Abstract
The chaperone ClpB in bacteria is responsible for the reactivation of aggregated proteins in collaboration with the DnaK system. Association of these chaperones at the aggregate surface stimulates ATP hydrolysis, which mediates substrate remodeling. However, a question that remains unanswered is whether the bichaperone complex can be selectively activated by substrates that require remodeling. We find that large aggregates or bulky, native-like substrates activates the complex, whereas a smaller, permanently unfolded protein or extended, short peptides fail to stimulate it. Our data also indicate that ClpB interacts differently with DnaK in the presence of aggregates or small peptides, displaying a higher affinity for aggregate-bound DnaK, and that DnaK-ClpB collaboration requires the coupled ATPase-dependent remodeling activities of both chaperones. Complex stimulation is mediated by residues at the β subdomain of DnaK substrate binding domain, which become accessible to the disaggregase when the lid is allosterically detached from the β subdomain. Complex activation also requires an active NBD2 and the integrity of the M domain-ring of ClpB. Disruption of the M-domain ring allows the unproductive stimulation of the DnaK-ClpB complex in solution. The ability of the DnaK-ClpB complex to discrimínate different substrate proteins might allow its activation when client proteins require remodeling.
Highlights
DnaK, as other members of the Hsp[70] family, participates in diverse cellular functions, including protein folding, disaggregation and disassembly of specific complexes
Previous studies have shown that activation of the DnaK-ClpB complex occurs at the aggregate surface, where it provides the energy for substrate remodeling[10,29,30]
This work explores how client proteins modulate the activation of the bichaperone complex and the structural elements of both proteins involved in this process
Summary
DnaK, as other members of the Hsp[70] family, participates in diverse cellular functions, including protein folding, disaggregation and disassembly of specific complexes. When the substrate is a large, natively folded protein or a protein aggregate, the lid cannot close onto the β-sandwich, whereas it does when the client is a small peptide or a fully extended polypeptide[25,26,27] These conformations might not be the only states that the SBD can adopt, and others with intermediate degrees of lid closure could exist depending on the properties of the bound substrates. Our data show that the bichaperone complex is only stimulated in the presence of protein aggregates or bulky, native-like substrates that require remodeling, and not by a smaller, permanently unfolded proteins or short peptides This substrate dependence might be a sophisticated way to regulate protein remodeling by this chaperone complex in vivo. The DnaK region responsible for activation is located at the flexible β-SBD, where the substrate binding site is located, whose accessibility to ClpB is controlled by the allosteric displacement of the lid. The interaction between this DnaK region and ClpB could help substrate hand-off from DnaK to ClpB
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