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, σ32, 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. 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, σ32, 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. Central to the chaperone functions of Hsp70 proteins is the transient interaction of their C-terminal substrate-binding domain with short peptide stretches within substrate polypeptides (1Bukau B. Horwich A.L. Cell. 1998; 92: 351-366Abstract Full Text Full Text PDF PubMed Scopus (2425) Google Scholar, 2Mayer M.P. Rüdiger S. Bukau B. Biol. Chem. 2000; 381: 877-885Crossref PubMed Scopus (109) Google Scholar, 3Hartl F.U. Hayer-Hartl M. Science. 2002; 295: 1852-1858Crossref PubMed Scopus (2786) Google Scholar). This interaction is controlled by the nucleotide status of the N-terminal ATPase domain of Hsp70, which in turn is regulated by co-chaperones of the DnaJ family and for some Hsp70s by nucleotide exchange factors (4Cheetham M.L.E. Caplan A.J. Cell Stress Chaperones. 1998; 3: 28-36Crossref PubMed Scopus (488) Google Scholar, 5Laufen T. Zuber U. Buchberger A. Bukau B. Fink A.L. Goto Y. Molecular Chaperones in Proteins: Structure, Function, and Mode of Action. Marcel Dekker, Inc., New York1998: 241-274Google Scholar, 6Kelley W.L. Curr. Biol. 1999; 9: R305-R308Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 7Mayer M.P. Brehmer D. Gassler C.S. Bukau B. Adv. Protein Chem. 2002; 59: 1-44Google Scholar). It is still an unsolved question as to why for some Hsp70s nucleotide exchange factors are essential for their activity in vivo and in vitro while for others nucleotide exchange factors are not essential or do not exist (8Burkholder W.F. Gottesman M.E. Bukau B. Molecular Chaperones and Folding Catalysts. Regulation, Cellular Function and Mechanisms. Harwood Academic Publishers, Amsterdam1999: 105-138Google Scholar, 9Georgopoulos C. Liberek K. Zylicz M. Ang D. Morimoto R.I. Tissières A. Georgopoulos C. The Biology of Heat Shock Proteins and Molecular Chaperones. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1994: 209-250Google Scholar, 10Höhfeld J. Jentsch S. EMBO J. 1997; 16: 6209-6216Crossref PubMed Scopus (338) Google Scholar, 11Gässler C.S. Wiederkehr T. Brehmer D. Bukau B. Mayer M.P. J. Biol. Chem. 2001; 276: 32538-32544Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). It has been shown recently that the nucleotide dissociation rates of Hsp70 proteins vary over a range of almost 3 orders of magnitude (12Brehmer D. Rüdiger S. Gässler C.S. Klostermeier D. Packschies L. Reinstein J. Mayer M.P. Bukau B. Nat. Struct. Biol. 2001; 8: 427-432Crossref PubMed Scopus (189) Google Scholar). The exchange rate of the Hsp70 homologue with the highest exchange rate was nearly as high as the exchange rate of the Hsp70 with the lowest basal exchange rate in the presence of saturating amounts of its exchange factor. This demonstrates that the molecular structure of Hsp70s does not have to be limiting for nucleotide exchange rates and suggests that an optimal exchange rate has been evolved for each system. Therefore, the question arises why nucleotide exchange factors exist and whether they have additional functions. For the essential nucleotide exchange factor of Escherichia coli DnaK (GrpE), it was suggested that it also accelerates substrate release in a nucleotide-independent manner (13Harrison C.J. Hayer-Hartl M. Di Liberto M. Hartl F.-U. Kuriyan J. Science. 1997; 276: 431-435Crossref PubMed Scopus (413) Google Scholar). This hypothesis was conceived first on the basis of the unusual structure of GrpE, the N-terminal part of which forms a long α-helix of 100 Å in length (amino acids 34–106) (13Harrison C.J. Hayer-Hartl M. Di Liberto M. Hartl F.-U. Kuriyan J. Science. 1997; 276: 431-435Crossref PubMed Scopus (413) Google Scholar). This helix extends well beyond the limits of the ATPase domain, suggesting that it might interact with the substrate-binding domain of DnaK (13Harrison C.J. Hayer-Hartl M. Di Liberto M. Hartl F.-U. Kuriyan J. Science. 