Structural basis of the interspecies interaction between the chaperone DnaK ( Hsp 70 ) and the co-chaperone GrpE of archaea and bacteria *

Hsp70s are chaperone proteins that are conserved in evolution and present in all prokaryotic and eukaryotic organisms. In the archaea, which form a distinct kingdom, the Hsp70 chaperones have been found in some species only, including Methanosarcina mazei. Both the bacterial and archaeal Hsp70(DnaK) chaperones cooperate with a GrpE co-chaperone which stimulates the ATPase activity of the DnaK protein. It is currently believed that the archaeal Hsp70 system was obtained by the lateral transfer of chaperone genes from bacteria. Our previous finding that the DnaK and GrpE proteins of M. mazei can functionally cooperate with the Escherichia coli GrpE and DnaK supported this hypothesis. However, the cooperation was surprising, considering the very low identity of the GrpE proteins (26%) and the relatively low identity of the DnaK proteins (56%). The aim of this work was to investigate the molecular basis of the observed interspecies chaperone interaction. Infrared resolution-enhanced spectra of the M. mazei and E. coli DnaK proteins were almost identical, indicating high similarity of their secondary structures, however, some small differences in band position and in the intensity of amide I’ band components were observed and discussed. Profiles of thermal denaturation of both proteins were similar, although they indicated a higher thermostability of the M. mazei DnaK compared to the E. coli DnaK. Electrophoresis under non-denaturing conditions demonstrated that purified DnaK and GrpE of E. coli and M. mazei formed mixed complexes. Protein modeling revealed high similarity of the 3-dimensional structures of the archaeal and bacterial DnaK and GrpE proteins.

The DnaK molecule has specialized domains for recognizing and binding substrates (polypeptides), binding and hydrolyzing ATP, and interacting with DnaJ and GrpE.These domains have been identified and their properties have been studied in several DnaKs, particularly that from Escherichia coli.DnaK consists of an about 44 kDa amino-terminal ATPase domain and an about 27 kDa carboxy-terminal substrate binding domain (SBD).The three-dimensional architecture of the ATPase domain (Harrison et al., 1997) and that of the SBD (Zhu et al., 1996) have been determined.The ATPase domain is composed of two lobes that form a cleft for ATP binding.In addition, this domain binds the nucleotide-exchange factor GrpE.The SBD consists of two separated regions: the β-sandwich subdomain with a cavity which accommodates the polypeptide, and the α-helical subdomain, forming a latch segment or lid, closing on top of the substrate-binding cavity (Zhu et al., 1996).
DnaK has a weak ATPase activity and it cycles between ATP-and ADP-bound stages, with its affinity for the polypeptide substrate being lower in the former than in the ADP-bound stage.The cycling of DnaK between these stages is regulated by the co-chaperone DnaJ and the nucleotide-exchange factor GrpE (the latter functions as a homodimer).The DnaJ protein binds to DnaK and accelerates hydrolysis of ATP by DnaK, thus facilitating the binding of the substrate polypeptide.GrpE induces release of ADP from DnaK and, upon rebinding of ATP, the DnaK-polypeptide complex dissociates and the folded protein is released.This completes the reaction cycle and leaves the substrate-binding cavity free and open to receive another polypeptide (Bukau et al., 2000;2006;Mayer et al., 2000;Deuerling & Bukau, 2004;Erbse et al., 2004;Young et al., 2004).
Archaeal organisms form an independent kingdom and possess a mixture of eukaryotic and prokaryotic features.They frequently inhabit environments with extremely high temperatures or very high saline content.Surprisingly, the Hsp70 system is not present in all the archaeal species -it has been found mainly in those which live at moderate temperatures, including the methanogenic archaeon Methanosarcina mazei.It is currently believed that the archaea which possess the DnaK-DnaJ-GrpE chaperoning system gained it by the lateral transfer of the genes from bacteria (Gribaldo et al., 1999;Ma-cario & Conway De Macario, 1999;2001;Macario et al., 2004).This theory is based on the DNA sequence analysis and recently a functional similarity of the M. mazei and E. coli DnaK systems has been shown, which supports the theory.We have found that M. mazei DnaK (DnaK Mm ) and GrpE (GrpE Mm ) are able to function efficiently with the E. coli cochaperone GrpE (GrpE Ec ) and DnaK (DnaK Ec ) in the reactivation of thermally denatured luciferase, and that GrpE Mm can replace GrpE Ec in vivo in the heat shock response and in promotion of bacteriophage λ growth (Zmijewski et al., 2004).The demonstration of these functional in vivo and in vitro interactions indicated that DnaK Mm was able to physically interact with GrpE Ec and, vice versa, DnaK Ec could also interact with GrpE Mm .This was rather surprising, considering the very low identity of the GrpE proteins (26%) and the relatively low identity of the DnaK proteins (56%).
