Crystal Structure of a Thermophilic GrpE Protein: Insight into Thermosensing Function for the DnaK Chaperone System

A homodimeric GrpE protein functions as a nucleotide exchange factor of the eubacterium DnaK molecular chaperone system. The co-chaperone GrpE accelerates ADP dissociation from, and promotes ATP binding to, DnaK, which cooperatively facilitates the DnaK chaperone cycle with another co-chaperone, DnaJ. GrpE characteristically undergoes two-step conformational changes in response to elevation of the environmental temperature. In the first transition at heat-shock temperatures, a fully reversible and functionally deficient structural alteration takes place in GrpE, and then the higher temperatures lead to the irreversible dissociation of the GrpE dimer into monomers as the second transition. GrpE is also thought to be a thermosensor of the DnaK system, since it is the only member of the DnaK system that changes its structure reversibly and loses its function at heat-shock temperatures of various organisms. We here report the crystal structure of GrpE from Thermus thermophilus HB8 (GrpE_Tth) at 3.23 Å resolution. The resolved structure is compared with that of GrpE from mesophilic Escherichia coli (GrpE_Eco), revealing structural similarities, particularly in the DnaK interaction regions, and structural characteristics for the thermal stability of GrpE_Tth. In addition, the structure analysis raised the possibility that the polypeptide chain in the reported GrpE_Eco structure was misinterpreted. Comparison of these two GrpE structures combined with the results of limited proteolysis experiments provides insight into the protein dynamics of GrpE_Tth correlated with the shift of temperature, and also suggests that the localized and partial unfolding at the plausible DnaK interaction sites of GrpE_Tth causes functional deficiency of nucleotide exchange factor in response to the heat shock.     

Thermostability of two cyanobacterial GrpE thermosensors

GrpE proteins act as co-chaperones for DnaK heat-shock proteins. The dimeric protein unfolds under heat stress conditions, which results in impaired interaction with a DnaK protein. Since interaction of GrpE with DnaK is crucial for the DnaK chaperone activity, GrpE proteins act as a thermosensor in bacteria. Here we have analyzed the thermostability and function of two GrpE homologs of the mesophilic cyanobacterium Synechocystis sp. PCC 6803 and of the thermophilic cyanobacterium Thermosynechococcus elongatus BP1. While in Synechocystis an N-terminal helix pair of the GrpE dimer appears to be the thermosensing domain and mainly mediates GrpE dimerization, the C-terminal four-helix bundle is involved in additional stabilization of the dimeric structure. The four-helix bundle domain has a key role in the thermophilic cyanobacterium, since dimerization of the Thermosynechococcus protein appears to be mediated by the four-helix bundle domain, and melting of this domain is linked to monomerization of the GrpE protein. Thus, in two related cyanobacteria the GrpE thermosensing function might be mediated by different protein domains.     

GrpE, a nucleotide exchange factor for DnaK

The cochaperone GrpE functions as a nucleotide exchange factor to promote dissociation of adenosine 5'-diphosphate (ADP) from the nucleotide-binding cleft of DnaK. GrpE and the DnaJ cochaperone act in concert to control the flux of unfolded polypeptides into and out of the substrate-binding domain of DnaK by regulating the nucleotide-bound state of DnaK. DnaJ stimulates nucleotide hydrolysis, and GrpE promotes the exchange of ADP for adenosine triphosphate (ATP) and also augments peptide release from the DnaK substrate-binding domain in an ATP-independent manner. The eukaryotic cytosol does not contain GrpE per se because GrpE-like function is provided by the BAG1 protein, which acts as a nucleotide exchange factor for cytosolic Hsp70s. GrpE, which plays a prominent role in mitochondria, chloroplasts, and bacterial cytoplasms, is a fascinating molecule with an unusual quaternary structure. The long alpha-helices of GrpE have been hypothesized to act as a thermosensor and to be involved in the decrease in GrpE-dependent nucleotide exchange that is observed in vitro at temperatures relevant to heat shock. This review describes the molecular biology of GrpE and focuses on the structural and kinetic aspects of nucleotide exchange, peptide release, and the thermosensor hypothesis.     

