The nucleotide exchange factor, GrpE, modulates substrate affinity by interaction of its N-terminal tails with the DnaK substrate–binding domain

The 70-kDa heat shock proteins assist in protein folding through allosteric communication between their nucleotide-binding domains and substrate-binding domains (SBDs), which are connected by an interdomain linker. Their nucleotide-dependent allosteric cycle is modulated by ligand binding and cochaperones, including nucleotide exchange factors. GrpE, the nucleotide exchange factor for the Escherichia coli 70-kDa heat shock protein, DnaK, has been proposed to have a dual effect on the chaperone, facilitating the exchange of ADP for ATP in the nucleotide-binding domain in a temperature-dependent fashion and promoting substrate release from the SBD. We recently reported NMR-based evidence that GrpE binding to DnaK has a direct structural effect on the SBD. Here, we built on these findings and provide new evidence supporting a model in which the disordered N-terminal tails of GrpE facilitate peptide dissociation from the nucleotide-free DnaK-GrpE complex by transiently binding to the canonical substrate-binding site in the SBD. This GrpE-SBD interaction, while weak, is favored by the high local concentration of the tails around the SBD after complex formation and provides a direct mechanism to facilitate substrate release in addition to the potential more indirect allosteric mechanism arising from a GrpE-SBD conformational shift. Moreover, we identified the DnaK binding motif in GrpE's N-terminal disordered tails as ¹⁷IIM¹⁹, which is highly conserved in bacteria. Excitingly, our data further suggest a mechanism for the temperature dependence of GrpE's modulation of DnaK's refolding activity: as the temperature increases, unfolding of GrpE's coiled-coil weakens its contacts with the SBD, reducing N-terminal tail binding, thus increasing the affinity of DnaK to substrates.