The aim of this work was to further investigate the molecular basis of the interspecies cooperation of the DnaK and GrpE proteins of E. coli and M. mazei.Using Fourier-transform infrared (FT-IR) spectroscopy we were able to show a high similarity of the secondary structures of the bacterial and archaeal DnaKs.Formation of the interspecies DnaK-GrpE complexes was demonstrated by native electrophoresis.Molecular modeling of the M. mazei DnaK domains and of GrpE, basing on the solved structures of their E. coli counterparts, was performed to better understand the experimentally shown similarities and interactions.

MATERIALS AND METHODS
Chemicals.Deuterium oxide (99.9% 2 H 2 O), 2 HCl, and NaO 2 H were purchased from Aldrich (Sigma-Aldrich S.r.l., Milan, Italy).All other chemicals were commercial products of the purest quality purchased from Sigma (Poznań, Poland), or were obtained as indicated in the text.
Proteins and native electrophoresis.DnaK and GrpE proteins from M. mazei and E. coli were purified as described previously (Zmijewski et al., 2004).All proteins were dialyzed against 25 mM Hepes, 100 mM KCl, 10% glycerol, pH 7.2.Protein (> 95% pure) concentrations were determined by the Bradford method (Bradford, 1976) and were confirmed by densitometry of Coomassie-stained sodium dodecyl sulphate polyacrylamide-gel electrophoresis (SDS/PAGE) gels.DnaK preparations were free of ATP, as tested by the malachite green method as we described before (Zmijewski et al., 2004).Native gel electrophoresis was performed using the Laemmli system (Laemmli, 1970) without SDS and without a stacking gel, in 10% resolving gels.Before the native electrophoresis, DnaK Mm , DnaK Ec , GrpE Mm or GrpE Ec were incubated for 30 min in 50 mM Hepes, pH 7.5, 50 mM KCl, and 10 mM MgCl 2 buffer in different combinations as indicated in figure legends.
Protein modeling.Modeling of M. mazei chaperone proteins was done by the Swiss Model server and Deep View/Swiss-PdbViewer (Peitsch, 1996;Guex & Peitsch, 1997).Models of the domains of the DnaK Mm protein were done with E. coli structures used as templates (1DKGD for the ATPase domain, and 1DKXA for the SBD).The model of the GrpE Mm dimer was based on the structure of the E. coli GrpE complex with the ATPase domain of DnaK Ec , and each molecule of the dimer was folded separately (1DKGA, 1DKGB).After modeling, the structures of the M. mazei proteins were minimized with the GROMOS96 force field implementation of Swiss-PdbViewer.
Preparation of samples for infrared spectroscopy.Typically, 1.5 mg of protein, dissolved in the buffer used for its purification, was centrifuged in a "30 K Centricon" micro concentrator (Millipore) at 5 000 × g at 4°C and was concentrated into a volume of 40 µl.Then, 300 µl of 25 mM Hepes, 50 mM NaCl, 3 mM dithiothreitol buffer, prepared in 2 H 2 O p 2 H 7.2, was added and the sample was concentrated again.The p 2 H value corresponds to the pH meter reading +0.4 (Salooma et al., 1964).The concentration-and-dilution procedure was repeated several times in order to completely replace the original buffer with the Hepes buffer.In the last washing, the protein solution was concentrated by decreasing its volume down to 35 µl and was used for FT-IR analysis.
Fourier-transform infrared spectroscopy.The concentrated protein samples were placed in a thermostated Graseby Specac 20500 cell (Graseby-Specac Ltd, Orpington, Kent, UK) fitted with CaF 2 windows and a 25-µm Teflon spacer.FT-IR spectra were recorded by means of a Perkin-Elmer 1760-x Fourier transform infrared spectrometer using a deuterated triglycine sulphate detector and a normal Beer-Norton apodization function.For at least 24 h before and during data acquisition, the spectrometer was continuously purged with dry air at a dew point of −40°C.Spectra of buffers and samples were acquired at 2 cm −1 resolution under the same scanning and temperature conditions.In the thermal denaturation experiments, the temperature was raised in 5°C steps from 20°C to 95°C.The cell was maintained at the desired temperature using an external bath circulator (HAAKE F3), and the actual temperature in the cell was controlled by a thermocouple placed directly onto the window.Spectra were collected and processed using the "Spectrum" software from Perkin Elmer.