A structure-based interpretation of E.coli GrpE thermodynamic properties

GrpE is the nucleotide exchange factor for the Escherichia coli molecular chaperone DnaK, the prokaryotic homologue of Hsp70. Thermodynamic properties of GrpE structural domains were characterized by examining a number of structural and point mutants using circular dichroism, differential scanning calorimetry and analytical ultracentrifugation. These structural domains are the long paired N-terminal helices, the central four-helix bundle, and the C-terminal compact beta-domains. We show that the central four-helix bundle (t(m) approximately 75 degrees C) provides a stable platform for the association of the long paired N-terminal helices (t(m) approximately 50 degrees C), which can then function as a temperature sensor. The stability of the N-terminal helices is linked to the presence of the C-terminal compact beta-domains of GrpE, providing a potential mechanism for coupling of DnaK-binding activity of GrpE with temperature. On the basis of our thermodynamic analysis of E.coli GrpE, we present a structure-based model for the melting properties of the nucleotide exchange factor, wherein the long paired helices function as a molecular thermocouple.     

Deletion of DnaK's lid strengthens binding to the nucleotide exchange factor,GrpE: a kinetic and thermodynamic analysis

In this study, we have used surface plasmon resonance (SPR) and isothermal microtitration calorimetry (ITC) to study the mechanism of complex formation between the Hsp70 molecular chaperone, DnaK, and its cochaperone, GrpE, which is a nucleotide exchange factor. Experiments were geared toward understanding the influence of DnaK's three domains, the ATPase (residues 1-388), substrate-binding (residues 393-507), and lid (residues 508-638) domains, on complex formation with GrpE. We show that the equilibrium dissociation constants for the interaction of GrpE with wtDnaK, lidless DnaK(2-517), the ATPase domain (2-388), and the substrate-binding fragment (393-507) are 64 (+/-16) nM, 4.0 (+/-1.5) nM, 35 (+/-10) nM, and 67 (+/-11) microM, respectively, and that the on-rate constant for the different reactions varies by over 4 orders of magnitude. SPR experiments revealed that GrpE-DnaK(393-507) complex formation is inhibited by added peptide and abolished when the 33-residue flexible "tail" of GrpE is deleted. Such results strongly suggest that the 33-residue flexible N-terminal tail of GrpE binds in the substrate-binding pocket of DnaK. This unique mode of binding between GrpE's tail and DnaK contributes to, but does not fully explain, the decrease in K(d) from 64 to 4 nM upon deletion of DnaK's lid. The possibility that deletion of DnaK's lid creates a more symmetrically shaped molecule, with enhanced affinity to GrpE, is also discussed. Our results reveal a complex set of molecular interactions between DnaK and its cochaperone GrpE. We discuss the impact of each domain on complex formation and dissociation.     

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.     

A GrpE mutant containing the NH(2)-terminal "tail" region is able to displace bound polypeptide substrate from DnaK

A key feature to the dimeric structure for the GrpE heat shock protein is the pair of long helices at the NH(2)-terminal end followed by a presumable extended segment of about 30 amino acids from each monomer. We have constructed a GrpE deletion mutant protein that contains only the unique tail portion (GrpE1-89) and another that is missing this region (GrpE88-197). Circular dichroism analysis shows that the GrpE1-89 mutant still contains one-third percent alpha-helical secondary structure. Using an assay that measures bound peptide to DnaK we show that the GrpE1-89 is able to lower the amount of bound peptide, whereas GrpE88-197 has no effect. Additionally, when the same peptide binding assay is carried out with the COOH-terminal domain of DnaK, the full-length GrpE and the two GrpE deletion mutants show little to no effect on peptide release. Furthermore, the GrpE88-197 mutant is able to enhance the off-rate of nucleotide from DnaK and the 1-89 mutant has no effect on the nucleotide release. Similar results of nucleotide release are observed with the NH(2)-terminal ATPase domain mutant of DnaK. The results presented show directly that there is interaction between the GrpE protein's "tail" region and the substrate COOH-terminal peptide binding domain of DnaK, although the effect is only fully manifest with an intact full-length DnaK molecule.     

