Abstract
The 70-kDa heat shock proteins (Hsp70s) assist in protein folding through allosteric communication between their nucleotide-binding domains (NBDs) and substrate-binding domains (SBDs), which are connected by an interdomain linker. Their nucleotide-dependent allosteric cycle is modulated by ligand binding and co-chaperones, including nucleotide exchange factors (NEFs). GrpE, the NEF for the E. coli Hsp70, DnaK, has been proposed to have a dual effect on the chaperone, facilitating the exchange of ADP for ATP in the NBD 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 expanded on these findings and obtained new evidence for 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. Moreover, we identified the DnaK binding motif in GrpE's N-terminal disordered tails as (17)IIM(19), which is conserved across many bacterial species. 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, and thus increasing DnaK affinity to substrates.