Correct subtraction of 2 H 2 O was adjusted to the removal of the 2 H 2 O bending absorption close to 1220 cm -1 (Tanfani et al., 1997).The deconvoluted parameters for the amide I' band were set with a gamma value of 2.5 and a smoothing length of 50.Second-derivative spectra were calculated over a 9data-point range (9 cm −1 ).The midpoint transition in thermal denaturation was calculated as described (Meersman et al., 2002).

Secondary structure and thermal stability of DnaK proteins
We expected that the interspecies interaction of the DnaK and GrpE proteins should be based on a three-dimensional similarity of the archaeal and bacterial proteins.In order to learn more about the overall secondary structures of the DnaK chaperones, we applied Fourier-transform infrared (FT-IR) spectroscopy.
The amide I' band of the spectrum of a protein is broad (1700-1620 cm −1 ), and it is composed of various bands due to the absorption of different secondary-structural elements (Byler & Susi, 1986;Arrondo et al., 1993).The amide I' component bands can be revealed through application of resolutionenhancement methods (deconvolution and/or second derivation) to the original absorbance spectrum (Byler & Susi, 1986;Arrondo et al., 1993).Figure 1A displays the second derivative and deconvolved infrared spectra of DnaK Mm and DnaK Ec in the 1750-1500 cm −1 range.In the 1700-1620 cm −1 interval, the spectra show the same amide I' component bands, which indicates very high similarity of the secondary structures of the DnaKs of M. mazei and E. coli.However, there are some minor differences in the position and intensity of the bands, suggesting small differences in the secondary-structural composition of the two proteins.The 1638.6 (in DnaK Ec ) and 1636.0 cm −1 (in DnaK Mm ) bands are due to β-sheets, whilst the 1650.9(in DnaK Ec ) and 1652.8 cm −1 (in DnaK Mm ) bands are due to α-helices (Arrondo et al., 1993).The position of the β-sheet band in the DnaK Mm spectrum, lower than in the DnaK Ec spectrum, suggests that β-sheets, or portions of them, are more exposed to the solvent ( 2 H 2 O) (Pedone et al., 2003) in DnaK Mm than in DnaK Ec .The position of the α-helix band in the DnaK Ec spectrum, lower than in the DnaK Mm spectrum, suggests that α-helices, or portions of them, are more exposed to the solvent in DnaK Ec than in DnaK Mm .The 1670.1 cm −1 band and the bands close to 1688 and 1680 cm −1 may be due to β-sheets and/or turns (Krimm & Bandekar, 1986;Arrondo et al., 1993).The 1628 cm −1 shoulder, which is absent in the DnaK Mm spectrum, could be due to protein intermolecular interactions, to an unusually strongly hydrogen-bonded β-sheet, or to β-struc-tures interacting strongly with the solvent (particularly solvent-exposed β-strands), in DnaK Ec (Jackson & Mantsch, 1992;Arrondo et al., 1993).Hence, the lack of the 1628 cm −1 band in the DnaK Mm spectrum indicates that the above-mentioned phenomena are absent or very minor in this protein.
The peak at 1547.2 cm −1 represents the residual amide II band (encompassing the 1600-1500 cm −1 interval) absorption, i.e., the absorption of the amide II band after 1 H/ 2 H exchange of the amide hydrogens of the polypeptide chain.The higher intensity of the residual amide II band in the DnaK Mm spectrum indicates that the 1 H/ 2 H exchange in DnaK Mm was less complete than in DnaK Ec .The other bands shown in the 1620-1500 cm −1 interval are due to amino acid side-chain absorption (Barth, 2000).