Thermodynamic linkage in the GrpE nucleotide exchange factor,a molecular thermosensor

GrpE is the nucleotide exchange factor for the Escherichia coli molecular chaperone DnaK, the bacterial homologue of Hsp70. In the temperature range of the bacterial heat shock response, the long helices of GrpE undergo a helix-to-coil transition, and GrpE exhibits non-Arrhenius behavior with respect to its nucleotide exchange function. It is hypothesized that GrpE acts as a thermosensor and that unwinding of the long helices of E. coli GrpE reduces its activity as a nucleotide exchange factor. In turn, it was proposed that temperature-dependent down-regulation of the activity of GrpE may increase the time in which DnaK binds its substrates at higher temperatures. A combination of thermodynamic and hydrodynamic techniques, in concert with the luciferase refolding assay, were used to characterize a molecular mechanism in which the long helices of GrpE are thermodynamically linked with the beta-domains via an intramolecular contact between Phe86 and Arg183. These "thermosensing" long helices were found to be necessary for full activity as a nucleotide exchange factor in the luciferase refolding assay. Point mutations in the beta-domains and in the long helices of GrpE destabilized the beta-domains. Engineered disulfide bonds in the long helices alternately stabilized the long helices and the four-helix bundle. This allowed the previously reported 75 degrees C thermal transition seen in the excess heat capacity function as monitored by differential scanning calorimetry to be further characterized. The observed thermal transition represents the unfolding of the four-helix bundle and the beta-domains. The thermal transitions for these two domains are superimposed but are not thermodynamically linked.     

Investigating the functional role of a buried interchain aromatic cluster in Escherichia coli GrpE dimer

Aromatic clusters in the core of proteins are often involved in imparting structural stability to proteins. However, their functional importance is not always clear. In this study, we investigate the thermosensing role of a phenylalanine cluster present in the GrpE homodimer. GrpE, which acts as a nucleotide exchange factor for the molecular chaperone DnaK, is well known for its thermosensing activity resulting from temperature-dependent structural changes that allow control of chaperone function. Using mutational analysis, we show that an interchain phenylalanine cluster in a four-helix bundle of the GrpE homodimer assists in the thermosensing ability of the co-chaperone. Substitution of aromatic residues with hydrophobic ones in the core of the four-helix bundle reduces the thermal stability of the bundle and that of a connected coiled-coil domain, which impacts thermosensing. Cell growth assays and SEM images of the mutants show filamentous growth of Escherichia coli cells at 42°C, which corroborates with the defect in thermosensing. Our work suggests that the interchain edge-to-face aromatic cluster is important for the propagation of the structural signal from the coiled-coil domain to the four-helical bundle of GrpE, thus facilitating GrpE-mediated thermosensing in bacteria.     

Crystal structure of DnaK protein complexed with nucleotide exchange factor GrpE in DnaK chaperone system: insight into intermolecular communication

The conserved, ATP-dependent bacterial DnaK chaperones process client substrates with the aid of the co-chaperones DnaJ and GrpE. However, in the absence of structural information, how these proteins communicate with each other cannot be fully delineated. For the study reported here, we solved the crystal structure of a full-length Geobacillus kaustophilus HTA426 GrpE homodimer in complex with a nearly full-length G. kaustophilus HTA426 DnaK that contains the interdomain linker (acting as a pseudo-substrate), and the N-terminal nucleotide-binding and C-terminal substrate-binding domains at 4.1-Å resolution. Each complex contains two DnaKs and two GrpEs, which is a stoichiometry that has not been found before. The long N-terminal GrpE α-helices stabilize the linker of DnaK in the complex. Furthermore, interactions between the DnaK substrate-binding domain and the N-terminal disordered region of GrpE may accelerate substrate release from DnaK. These findings provide molecular mechanisms for substrate binding, processing, and release during the Hsp70 chaperone cycle.     

