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. 2015 Apr 17;290(16):10083-92.
doi: 10.1074/jbc.M114.623371. Epub 2015 Mar 4.

Modulation of the chaperone DnaK allosterism by the nucleotide exchange factor GrpE

Roberto Melero  1 , Fernando Moro  2 , María Ángeles Pérez-Calvo  3 , Judit Perales-Calvo  2 , Lucía Quintana-Gallardo  3 , Oscar Llorca  4 , Arturo Muga  5 , José María Valpuesta  6
Affiliations

Modulation of the chaperone DnaK allosterism by the nucleotide exchange factor GrpE

Roberto Melero et al. J Biol Chem. .

Abstract

Hsp70 chaperones comprise two domains, the nucleotide-binding domain (Hsp70NBD), responsible for structural and functional changes in the chaperone, and the substrate-binding domain (Hsp70SBD), involved in substrate interaction. Substrate binding and release in Hsp70 is controlled by the nucleotide state of DnaKNBD, with ATP inducing the open, substrate-receptive DnaKSBD 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 DnaKNBD. DnaKSBD location in this complex is debated; there is evidence that it interacts with the GrpE N-terminal disordered region, far from DnaKNBD. Although we confirmed this interaction using biochemical and biophysical techniques, our EM-based three-dimensional reconstruction of the DnaK-GrpE complex located DnaKSBD near DnaKNBD. 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 DnaKSBD, whereas the DnaKNBD-DnaKSBD linker contacts the GrpE helical region. We suggest that these interactions define a more complex role for GrpE in the control of DnaK function.

Keywords: 70-Kilodalton Heat Shock Protein (Hsp70); Chaperone; Chaperone DnaK (DnaK); Electron Microscopy (EM); GrpE; Nucleotide Exchange Factor; Protein Folding.

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Figures

FIGURE 1.
FIGURE 1.
Crystal structures of DnaK, GrpE, and DnaKNBD-GrpE. A, crystal structure of DnaK (PDB code 2KHO). In both the crystal structure (bottom) and the sequence (top), the DnaKNBD and DnaKSBD domains are connected by a linker. B, crystal structure of GrpE. Top, scheme of the GrpE sequence; bottom, crystal structure of the GrpE dimer (extracted from PDB code 1DKG), showing the two structural regions (head and tail). C, crystal structure of the DnaKNBD-GrpE complex (PDB code 1DKG).
FIGURE 2.
FIGURE 2.
Purification of the DnaK-GrpE complexes. A, native gel showing the formation of the DnaK-GrpE complexes. DnaK is incubated alone or with GrpE, GrpE(34–197) or GrpE(69–197). B, native gel showing DnaKR151A-GrpE complex formation. DnaKR151A is incubated alone or with GrpE, GrpE(34–197), or GrpE(69–197).
FIGURE 3.
FIGURE 3.
Structure of the DnaKNBD-GrpE complex. A, two-dimensional reference-free averages of DnaKNBD·GrpE. The top image shows the two structural regions of the complex, the head and the tail. B, three orthogonal views of the three-dimensional reconstruction of DnaKNBD·GrpE. C, the same three views with docking of the atomic structure of the DnaKNBD-GrpE complex (PDB code 1DKG) (9) into the three-dimensional reconstruction of the same complex. D, the same docking after a flexible fitting on the same atomic structure. Bars in A and B = 50 Å.
FIGURE 4.
FIGURE 4.
Structure of the DnaK-GrpE and DnaKR151A-GrpE complexes. A, two-dimensional reference-free averages of the DnaK-GrpE corresponding to the two main populations obtained after three-dimensional classification. B, location of DnaKSBD in the DnaK-GrpE structure. (i) Side view of one conformation of DnaK-GrpE. (ii) The same view with the difference map between the three-dimensional reconstructions of DnaK-GrpE and DnaKNBD-GrpE (green mesh) docked into the structure. (iii) The same view with the atomic structure of DnaKSBD (PDB code 1DKG; yellow) docked in the difference map. C, three orthogonal views of the structure of the two conformations. The atomic structure of DnaKNBD-GrpE with flexible fitting as in Fig. 1C, and that of DnaKSBD as shown in B, are docked into the two three-dimensional reconstructions. Asterisks indicate the area where the DnaKNBD-DnaKSBD linker is predicted to be located. D, two-dimensional reference-free average of DnaKR151A-GrpE. In addition to the two structural regions described above (head and tail), an extra thin mass protrudes from the head (arrow). E, three orthogonal views of the structure of DnaKR151A-GrpE. F, side view of DnaKR151A-GrpE with the difference map between the three-dimensional reconstructions of DnaKR151A-GrpE and DnaKNBD-GrpE complexes (green mesh) docked into the structure. G, the same three views as in E, with the atomic structure of the DnaKNBD-GrpE complex with flexible fitting, and that of DnaKSBD as shown in B, are docked into the three-dimensional reconstruction. Bars in A, B, D, and E = 50 Å.
FIGURE 5.
FIGURE 5.
Kinetics of peptide binding to different DnaK-GrpE complexes. A, binding kinetics of dNR to DnaK (in the presence of purified ADP), alone (black line) or with GrpE (green), GrpE(34–197) (blue), or GrpE(69–197) (magenta). Binding of dNR (0.5 μm) was followed by monitoring the increase in dansyl moiety fluorescence at 25 °C (535 nm). B, kinetics was determined as in A for dNR binding to DnaKR151A and to the corresponding complexes with GrpE or its mutants. C, binding constants (Kobs) for the kinetics shown in A and B. In addition, binding curves to DnaK and complexes with GrpE and GrpE(34–197) were performed at 45 °C. Curves were fitted to a single exponential equation: F = F0 + ΔF exp(−Kobs/t). D, GrpE(1–33) competition with dNR for DnaK binding. Titration of DnaK-dNR (5:1 μm) complexes with increasing GrpE(1–33) concentration (closed circles). As a control, the same titration was performed with peptide NR (open circles). E, determination of the on- and off-rate constants for dNR binding to DnaK and DnaK-GrpE(34–197). Binding curves were performed as in A at increasing DnaK concentrations alone (closed circles) or with 5 μm GrpE(34–197) (open circles). Experiments were repeated at least three times (data shown as mean ± S.E.). On- (k+1) and off-rate (k−1) constants were obtained, respectively, from the slope and y intercept of the linear plots of Kobs versus DnaK concentration. For DnaK, k+1 and k−1 were 1100 ± 100 m−1 s−1 and 0.001 ± 0.0001 s−1, respectively. For DnaK-GrpE(34–197), values of k+1 = 3800 ± 300 m−1 s−1 and k−1 = 0.002 ± 0.0001 s−1 were obtained. F, refolding of luciferase aggregates (25 nm) by 1 μm DnaK, 1 μm DnaJ, and 1 μm GrpE (green circles), GrpE(34–197) (blue), or GrpE(69–197) (magenta). Initial refolding rates (% refolding min−1) were obtained by linear regression of initial time points (gray lines).
FIGURE 6.
FIGURE 6.
Proposed action mechanism for the DnaK-GrpE complex. A, movement of the GrpE tail in the DnaK-GrpE complex. The N-terminal region of the GrpE dimer is flexible and can contact the head of the DnaK-GrpE, near DnaKSBD. B, dual action of GrpE as a NEF. The model is described in the text.
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