To obtain more information on DnaK Mm and DnaK Ec structures we compared the thermal denaturation patterns of these proteins.We collected infrared spectra as a function of temperature (in the range 20-95 o C). Figure 1B shows as an example the infrared absorbance spectra of DnaK Mm collected at 20 and 90 o C. The amide I' band intensity (absorbance) decreases with an increase in temperature, whereas the amide I'-band width (wavenumber) increases.An amide I' band shift also occurs.The Circles and squares, DnaK Mm and DnaK Ec , respectively.The t m was calculated from the curves as described (Meersman et al., 2002).The t m for DnaK Mm is 61.0 and that for DnaK Ec is 56.4°C.All determinations and the calculation of arbitrary units (a.u.) were done as described in Materials and Methods.Interaction of DnaK and GrpE proteins from archaea and bacteria thermal denaturation can be followed by measuring the amide I' bandwidth at ¾ height, as marked in Fig. 1B.These parameters were used to calculate the thermal denaturation curves of the proteins, dis-played in Fig. 1C, as done previously (D' Auria et al., 2004).The curves were similar but DnaK Mm was more thermostable than was DnaK Ec : the t m values of the proteins were 61.0°C and 56.4°C, respectively  (Fig. 1C).Since the secondary structures of the two DnaKs analyzed were very similar, the higher t m value of DnaK Mm may reflect the differences in tertiary or quaternary structure with respect to DnaK Ec .The formation of intermolecular interactions (like during aggregation process) is usually accompanied by an increased amide I' bandwidth (D'Auria et al., 2004).Thus, the higher value of the amide I' bandwidth of DnaK Mm at 20 o C as compared to DnaK Ec (Fig. 1C) may indicate that DnaK Mm forms oligomers.However, we must stress that the observed differences are small and the existence of the putative DnaK Mm oligomers requires further studies.
In conclusion, the FT-IR results showed a very high degree of similarity of the secondary structures of the DnaK proteins of M. mazei and E. coli, but with some small differences.

Physical cooperation of DnaK and GrpE
To assess the existence of interspecies "hybrid" DnaK-GrpE complexes, we incubated purified bacterial and archaeal DnaKs and GrpEs together, and then resolved the mixtures by native gel electrophoresis.Results presented in Fig. 2 showed that DnaK Ec formed a complex with GrpE Mm (lane 4), and a similar complex was observed for DnaK Mm and GrpE Ec (lane 8).Incidentally, the formation of the DnaK Ec -GrpE Mm complex was used by us during purification of GrpE Mm ; the latter protein bound efficiently to DnaK Ec -Sepharose affinity columns and, subsequently, the archaeal protein GrpE Mm was released in the presence of ATP (Zmijewski et al., 2004;and this work).DnaK Mm complexed more efficiently with GrpE Mm than with GrpE Ec (Fig. 2, lanes 2 and 8); this could contribute to the species specificity of DnaK Mm observed in vivo (Zmijewski et al., 2004).DnaK Mm migrated more slowly than the lowestmolecular-mass form of DnaK Ec (Fig. 2, lanes 1 and 5).It should be noted that DnaK Mm tends to form highly oligomeric forms, which is visible in Fig. 2, lane 1.Since DnaK Mm has a lower molecular mass than DnaK Ec (DnaK Mm = 66 288 Da; DnaK Ec = 69 076 Da), the slower migration of the lowest molecular form of DnaK Mm is probably due, at least partially, to differences in the shape of the molecules, since the calculated isoelectric points are quite similar (pI of DnaK Mm and DnaK Ec is 4.89 and 4.83, respectively).

DISCUSSION
It is currently believed that the archaeal Hsp70 system arose by lateral transfer of the chaperone genes from bacteria.This hypothesis is based on the sequence analysis of the known Hsp70 genes (Gupta & Singh, 1992;Gribaldo et al., 1999;Macario & Conway De Macario, 1999;Macario et al., 2004).Our previous finding that the DnaK and GrpE proteins of M. mazei can functionally cooperate with the E. coli GrpE and DnaK proteins supported this hypothesis (Zmijewski et al., 2004).The aim of this work was to investigate the molecular basis of the observed interspecies chaperone-co-chaperone interaction.
We have shown, using FT-IR spectroscopy, a high similarity of the secondary structures of the archaeal and bacterial DnaK proteins (Fig. 1A).This forms a good foundation for the observed DnaK-GrpE interspecies cooperation.