Influence of GrpE on DnaK-substrate interactions

The DnaK chaperone of Escherichia coli assists protein folding by an ATP-dependent interaction with short peptide stretches within substrate polypeptides. This interaction is regulated by the DnaJ and GrpE co-chaperones, which stimulate ATP hydrolysis and nucleotide exchange by DnaK, respectively. Furthermore, GrpE has been claimed to trigger substrate release independent of its role as a nucleotide exchange factor. However, we show here that GrpE can accelerate substrate release from DnaK exclusively in the presence of ATP. In addition, GrpE prevented the association of peptide substrates with DnaK through an activity of its N-terminal 33 amino acids. A ternary complex of GrpE, DnaK, and a peptide substrate could be observed only when the peptide binding to DnaK precedes GrpE binding. Furthermore, we demonstrate that GrpE slows down the release of a protein substrate, sigma(32), from DnaK in the absence of ATP. These findings suggest that the ATP-triggered dissociation of GrpE and substrates from DnaK occurs in a concerted fashion.     

Mutational analysis of the energetics of the GrpE.DnaK binding interface: equilibrium association constants by sedimentation velocity analytical ultracentrifugation

DnaK, the prokaryotic Hsp70 molecular chaperone, requires the nucleotide exchange factor and heat shock protein GrpE to release ADP. GrpE and DnaK are tightly associated molecules with an extensive protein-protein interface, and in the absence of ADP, the dissociation constant for GrpE and DnaK is in the low nanomolar range. GrpE reduces the affinity of DnaK for ADP, and the reciprocal linkage is also true: ADP reduces the affinity of DnaK for GrpE. The energetic contributions of GrpE side-chains to GrpE-DnaK binding were probed by alanine-scanning mutagenesis. Sedimentation velocity (SV) analytical ultracentrifugation (AUC) was used to measure the equilibrium constants (Keq) for GrpE binding to the ATPase domain of DnaK in the presence of ADP. ADP-bound DnaK is the natural target of GrpE, and the addition of ADP (final concentration of 5 microM) to the preformed GrpE-DnaK(ATPase) complexes allowed the equilibrium association constants to be brought into an experimentally accessible range. Under these experimental conditions, the substitution of one single GrpE amino acid residue, arginine 183 with alanine, resulted in a GrpE-DnaK(ATPase) complex that was weakly associated (Keq =9.4 x 10(4) M). This residue has been previously shown to be part of a thermodynamic linkage between two structural domains of GrpE: the thermosensing long helices and the C-terminal beta-domains. Several other GrpE side-chains were found to have a significant change in the free energy of binding (DeltaDeltaG approximately 1.5 to 1.7 kcal mol(-1)), compared to wild-type GrpE.DnaK(ATPase) in the same experimental conditions. Overall, the strong interactions between GrpE and DnaK appear to be dominated by electrostatics, not unlike barnase and barstar, another well-characterized protein-protein interaction. GrpE, an inherent thermosensor, exhibits non-Arrhenius behavior with respect to its nucleotide exchange function at bacterial heat shock temperatures, and mutation of several solvent-exposed side-chains located along the thermosensing indicated that these residues are indeed important for GrpE-DnaK interactions.     

The NH2-terminal domain of the chloroplast GrpE homolog CGE1 is required for dimerization and cochaperone function in vivo

