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

Protein Expression and Purification

DnaK and DnaK_NBD (DnaK(1–385)) were cloned, expressed, and purified as described (7). The DnaK_R151A mutant was expressed and purified following the protocol developed by Taneva et al. (21). GrpE was cloned, expressed, and purified as described (22). The GrpE(34–197) mutant was generated by digestion of wtGrpE with papain (125:1 (w:w), 90 min, 25 °C), followed by chromatography purification in a HiLoad Superdex 75 16/60 column (GE Healthcare) (7). The sequence of the resulting protein was confirmed by MS and N-terminal sequencing. GrpE(69–197) was cloned, expressed, and purified by the protocol of Moro et al. (7).

Interaction in Native PAGE

To test their complex-forming ability, the DnaK and GrpE variants were mixed in a 1:2.5 DnaK:GrpE (monomer) molar ratio in 20 mm HEPES, pH 7.6, 50 mm KCl, 2.5 mm MgCl₂, 0.5 mm ADP (20 min, 25 °C). Aliquots of samples were loaded on a precast Native PAGE Novex 4–16% BisTris gel (Life Technologies), run at 150 V (2 h), and stained with Coomassie Blue.

Peptide Binding Kinetics

As described above, combinations of the DnaK and GrpE variants were incubated to form stable complexes in 25 mm HEPES, pH 7.6, 50 mm KCl, 5 mm MgCl₂, 1 mm ADP. Trace ATP was removed from ADP solutions as described (23). The peptides used in these experiments were fluorescent α-N, dansyl-NRLLLTG (dNR) (Neosystem), unlabeled NR, and one corresponding to the first 33 GrpE residues (GrpE(1–33); ProteoGenix). Peptides were bound by adding dNR (0.5 μm) to the DnaK-GrpE complexes; binding was measured in a FluoroMax-3 spectrofluorimeter (Jobin Ibon), with a 335-nm excitation wavelength and a 535-nm emission wavelength (excitation and emission slit widths of 4 nm). Competition of NR and GrpE(1–33) with dNR for DnaK binding was analyzed by titrating DnaK-dNR (5:1 μm) complexes with increasing peptide concentrations. In these experimental conditions, 80% dNR is bound to DnaK (K_d ∼ 1 μm).

Luciferase Refolding Experiments

Luciferase (2.5 μm) was denatured in 6 m guanidine hydrochloride, 30 mm Tris/HCl, pH 7.5, 5 mm DTT (dithiothreitol) (2 h, 25 °C). Refolding was started after 100-fold dilution in 20 mm HEPES/KOH, pH 7.4, 50 mm KCl, 5 mm MgCl₂, 5 mm DTT, 2 mm ATP, 20 ng ml⁻¹ of pyruvate kinase, 4 mm phosphoenolpyruvate, and addition of 1 μm DnaK, 1 μm DnaJ, 1 μm of the corresponding GrpE variant. Samples were incubated for various times (30 °C) and luciferase activity was measured in a Synergy HT luminometer (Biotek) using the Luciferase Assay System (Promega E1500).

Electron Microscopy and Three-dimensional Reconstruction

The complexes (DnaK-GrpE, DnaK_R151A-GrpE, DnaK_NBD-GrpE) were purified by gel filtration on a Superdex 200 column (GE Healthcare). Aliquots of purified complexes were applied to carbon-coated copper grids (30 s) and stained with 2% uranyl acetate. Micrographs were taken in minimal dose conditions in a JEM1200EX-II microscope (JEOL) operated at 100 kV. Micrographs of the DnaK-GrpE and DnaK_NBD-GrpE complexes were recorded on Kodak SO-163 plates at 60,000 magnification and digitized on a Photoscan TD scanner (Zeiss). Images of DnaK_R151A-GrpE complex were obtained in a JEOL 1230 transmission electron microscope operated at 100 kV, using a low-dose protocol and a 4k × 4k TVIPS CMOS detector under the control of EM-TOOLS software (TVIPS). Final magnification of the CMOS images was ×54,926. A total of 30,223, 15,438, and 21,910 particles of DnaK-GrpE, DnaK_NBD-GrpE, and DnaK_R151A-GrpE, respectively, were selected, normalized, and CTF-corrected using procedures implemented in the XMIPP package (24–26). All particles were initially classified using clustering reference-free methods in XMIPP (27). Angular refinement was performed using EMAN and XMIPP (24, 27, 28). For three-dimensional reconstructions, different starting templates were generated using the EMAN startcsym program, by common lines or using artificial noisy models and Gaussian blobs with the rough dimension of the proteins (28), subsequently refined using XMIPP. The different strategies converged to similar solutions.