To better understand the molecular background of this interaction, we modeled the ATPase (Fig. 3B and C) and SBD (Fig. 3A) domains of DnaK Mm , basing on the solved crystal structures of the respective DnaK Ec domains.We found that superimposition of the ATPase domains from DnaK Mm and DnaK Ec gave a calculated root-mean-square deviation (rmsd) for 347 equivalent backbone atoms (N, Cα, C) of the ATPase domain of DnaK Mm of 0.26 Å (0.22 for Cα).Superimposition of the modeled DnaK Mm SBD and the solved structure of the DnaK Ec SBD gave a calculated rmsd of 0.1 Å for 205 equivalent backbone atoms (N, Cα, C), and 0.09 for the Cα atoms of DnaK Mm SBD.These values indicate a high degree of similarity between DnaK Mm and DnaK Ec both in the ATPase domain (Fig. 3B and C) as well as in the SBD (Fig. 3A).
Since the functional cooperation of DnaK and GrpE requires a physical interaction of DnaK with a GrpE dimer (Schonfeld et al., 1995;Harrison et al., 1997;Harrison, 2003), we have analyzed the formation of the DnaK-GrpE complexes by electrophoresis under non-denaturing conditions.This experiment revealed that indeed the hybrid DnaK Mm -GrpE Ec and DnaK Ec -GrpE Mm complexes were formed efficiently (Fig. 2).Interaction of DnaK Ec with GrpE Ec involves several contact regions in both molecules, as seen from the solved structure of the GrpE Ec dimer bound to the ATPase domain of DnaK Ec (Harrison et al., 1997).DnaK Mm , like bacterial DnaK but unlike eukaryotic Hsp70, possesses in its ATPase domain a conserved loop (circled in Fig. 3B and C), which in bacteria plays a key role in GrpE binding (Buchberger et al., 1994).Other sites involved in DnaK-GrpE interactions are also conserved between the two molecules; for example, the loop formed by amino acids 43 to 47, and the amino acid glycine 32 (not shown), are present both in DnaK Ec and DnaK Mm .Furthermore, the model of GrpE Mm (Fig. 4B), based on the solved structure of the GrpE Ec dimer (Fig. 4A), predicted that the structure of GrpE Mm was very similar, lending support to the observed fact that the archaeal and the bacterial molecules can interact and assemble into a functional chaperone machine, even though the two GrpEs have relatively few amino acids in common.It is worth noting here that the mitochondrial and bacterial GrpEs, in spite of low sequence homology, can be exchanged (Choglay et al., 2001).A comparison of the sequence homology showed that the M. mazei GrpE, like that of the E. coli GrpE, is more similar to isoform 2 than to isoform 1 of the mammalian mitochondrial GrpE.
Apart from the discussed similarity of the DnaK Mm and DnaK Ec structures, we have found some differences.As predicted by sequence data and modeling (Fig. 3B and C), the ATPase domains differ in one significant structural feature.In the DnaK Ec ATPase domain, there is a 24-amino-acid segment (positions 75-98) that forms a loop consisting of two β-strands separated by an α-helix (Fig. 3B  and C).This segment is present in the DnaKs from Gram-negative bacteria and in the Hsp70 of eukaryotes, but it is missing in the DnaKs from Grampositive bacteria and archaea (Macario et al., 1991;Gupta & Singh, 1992).The physiological role of this region is unknown and, to our knowledge, there are no genetic or biochemical data indicating that it is involved in the GrpE binding.
There were some small differences shown by FT-IR spectroscopy.(1) In the DnaK Mm spectrum, one component (representing β-structures) was lacking at 1628 cm −1 , which was present in DnaK Ec (Fig. 1A).This difference may be due to the absence, in the GrpE-binding loop of the DnaK Mm ATP-binding domain, of the β-structure that is present at this location in DnaK Ec (Fig. 3B and C).This putative structural difference might explain why DnaK Mm complexed more efficiently with GrpE Mm than with GrpE Ec (Fig. 2).( 2) The band at 1652.8 cm −1 , representing α-helices, was shifted towards the higher wavenumbers in the DnaK Mm spectrum (Fig. 1A), indicating that these structures are less exposed to the solvent in DnaK Mm than in DnaK Ec .Modeling of the ATPase domain revealed that the 24-amino-acid segment absent in the archaeal protein corresponds to an α-helix that is exposed to the surface (Fig. 3B and  C).The lack of this 24-amino-acid segment may have contributed to the observed band shift.(3) The band at 1636 cm −1 , representing β-structures, was shifted towards the lower wavenumbers in the DnaK Mm spectrum; these β-structures are more exposed to the solvent in DnaK Mm than in DnaK Ec .This difference between the spectra could be due to the exposure of the β-sheets in the ATPase domain of DnaK Mm caused by the absence of the 24-amino-acid α-helix discussed above.It is also possible that the SBD, composed mainly of β-sheets, is more widely open in DnaK Mm than in DnaK Ec .(4) The band at 1547.2 cm −1 , representing the residual amide II band, was higher in the spectrum of DnaK Mm than in the spectrum of DnaK Ec (Fig. 1A), indicating less deuterium exchange for the archaeal protein.DnaK Mm appears to be generally more compact and/or less flexible than DnaK Ec , a possibility supported by the results of thermal-stability measurements that showed a higher temperature stability for DnaK Mm (Fig. 1C).It is possible that the higher thermostability of DnaK Mm might be caused by the fact that DnaK Mm tends to form oligomers (Fig. 2, and results not shown).