GrpE proteins function as nucleotide exchange factors for DnaK-type Hsp70s. We have previously identified a chloroplast homolog of GrpE in Chlamydomonas reinhardtii, termed CGE1. CGE1 exists as two isoforms, CGE1a and CGE1b, which are generated by temperature-dependent alternative splicing. CGE1b contains additional valine and glutamine residues in its extreme NH2-terminal region. Here we show that CGE1a is predominant at lower temperatures but that CGE1b becomes as abundant as CGE1a at elevated temperatures. Coimmunoprecipitation experiments revealed that CGE1b had a approximately 25% higher affinity for its chloroplast chaperone partner HSP70B than CGE1a. Modeling of the structure of CGE1b revealed that the extended alpha-helix formed by GrpE NH2 termini is 34 amino acids longer in CGE1 than in Escherichia coli GrpE and appears to contain a coiled coil motif. Progressive deletions of this coiled coil increasingly impaired the ability of CGE1 to form dimers, to interact with DnaK at elevated temperatures, and to complement temperature-sensitive growth of a DeltagrpE E. coli strain. In contrast, deletion of the four-helix bundle required for dimerization of E. coli GrpE did not affect CGE1 dimer formation. Circular dichroism measurements revealed that CGE1, like GrpE, undergoes two thermal transitions, the first of which is in the physiologically relevant temperature range (midpoint approximately 45 degrees C). Truncating the NH2-terminal coiled coil shifted the second transition to lower temperatures, whereas removal of the four-helix bundle abolished the first transition. Our data suggest that bacterial GrpE and chloroplast CGE1 share similar structural and biochemical properties, but some of these, like dimerization, are realized by different domains.     

Crystal structure of human GGA1 GAT domain complexed with the GAT-binding domain of Rabaptin5

GGA proteins coordinate the intracellular trafficking of clathrin-coated vesicles through their interaction with several other proteins. The GAT domain of GGA proteins interacts with ARF, ubiquitin, and Rabaptin5. The GGA-Rabaptin5 interaction is believed to function in the fusion of trans-Golgi-derived vesicles to endosomes. We determined the crystal structure of a human GGA1 GAT domain fragment in complex with the Rabaptin5 GAT-binding domain. In this structure, the Rabaptin5 domain is a 90-residue-long helix. At the N-terminal end, it forms a parallel coiled-coil homodimer, which binds one GAT domain of GGA1. In the C-terminal region, it further assembles into a four-helix bundle tetramer. The Rabaptin5-binding motif of the GGA1 GAT domain consists of a three-helix bundle. Thus, the binding between Rabaptin5 and GGA1 GAT domain is based on a helix bundle-helix bundle interaction. The current structural observation is consistent with previously reported mutagenesis data, and its biological relevance is further confirmed by new mutagenesis studies and affinity analysis. The four-helix bundle structure of Rabaptin5 suggests a functional role in tethering organelles.     

Physical interactions between members of the DnaK chaperone machinery: characterization of the DnaK.GrpE complex

Many of the functions of the Escherichia coli Hsp 70, DnaK, require two cofactors, DnaJ and GrpE. GrpE acts as a nucleotide exchange factor in the DnaK reaction cycle but the details of its mechanism remain unclear. GrpE has high affinity for monomeric native DnaK, with a K_d estimated at ≤50 nM. GrpE is a very asymmetric molecule and exists as either a dimer or trimer in its native state. The stoichiometry of GrpE to DnaK in the isolated complex was 3:1, suggesting a trimer. Formation of the complex is quite fast (k_on >1 S⁻¹, whereas the off-rate is very slow on the HPLC timescale (k_off ≤ 10⁻⁴ S⁻¹). GrpE has no affinity for ATP or ADP, nor the oligomeric and moltn globule states of DnaK. The complex is much more thermally stable than either GrpE or DnaK alone, and prevents the formation of the molten globule-like state of DnaK at physiologically relevant temperatures. Formation of the complex does not cause any change in secondary structure, as determined by the lack of change in the circular dichroism spectrum. However, binding of GrpE induces a similar tertiary strcutral change in DnaK to that induced by binding of ATP₁ based on the blue shift in λ_max from the fluroscence of the single tryptophan in DnaK. The nucleotide exchange properties of GrpE can be explained by the conformational change which may represent the opening of the nucleotide cleft on DnaK, subsequently inducing a low affinity state for ADP.     