For reconstruction of the two co-existing DnaK-GrpE conformations, the entire dataset was first processed assuming the complex displayed a unique conformation. The resulting structure and its projections, as well as comparison with reference-free averages, suggested that the reconstruction obtained is a mixture of two conformations. To test this hypothesis, two reconstructions were generated with two sets of reference-free averages corresponding to each of the putative conformations, using their current angular assignments. These structures were then used as templates for three-dimensional classification using maximum-likelihood methods implemented in XMIPP (29). After refinement, particles were split into groups corresponding to the two conformations, which were further refined separately. As a control, particles of each group were refined using the opposite template, and were found to converge into the structure defined by the group they belong to and not the template. Each structure and each particle subset generated compatible projections and averages, also consistent with the reference-free averages. Resolution was estimated using Fourier shell correlation at 0.5 criteria. The atomic structures were first fitted manually into the EM density maps and fine docking was performed using the automatic option in the UCSF Chimera package (30). Flexible fitting was performed using iMODfit (31). Handedness of the structures was defined as that which generated the best cross-correlation with the crystal structures.

DnaK-GrpE Complex Formation with the DnaK and GrpE Variants

To analyze the interaction between the chaperone DnaK and its NEF GrpE, we generated several variants of the two proteins. For the chaperone, we expressed and purified wtDnaK, a deletion mutant corresponding to the nucleotide-binding domain alone (residues 1–385; DnaK_NBD), and a mutant that lacks allosteric communication between DnaK_NBD and DnaK_SBD (DnaK_R151A; 32). For GrpE, we generated the full-length version and two deletion mutants (7); one lacked the first 33 residues corresponding to the disordered region (GrpE(34–197)), and the other also lacked part of the long α-helix (GrpE(69–197)) but maintained those residues of this region involved in DnaK_NBD binding (Asn-71, Arg-73, and Arg-74) (17). The atomic structure of the DnaK_NBD-GrpE complex predicts that both GrpE mutants would generate a stable complex with DnaK; this was confirmed by native electrophoresis (Fig. 2A), as it was for the allosteric mutant DnaK_R151A (Fig. 2B).

DnaK-GrpE Complex Structure

There are high-resolution data for the DnaK-GrpE complex, although in one case they are derived from a complex lacking the DnaK_SBD domain (17), and in the other, the DnaK-GrpE complex has a 2:2 symmetry, which has not been observed in solution (20). We thus used EM and image processing to determine the DnaK-GrpE complex structure. As this complex is small by EM standards (125 kDa), as a control we first performed a three-dimensional reconstruction of the known DnaK_NBD-GrpE complex. We generated the complex and purified it by gel filtration, and negatively stained one aliquot. The sample appeared to be heterogeneous, probably due to the distinct DnaK_NBD-GrpE views on the grid. The most common view showed a circular mass from which a stem protruded (see Fig. 3A). We selected and processed 15,438 particles and generated a structure (23-Å resolution; Fourier shell correlation 0.5) that can be described as composed of a head and a tail (Fig. 3B). The structure is ∼130 Å long, with a head length of ∼70 Å and a width of ∼70 × ∼90 Å. The overall complex shape is very similar to the atomic structure of the DnaK_NBD-GrpE complex (Protein Data Bank 1DKG; Ref. 17), and docking of the latter into the former is good in the complex head (Fig. 3C). There are nonetheless notable differences between the tails of the two structures. The tail is longer in the complex visualized by EM, which can be explained by the first 33 residues, which were not determined in the crystal structure. The other difference is in tail shape, which is bent in the EM reconstruction and straight in the crystal structure. A flexible fitting performed on the tail structure helped to dock it within our three-dimensional reconstruction (Fig. 3D).

We then determined the structure of the DnaK-GrpE complex, for which we generated and purified the complex using the same strategy as for DnaK_NBD-GrpE. Aliquots of the purified complex were negatively stained and observed by EM. Heterogeneity was again indicated, which we associated to the different views of the complex. In most cases, the particles showed a circular structure, larger than that observed for DnaK_NBD-GrpE, and a stem protruding from the main mass with different lengths and angles. We selected 30,223 particles and classified them two- and three-dimensionally (see “Experimental Procedures”). Our classification and processing strategy allowed us to isolate two subsets of molecule images corresponding to two distinct, albeit homogenous conformations. The two volumes generated were very similar except for tail shape, which had a different degree of bending; one was similar to that for DnaK_NBD-GrpE (Fig. 4, A and C, conformation 1) and in the other, much more curved, the tip of the tail contacted the head (Fig. 4, A and C, conformation 2).