The above-discussed small differences between DnaK Mm and DnaK Ec , shown by FT-IR, do not preclude GrpE Ec binding by DnaK Mm , however, they may be one of the reasons why E. coli dnaK mutants are not complemented by the M. mazei dnaK gene (Zmijewski et al., 2004).
In conclusion, the experimental data supported by modeling showed a high similarity of the secondary structures of DnaK Mm and DnaK Ec .In addition, modeling suggested a similarity of the 3dimensional structures of these chaperones, and of the archaeal and bacterial GrpE proteins.We believe that these similarities form the structural basis for the formation of the DnaK-GrpE interspecies hybrid complexes.Our results, showing an overall structural similarity of the bacterial and archaeal DnaK proteins and suggesting such similarity of the GrpE proteins are a further support for the theory of lateral gene transfer from bacteria to archaea.

Figure 1 .
Figure 1.Comparative analyses of DnaK Mm and DnaK Ec secondary structures -FT-IR spectroscopy data.A. Resolution-enhanced spectra of DnaK Mm and DnaK Ec over the range of infrared wavenumbers shown on the horizontal axis.Deconvolved (top graph) and second-derivative (bottom graph) spectra of DnaK Ec (continuous line) and DnaK-Mm (dashed line) at 20°C and p 2 H 7.2.B. Temperature-dependent changes of DnaK Mm infrared spectrum.The absorbance spectra of DnaK Mm at 20 and 90°C are shown.Amide I' bandwidths (measured at ¾ of height) are indicated.C. Thermal denaturation curves of DnaK Mm and DnaK Ec (obtained by measurment of the amide I' bandwidths at ¾ of height).Circles and squares, DnaK Mm and DnaK Ec , respectively.The t m was calculated from the curves as described(Meersman et al., 2002).The t m for DnaK Mm is 61.0 and that for DnaK Ec is 56.4°C.All determinations and the calculation of arbitrary units (a.u.) were done as described in Materials and Methods.

Figure 2 .
Figure 2. Formation of interspecies DnaK-GrpE complexes between the proteins from E. coli (DnaK Ec and GrpE Ec ) and M. mazei (GrpE Mm and DnaK Mm ).The DnaK-GrpE complexes were studied by 10% PAGE under non-denaturing conditions.Three micromoles of DnaK Mm or DnaK Ec were incubated alone, or with GrpE Mm or GrpE Ec (3 µM dimer), in 50 mM Tris pH 7.5, 50 mM KCl and 10 mM MgCl 2 buffer, in combinations shown above the corresponding gel lanes, at 25 o C for 15 min.

Figure 3 .
Figure 3.Comparison of structure of DnaK Ec to model of DnaK Mm .Models of the DnaK Mm (yellow) ATPase and SBD domains were superimposed on the equivalent DnaK Ec domain structure (violet).A. The SBDs of DnaK Mm (yellow) and DnaK Ec (violet).The DnaK Ec SBD (1DKX) was used as a template for modeling of the DnaK Mm SBD.B and C. The predicted DnaK Mm ATPase domain possesses a GrpE-binding loop like that in the DnaK Ec .The ATPase domains of the archaeal and bacterial (1DKGD, used as a template for modeling of the archaeal domain) chaperones, in ribbon display, are shown viewed from the back (B) (as per Harrison et al., 1997), and from the side (C); the GrpE-binding loops are encircled.The 24-amino acid segment of DnaK Ec , shown in red, is absent in DnaK Mm .

Figure 4 .
Figure 4. Comparison of structure of GrpE from E. coli to model of GrpE Mm .The GrpE Ec dimer structure (A).The model of GrpE Mm dimer constructed based on the solved structure of the E. coli DnaK-GrpE complex (1DKG) (B).