Regulation of ATPase and chaperone cycle of DnaK from Thermus thermophilus by the nucleotide exchange factor GrpE

The nucleotide binding and release cycle of the molecular chaperone DnaK is regulated by the accessory proteins GrpE and DnaJ, also called co-chaperones. The concerted action of the nucleotide exchange factor GrpE and the ATPase-stimulating factor DnaJ determines the ratio of the two nucleotide states of DnaK, which differ in their mode of interaction with unfolded proteins. In the Escherichia coli system, the stimulation by these two antagonists is comparable in magnitude, resulting in a balance of the two nucleotide states of DnaK(Eco) in the absence and the presence of co-chaperones.The regulation of the DnaK chaperone system from Thermus thermophilus is apparently substantially different. Here, DnaJ does not stimulate the DnaK-mediated ATP hydrolysis and thus does not appear to act as an antagonist of the nucleotide exchange factor GrpE(Tth). This raises the question of whether T. thermophilus GrpE stimulates nucleotide exchange to a smaller degree as compared to the E. coli system and how the corresponding rates relate to intrinsic ATPase and ATP binding as well as luciferase refolding kinetics of T. thermophilus DnaK.We determined dissociation constants as well as kinetic constants that describe the interactions between the T. thermophilus molecular chaperone DnaK, its nucleotide exchange factor GrpE and the fluorescent ADP analogue N8-(4-N'-methylanthraniloylaminobutyl)-8-aminoadenosine-5'-diphosphate by isothermal equilibrium titration calorimetry and stopped-flow kinetic experiments and investigated the influence of T. thermophilus DnaJ on the DnaK nucleotide cycle.The interaction of GrpE with the DnaK.ADP complex versus nucleotide-free DnaK can be described by a simple equilibrium system, where GrpE reduces the affinity of DnaK for ADP by a factor of about 10. Kinetic experiments indicate that the maximal acceleration of nucleotide release by GrpE is 80,000-fold at a saturating GrpE concentration.Our experiments show that in T. thermophilus, although the thermophilic DnaK system displays no stimulation of the DnaK-ATPase activity by DnaJ, nucleotide exchange is still efficiently stimulated by GrpE. This indicates that two counteracting factors are not absolutely necessary to maintain a functional and regulated chaperone cycle. This conclusion is corroborated by data that show that the slower ATPase cycle of the DnaK system as well as of heterologous T. thermophilus DnaK/E. coli DnaK systems is directly reflected in altered refolding kinetics of firefly luciferase but not necessarily in refolding yields.     

Folding properties of the nucleotide exchange factor GrpE from Thermus thermophilus: GrpE is a thermosensor that mediates heat shock response.

Hsp70 proteins like DnaK bind unfolded polypeptides in a nucleotide-dependent manner. The switch from high-affinity ADP-state to low- affinity ATP-state with concomitant substrate release is accelerated significantly by GrpE proteins. GrpE thus fulfils an important role in regulation of the chaperone cycle. Here, we analysed the thermal stability of GrpE from Thermus thermophilus using differential scanning calorimetry and CD-spectroscopy. The protein exhibits unusual unfolding characteristics with two observable thermal transitions. The first transition is CD-spectroscopically silent with a transition midpoint at 90 degrees C. The second transition, mainly constituting the CD-signal, ranges between 100 and 105 degrees C depending on the GrpE(Tth) concentration, according to the model N(2) <==> I(2) <==> 2U. Using a C-terminally truncated version of GrpE(Tth) it was possible to assign the second thermal transition to the dimerisation of GrpE(Tth), while the first transition represents the completely reversible unfolding of the globular C-terminal domain. The unfolding of this domain is accompanied by a distinct decrease in nucleotide exchange rates and impaired binding to DnaK(Tth). Under heat shock conditions, the DnaK-ADP-protein-substrate complex is thus stabilised by a reversibly inactivated GrpE-protein that refolds under permissive conditions. In combination with studies on GrpE from Escherichia coli presented recently by Christen and co-workers, it thus appears that the general role of GrpE is to function as a thermosensor that modulates nucleotide exchange rates in a temperature-dependent manner to prevent substrate dissociation at non-permissive conditions.     