The main difference between the two DnaK-GrpE structures and that of DnaK_NBD-GrpE was the head, which for DnaK-GrpE was larger (∼70 Å long, ∼100 Å wide) due to the presence of DnaK_SBD. The difference map between DnaK-GrpE and DnaK_NBD-GrpE located the DnaK_SBD position more precisely (Fig. 4B, ii); we therefore docked the atomic structure of this domain (PDB code 4EZX; Ref. 33) to this position (Fig. 4B, iii). In this manner, DnaK_SBD was located in the head of the DnaK-GrpE complex, in close proximity to DnaK_NBD; the two domains embraced the head of the GrpE structure in an apparently symmetric manner (Fig. 4C). The complex tail is longer than that determined in the atomic structure of DnaK_NBD-GrpE, confirming visualization of the first 33 GrpE residues in our EM structures. In one conformation, the tip of the tail contacted with the head of the structure, near DnaK_SBD (Fig. 4C, conformation 2). The locations of DnaK_NBD and DnaK_SBD in the DnaK-GrpE complex leave the interdomain linker (not observed in the three-dimensional reconstruction, Fig. 4C, black asterisk), near the central region of the long GrpE α-helix (residues 65–75). This finding confirms proteolysis protection results that suggested interaction between the DnaK linker and the long GrpE α-helix N-terminal region (7).

DnaK Allosterism Influences the Structure of the DnaK-GrpE Complex

As GrpE binding appears to modify the conformational equilibrium of the chaperone, we explored the relationship between DnaK allosterism and complex structure. We generated a complex between GrpE and DnaK_R151A, a mutant that harbors a single substitution resulting in defective interdomain communication. We purified the complex by gel filtration and negatively stained one aliquot. EM analysis showed different particles, the majority with two thin masses protruding from a central, round mass (Fig. 4D). We selected and processed 21,910 particles to generate a three-dimensional reconstruction of DnaK_R151A-GrpE (Fig. 4E), which confirmed two long masses protruding from the central mass. The volume generated was reminiscent of that of DnaK_NBD-GrpE except for an extra protruding mass, which we associated to DnaK_SBD, which is not present in DnaK_NBD-GrpE (Fig. 4F). Docking of the modified atomic structure of the DnaK_NBD-GrpE complex used in Fig. 3D was good in this three-dimensional reconstruction, as was docking of the DnaK_SBD atomic structure in the extra protruding mass (Fig. 4G). The atomic model placed the DnaK_SBD far from the main structure of the complex, and strongly suggested lack of interaction between the DnaK linker and the GrpE tail. Defective interdomain communication thus maintains DnaK in an ADP-like, extended conformation when in complex with GrpE.

GrpE Modifies DnaK_SBD Dynamics

EM reconstruction of the DnaK-GrpE complex showed that the ADP-bound, high substrate affinity conformation of DnaK is modified by GrpE, which displaces the DnaK_SBD to a position nearer DnaK_NBD and the GrpE head. To determine whether this conformational change modulates DnaK_SBD dynamics, we analyzed the kinetics of substrate association to DnaK alone or with the GrpE variants (GrpE, GrpE(34–197), and GrpE(69–197)). As substrate we used dNR, a peptide modified with a dansyl fluorescent probe and high affinity for DnaK. The kinetics of peptide binding to DnaK was slow, with a K_obs value of 0.0017 s⁻¹ (t_½ 10 min) (Fig. 5, A and C), whereas association to the DnaK-GrpE complex was 3-fold faster. The lower fluorescence increase suggested that less peptide was bound to DnaK-GrpE than to free DnaK. In contrast, whereas the amount of dNR bound was similar for DnaK-GrpE(34–197) complex and isolated DnaK, the kinetics was 4-fold faster. These data suggest that the first 33 GrpE residues act as a pseudo-substrate that competes with dNR for the DnaK binding site, as reported (7, 34, 35). To further confirm this hypothesis, this GrpE fragment (GrpE(1–33)) was synthesized and its ability to interact with the DnaK binding site analyzed. Titration of DnaK-dNR complexes with GrpE(1–33) showed that within the 0–200 μm concentration range, the peptide was soluble and unable to displace DnaK-bound dNR (Fig. 5D). This finding is not surprising, because GrpE(1–33) does not have a high-affinity binding site for DnaK, as its sequence consists almost entirely of charged and polar residues, different from the hydrophobic residues normally recognized by the chaperone binding site. This apparent contradiction could be explained by our observation that DnaK-GrpE binding places the NEF N-terminal, disordered region near the DnaK substrate-binding pocket (Fig. 4, A and C), thus facilitating its interaction, as previously proposed (34). We therefore suggest that the DnaK binding site is more accessible in the presence of GrpE, due to the conformational changes induced in DnaK by GrpE binding. This last observation coincides well with the higher protease accessibility of residues in the DnaK lid subdomain when GrpE is present (7).

