact state; iii), stimulates its ATPase activity; and iv), favors association of the chaperone with substrate proteins and with aggregate-bound DnaK. These effects strongly enhance protein aggregate reactivation by the DnaK-ClpB network, highlighting the importance of volume exclusion in complex processes in which several proteins have to work in a sequential manner.

Sedimentation Equilibrium Analysis of ClpB Self-Association in Diluted and Crowded Solutions

“…Volume exclusion is also predicted to result in the acceleration of slow, transition-state-limited association reactions and the deceleration of fast, diffusion-limited association reactions (Zhou et al, 2008). It is furthermore anticipated that crowding will also lead to phase separation phenomena that can affect significantly the spatial arrangement and local distribution of ClpB complexes (Martin et al, 2014). Finally, crowding can contribute to the maintenance of functional activity of essential protein-nucleic acid and protein-protein complexes in Escherichia coli against large changes in the osmolarity of its environment (Cayley & Record, 2004).…”

“…The results obtained in the presence of Ficoll demonstrate that excluded volume effects shifted the ClpB association equilibrium toward the functional hexamer at salt conditions that otherwise would promote chaperone dissociation in dilute solutions, and that the N-terminal domain of the protein does not significantly alter this behavior. Shifting of the association equilibrium of ClpB toward the hexamer has important consequences in the activity of this hexameric disaggregase, as it also stimulates the ATPase activity of ClpB and promotes its interaction with substrate proteins and with aggregate-bound DnaK (Martin et al, 2014). These effects strongly enhance protein aggregate reactivation by the DnaK-ClpB network, highlighting the importance of volume exclusion in the regulation of the oligomerization state of ClpB, which in turn modulates its association with DnaK, a requirement to fulfill its biological function.…”

“…A detailed description of the production of wild-type ClpB and deletion mutants ClpB Δ(410-455) and ClpB Δ(410-520) in which the M domain was partially or totally deleted, respectively, and ClpB Δ(1-142) , which lacks the N-terminal domain, has already been described (del Castillo et al, 2011;Martin et al, 2014). All the experiments were done in 50 mM Tris-HCl, pH 7.5, 5 mM MgCl 2 buffer, containing different KCl concentrations (20-500 mM), in the absence or in the presence of 1 mM nucleotide (ADP or ATP).…”

Chaperone-assisted protein aggregate reactivation: Different solutions for the same problem

“…Volume exclusion generated by macromolecular crowding provides a non-specific force that facilitates processes leading to a reduction in excluded volume, namely protein compaction, association and aggregation [93], and thus could challenge the protein aggregate reactivation ability of the Hsp100/Hsp70 complex. A recent study showed that chaperones could reactivate aggregated proteins even more efficiently in a crowded and non-confined environment than in dilute conditions [94]. To explain this behavior, the sequential binding of chaperones to the aggregate surface must be considered.…”

“…Binding of the DnaK system and ClpB to the aggregate proceeds in two steps: in the first one, aggregate-bound DnaJ recruits DnaK molecules that in the second step drive the association of ClpB [23,26,27]. Crowding i) shifts the association equilibrium of ClpB toward the functional hexamer able to bind DnaK; ii) accelerates the functional cycle of the disaggregase; and iii) enhances the affinity of ClpB for aggregate-bound DnaK [94]. Therefore, crowding favors formation of chaperone functional complexes at the aggregate surface, and at the same time activates them so that they can exert more work to pull unfolded substrate molecules out of the aggregate during the reactivation reaction.…”