Modulation of the Chaperone DnaK Allosterism by the Nucleotide Exchange Factor GrpE

Hsp70 chaperones comprise two domains, the nucleotide-binding domain (Hsp70(NBD)), responsible for structural and functional changes in the chaperone, and the substrate-binding domain (Hsp70(SBD)), involved in substrate interaction. Substrate binding and release in Hsp70 is controlled by the nucleotide state of DnaK(NBD), with ATP inducing the open, substrate-receptive DnaK(SBD) conformation, whereas ADP forces its closure. DnaK cycles between the two conformations through interaction with two cofactors, the Hsp40 co-chaperones (DnaJ in Escherichia coli) induce the ADP state, and the nucleotide exchange factors (GrpE in E. coli) induce the ATP state. X-ray crystallography showed that the GrpE dimer is a nucleotide exchange factor that works by interaction of one of its monomers with DnaK(NBD). DnaK(SBD) location in this complex is debated; there is evidence that it interacts with the GrpE N-terminal disordered region, far from DnaK(NBD). Although we confirmed this interaction using biochemical and biophysical techniques, our EM-based three-dimensional reconstruction of the DnaK-GrpE complex located DnaK(SBD) near DnaK(NBD). This apparent discrepancy between the functional and structural results is explained by our finding that the tail region of the GrpE dimer in the DnaK-GrpE complex bends and its tip contacts DnaK(SBD), whereas the DnaK(NBD)-DnaK(SBD) linker contacts the GrpE helical region. We suggest that these interactions define a more complex role for GrpE in the control of DnaK function.