1997; 276: 431-435Crossref PubMed Scopus (413) Google Scholar). The N-terminal 33 amino acids of GrpE that were unstructured and had to be removed for crystallization were proposed to be responsible for the interaction with the substrate-binding domain of DnaK and for influencing substrate release (13Harrison C.J. Hayer-Hartl M. Di Liberto M. Hartl F.-U. Kuriyan J. Science. 1997; 276: 431-435Crossref PubMed Scopus (413) Google Scholar). In a later study (14Mally A. Witt S.N. Nat. Struct. Biol. 2001; 8: 254-257Crossref PubMed Scopus (35) Google Scholar), it was claimed that even in the absence of added ATP, GrpE was able to stimulate substrate dissociation and association by 200- and 60-fold under saturating conditions. Whereas Mally and Witt (14Mally A. Witt S.N. Nat. Struct. Biol. 2001; 8: 254-257Crossref PubMed Scopus (35) Google Scholar) found the GrpE-mediated substrate release in the absence of nucleotide and in the presence of ATP, Han and Christen (15Han W. Christen P. Biochem. J. 2003; 369: 627-634Crossref PubMed Scopus (29) Google Scholar) claim that the stimulatory effect only occurs in the presence of ADP and not in the absence of the nucleotide. Finally, it has been suggested that GrpE acts as thermometer, because its activity as a nucleotide exchange factor for DnaK is strongly thermosensitive (16Grimshaw J.P. Jelesarov I. Schönfeld H.J. Christen P. J. Biol. Chem. 2001; 276: 6098-6104Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 17Grimshaw J.P. Jelesarov I. Siegenthaler R.K. Christen P. J. Biol. Chem. 2003; 278: 19048-19053Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 18Groemping Y. Reinstein J. J. Mol. Biol. 2001; 314: 167-178Crossref PubMed Scopus (56) Google Scholar, 19Gelinas A.D. Langsetmo K. Toth J. Bethoney K.A. Stafford W.F. Harrison C.J. J. Mol. Biol. 2002; 323: 131-142Crossref PubMed Scopus (35) Google Scholar, 20Gelinas A.D. Toth J. Bethoney K.A. Langsetmo K. Stafford W.F. Harrison C.J. Biochemistry. 2003; 42: 9050-9059Crossref PubMed Scopus (23) Google Scholar). Accordingly, above 45 °C, GrpE would be poorly active, which leads to a stabilization of DnaK-substrate complexes. To further investigate potential novel functions of GrpE in more detail, we analyzed the effect of GrpE on the binding equilibrium and on the association and dissociation kinetics of the interaction of DnaK with peptide and proteins substrates. In addition, we removed the N-terminal 33 amino acids of GrpE (ΔNGrpE) to analyze the effects of the N terminus on peptide binding and release. Reagents—Unless indicated otherwise, all of the chemicals were of the highest purity available. ATP-agarose (A2767) and luciferase (L9506) were from Sigma. Luciferin was from Fluka. Peptides—Peptides σ32-Q132-Q144-C (QRKLFFNLRKTKQC (21McCarty J.S. Rüdiger S. Schönfeld H.-J. Schneider-Mergener J. Nakahigashi K. Yura T. Bukau B. J. Mol. Biol. 1996; 256: 829-837Crossref PubMed Scopus (95) Google Scholar)), NR (NRLLLTGC (22Gragerov A. Gottesman M.E. J. Mol. Biol. 1994; 241: 133-135Crossref PubMed Scopus (79) Google Scholar)), and vesicular stomatitis virus glycoprotein (VSV-GC) 1The abbreviations used are: VSV-GC, vesicular stomatitis virus glycoprotein; GdnCl, guanidine chloride; IPTG, isopropyl-1-thio-β-d-galactopyranoside; AANS, 2-(4-anilino)naphthalene-6-sulfonic acid. (KLIGVLSSLFRPK (23Flynn G.C. Chappell T.G. Rothman J.E. Science. 1989; 245: 385-390Crossref PubMed Scopus (579) Google Scholar, 24Landry S.J. Jordan R. McMacken R. Gierasch L.M. Nature. 1992; 355: 455-457Crossref PubMed Scopus (257) Google Scholar)) were synthesized chemically using standard chemistry and purified by high pressure liquid chromatography. The peptides were labeled using 2-(4-(iodoacetamido)anilino)naphthalene-6-sulfonic acid (Molecular Probes) (σ32-Q132-Q144-CAANS), dansyl chloride (D-NR, Molecular Probes), and N-succinimidyl-[2,3-3H]propionate (Amersham Biosciences) ([3H]VSV-GC) according to the manufacturer's recommendations and separated from the unreacted label by gel filtration on a G25-Sephadex column (5 × 300 mm). Proteins—DnaK, DnaJ, and GrpE were expressed and purified according to published protocols (25Buchberger A. Schröder M. A. Bukau B. Nat. Struct. Biol. 1994; PubMed Scopus Google Scholar, M. T. S. Georgopoulos C. J. Biol. Chem. Full Text PDF PubMed Google Scholar, H.-J. D. Schröder Bukau B. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, H.-J. D. M. Google Scholar). nucleotide was removed as Bukau B. Reinstein J. J. Mol. Biol. 1996; PubMed Scopus Google Scholar). was from purified GrpE by with according to Harrison (13Harrison C.J. Hayer-Hartl M. Di Liberto M. Hartl F.-U. Kuriyan J. Science. 1997; 276: 431-435Crossref PubMed Scopus (413) Google Scholar), purified by exchange and gel filtration as for GrpE, and by was in a T. T. Bukau B. M. K. T. Mol. 1998; PubMed Scopus Google Scholar) and purified as T. T. Bukau B. J. 2001; PubMed Scopus Google Scholar). and were labeled using N-succinimidyl-[2,3-3H]propionate a for label was removed by The protein was by the protein and the was by and the activity and was and dissociation kinetics using and peptides and were as C.S. Wiederkehr T. Brehmer D. Bukau B. Mayer M.P. J. Biol. Chem. 2001; 276: 32538-32544Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, M.P. Schröder Rüdiger S. K. T. Bukau B. Nat. Struct. Biol. 2000; PubMed Scopus Google Scholar, J.S. Buchberger A. Reinstein J. Bukau B. J. Mol. Biol. PubMed Scopus Google Scholar) with the that GrpE was added to the or with the peptide the indicated using a or a for the For of association peptides were added to DnaK in with or in the absence or presence of GrpE the indicated were as L. Buchberger A. Bukau B. Reinstein J. Biochemistry. 1997; PubMed Scopus Google Scholar) by of DnaK or with or peptides with Protein substrate dissociation equilibrium were according to a published M.P. T. K. J.S. Bukau B. J. Mol. Biol. 1999; PubMed Scopus Google Scholar). and DnaK were in the absence or presence of GrpE in a of of for and of was added and and were separated by gel filtration over a column (Amersham and in T. of in were and was by For dissociation and DnaK were in the absence or presence of GrpE in a of of for of were and the was further for the indicated by gel filtration as of and were as T. Hartl F.-U. Bukau B. EMBO J. PubMed Scopus Google Scholar) using with and a of was in the presence or absence of for °C, and was by the to were using a and a with and of or were by the with a rate of of or was by a of to with or were by from to 3 and 3 to of the protein were for For each and were a rate of and the of the was its of GrpE on investigate the of GrpE on the of DnaK-substrate we GrpE to a complex of with a which was labeled a C-terminal with 2-(4-(iodoacetamido)anilino)naphthalene-6-sulfonic acid (σ32-Q132-Q144-CAANS), and the of the shown in the of the peptide with of This could have GrpE could the or GrpE could the DnaK-substrate To the first we the with a peptide ([3H]VSV-GC) and separated and peptides by chromatography. This that the of complex with GrpE GrpE on the of in the of in the equilibrium of the DnaK-substrate complex could from an in the complex dissociation rate as claimed (13Harrison C.J. Hayer-Hartl M. Di Liberto M. Hartl F.-U. Kuriyan J. Science. 1997; 276: 431-435Crossref PubMed Scopus (413) Google Scholar, A. Witt S.N. Nat. Struct. Biol. 2001; 8: 254-257Crossref PubMed Scopus (35) Google Scholar, W. Christen P. Biochem. J. 2003; 369: 627-634Crossref PubMed Scopus (29) Google Scholar) or a in the association the dissociation rate of the complex of DnaK with in the absence and presence of GrpE protein and the and GrpE accelerate the substrate dissociation rate in the absence of nucleotide or in the presence of which had been purified from ATP by high pressure liquid A and when the complex was with GrpE ATP a of the substrate release was were when we used a peptide that was labeled its N terminus with a dansyl or the peptide not To demonstrate the of the ternary complex of a GrpE with DnaK and a we on a which is able to and GrpE was added to a complex of DnaK with and a analyzed by gel amounts of were found in the ternary complex in the presence of GrpE as in the complex in the absence of GrpE was the of the peptide with DnaK, only amounts of the of the and ternary complexes. GrpE the of with the association of the peptide with DnaK was by the presence of GrpE not by the presence of shown in the of that was in the of the association was with GrpE a of to DnaK, the of was of the in the absence