GrpE accelerates peptide binding and release from the high affinity state of DnaK

The Escherichia coli nucleotide exchange factor GrpE accelerates the rate of ADP dissociation from high affinity ADP-DnaK, thus enabling ATP binding and transition to the low affinity state. We show here that GrpE, in the absence of ATP, accelerates the rates of the forward and reverse reaction ADP-DnaK-P right harpoon over left harpoon ADP-DnaK + P, where P denotes peptide substrate. Specifically, the binding of GrpE to an ADP-DnaK-P (or DnaK-P) complex increases koff and kon by approximately 200-fold and approximately 60-fold, respectively. The results are consistent with a GrpE- induced conformational change in the C-terminal polypeptide binding domain of an ADP-DnaK molecule, which results in a unique low affinity intermediate from which peptide can dissociate. A simulation of peptide dissociation from DnaK as a function of the [ATP] / [ADP] ratio shows that GrpE induced peptide dissociation from ADP-DnaK is important at elevated cellular concentrations of ADP, which typically occur upon stress.     

Insights into dimerization and four-helix bundle formation found by dissection of the dimer interface of the GrpE protein from Escherichia coli

The GrpE heat shock protein from Escherichia coli has a homodimeric structure. The dimer interface encompasses two long alpha-helices at the NH(2)-terminal end from each monomer (forming a "tail"), which lead into a small four-helix bundle from which each monomer contributes two short sequential alpha-helices in an antiparallel topological arrangement. We have created a number of different deletion mutants of GrpE that have portions of the dimer interface to investigate requirements for dimerization and to study four-helix bundle formation. Using chemical crosslinking and analytical ultracentrifugation techniques to probe for multimeric states, we find that a mutant containing only the long alpha-helical tail portion (GrpE1-88) is unable to form a dimer, most likely due to a decrease in alpha-helical content as determined by circular dichroism spectroscopy, thus one reason for a dimeric structure for the GrpE protein is to support the tail region. Mutants containing both of the short alpha-helices (GrpE1-138 and GrpE88-197) are able to form a dimer and presumably the four-helix bundle at the dimer interface. These two mutants have equilibrium constants for the monomer-dimer equilibrium that are very similar to the full-length protein suggesting that the tail region does not contribute significantly to the stability of the dimer. Interestingly, one mutant that contains just one of the short alpha-helices (GrpE1-112) exists as a tetrameric species, which presumably is forming a four-helix bundle structure. A proposed model is discussed for this mutant and its relevance for factors influencing four-helix bundle formation.     

Interaction of the Hsp110 Molecular Chaperones from S. cerevisiae with Substrate Protein

Hsp110 proteins act as nucleotide exchange factors of the molecular chaperone Hsp70 in eukaryotes. In addition, they have been reported to stabilize unfolded proteins for subsequent refolding. Hsp110 proteins belong to the Hsp70 superfamily and, in analogy to Hsp70, the substrate-binding site was proposed to be located at the interface of the β-sandwich domain and the three-helix-bundle domain. Saccharomyces cerevisiae has two closely related cytosolic isoforms of Hsp110, Sse1p and Sse2p. Under normal growth conditions, Sse1p is the predominant form. Sse2p is induced under stress conditions, such as heat shock. Consistent with these findings, we find that Sse2p has increased temperature stability. Both Sse1p and Sse2p accelerate nucleotide exchange on the yeast Hsp70 Ssa1p. Furthermore, Sse1p and Sse2p effectively compete for binding of unfolded luciferase. In contrast to Sse1p, however, Sse2p fails to stabilize this model substrate under thermal stress for subsequent Hsp70-mediated refolding. Using a domain shuffling approach, we show that both the nucleotide-binding domain and the β-sandwich domain of Sse1p are required to preserve nonnative luciferase in a folding-competent state. Our findings suggest that Sse1p must undergo partial unfolding for efficient protection of luciferase, and that the β-sandwich domain of Sse1p acts as an intramolecular chaperone for refolding of the nucleotide-binding domain. Under extreme stress conditions, Sse2p appears to take over the nucleotide exchange factor function of Sse1p and might promote the controlled aggregation of stress-denatured proteins.