To better understand the GrpE effect in dNR binding kinetics, on- and off-rate constants were determined in association experiments with increasing DnaK concentrations (Fig. 5E). We used the GrpE(34–197) variant to avoid competition of the first 33 GrpE residues with dNR. Plots of K_obs versus DnaK concentration were linear, and on- and off-rate constants were obtained from the slope and y intercept, respectively. The values for isolated DnaK (k₊₁, 1100 ± 100 m⁻¹ s⁻¹ and k₋₁, 0.001 ± 0.0001 s⁻¹) were comparable with those reported (36), and increased 3.5- and 2-fold, respectively, for the complex with GrpE(34–197). These findings confirmed the greater DnaK binding site accessibility when the chaperone interacts with GrpE, and indicated that the nucleotide exchange factor increases affinity for the substrate only slightly (K_d = k₋₁/k₊₁); GrpE thus does not induce substrate release, as was postulated (8).

A distinct scenario is observed when DnaK is complexed with GrpE(69–197). This deletion variant constitutes the GrpE “head,” the main element responsible for interaction with DnaK_NBD (17). We observed no fluorescence increase for the DnaK-GrpE(69–197) complex (Fig. 5, A and C), which suggests that DnaK has adopted a conformation unable to bind dNR stably, in accordance with the strong stimulation of peptide dissociation from DnaK(ADP) induced by this mutant (7). Similar kinetic curves were obtained when we assayed dNR binding to DnaK in the presence of ATP (not shown), which induces docking of the chaperone domains and opening of the peptide binding site (37). These data suggest that DnaK_NBD interaction with the GrpE head (see Fig. 1C) promotes an open DnaK_SBD conformation.

We performed similar dNR binding experiments to DnaK_R151A in the presence of these GrpE variants (Fig. 5, B and C). dNR binding to DnaK_R151A(ADP) was almost 2-fold slower than to wtDnaK, reflecting the allosteric defect in the mutant. The presence of GrpE or GrpE(34–197) did not modify the K_obs of dNR binding to DnaK_R151A, whereas DnaK-GrpE(69–197) slightly increased this value (Fig. 5C). In accordance with the EM reconstruction of this complex, these results indicate that in the absence of interdomain communication, GrpE is unable to modify DnaK_SBD dynamics.

The data indicated that interaction of the GrpE N-terminal segments with DnaK has two main effects: 1) when tethered to the rest of the NEF molecule, the 33 disordered N-terminal residues compete with the substrate for DnaK binding, and 2) interaction of the N-terminal half of the GrpE tail (residues 34–68) with the DnaK linker and/or DnaK_SBD increases accessibility of the chaperone peptide binding site and stabilizes the DnaK-substrate complex. This helical GrpE segment unfolds reversibly at heat shock temperatures and modulates the functional cycle of DnaK (18). We therefore studied DnaK_SBD dynamics at 45 °C, alone, or with GrpE and GrpE(34–197). As predicted, binding kinetics was greatly accelerated for isolated DnaK(ADP), with a K_obs increase from 0.0017 s⁻¹ at 25 °C to 0.051 s⁻¹ at 45 °C. Neither GrpE nor GrpE(34–197) modified the K_obs value in these experimental conditions, in contrast to observations at 25 °C (Fig. 5C). The results suggest that unfolding of the long N-terminal helices in heat shock conditions blocks the GrpE-induced structural rearrangement of DnaK(ADP) that takes place at permissive temperatures.

The GrpE N-terminal Domain Regulates Chaperone Activity of the DnaK System

To address whether interaction of the N-terminal GrpE regions (the first 34 or 69 residues) with DnaK influenced the chaperone activity of the DnaK system, we measured activity after refolding of chemically denatured luciferase, using GrpE, GrpE(34–197), or GrpE(69–197) as NEF (Fig. 5F). Deletion of the first 33 residues induced a 25% reduction in the initial refolding rate (from 4.9 ± 0.2 to 3.7 ± 0.4% min⁻¹). A similar deletion variant was able to refold thermally denatured luciferase in the DnaK system as efficiently as wtGrpE (34), indicating that the chaperone activity depends on the conformational properties and stability of the unfolded and aggregated substrates. When GrpE(69–197) was used as NEF, the initial refolding rate was reduced by 65% (1.6 ± 0.2% min⁻¹), coinciding with data reported for the shorter GrpE(89–197) construct (38). Based on the ability of the GrpE(69–197) mutant to dissociate DnaK-substrate complexes (7), premature release of unfolded luciferase might hamper correct refolding of the substrate. These results indicate functional significance for the interaction between the GrpE tail, specifically the N-terminal part of the helical region, with the chaperone.