of GrpE, and a of to DnaK, almost was the association rates were not by the presence of GrpE were with peptides with This that GrpE to DnaK the association of peptide It why of GrpE to a in the of and observed equilibrium with the gel filtration demonstrate that GrpE slows down peptide association to DnaK and that a ternary complex only forms when peptide first to rate of association is independent of in a of GrpE on the of DnaK with Protein suggest that in the the N of the GrpE are in to the DnaK substrate-binding the N-terminal 33 amino acids of GrpE are to be they in a peptide the substrate-binding of GrpE the substrate-binding it the association of protein substrates to DnaK as To we coli factor with DnaK in the absence and presence of of and analyzed the by chromatography. In the absence of GrpE, of were the of and the of the In the presence of of GrpE, the the of the complex and an additional This to the ternary complex of a GrpE with DnaK and The the in the presence and absence of of the in all of the that the of and not as as in the of the peptides and from that the of DnaK for is as well in the presence of GrpE, not to the as found for peptide substrates. To investigate in more detail, we the dissociation rate of the complex in the absence and presence of shown in the in of DnaK for in the presence of GrpE is not the of an in the dissociation the GrpE a in the dissociation rate of σ32, a of This in was observed of the when GrpE was added whether it the of the with the labeled or with the The only observed was that the of was when GrpE was added the of the with the ATP was added with the σ32, or could be independent of whether GrpE was or to DnaK the of GrpE as with the of the suggest that the N terminus of GrpE is in of the substrate-binding Therefore, we that GrpE would be able to the status of the substrate-binding domain and that substrate could in have an on the nucleotide exchange activity of To we the or nucleotide exchange rates in the absence and presence of a peptide or protein For we DnaK with the labeled ADP Bukau B. Reinstein J. J. Mol. Biol. 1996; PubMed Scopus Google Scholar) in the absence or presence of the peptide substrate σ32-Q132-Q144-C or the protein substrate for and in a with ATP. in the nucleotide exchange rates were observed not that the status of the substrate-binding domain does not the nucleotide exchange of The of the N terminus of GrpE and the substrate that is in the substrate-binding domain of DnaK could have an on the chaperone of Therefore, we the of DnaK and DnaJ in the luciferase in the presence of GrpE or was for in the presence of DnaK, DnaJ, and GrpE or and to The of activity and the of activity to were GrpE and could be of the the of the N-terminal 33 amino acids in we to the of a by GrpE or from an °C, the the or the well all of the In the the not of 100 and °C, the the not The the in the absence of and not of of 100 or The the only in the presence of 100 or more To we the in vivo of GrpE and in the the through using found that GrpE was even in the absence of IPTG, because of of the with as observed in on 100 and the GrpE were by over the GrpE of the This of GrpE the because an of GrpE is to the chaperone of the DnaK L. Buchberger A. Bukau B. Reinstein J. Biochemistry. 1997; PubMed Scopus Google Scholar). In the of was even still the of GrpE in the absence of Therefore, the for the in their to the of the was to the of the of protein and not to the in their of the of a in a GrpE Folding by N-terminal were expressed from the in and structure not in the we that the N terminus of GrpE the folding of To we and and the of GrpE by and The kinetics of GrpE and not The Whereas GrpE under the more and only a of the and could be and not findings that GrpE and not the association of peptides to DnaK while it is of binding to a complex to a ternary complex could be in the N terminus of GrpE with the substrate-binding domain in a that the of the substrate-binding is the 33 N-terminal are in or interact with the substrate-binding The first hypothesis is because GrpE had on the dissociation of which would be GrpE would the substrate-binding domain in its The hypothesis is by the that the N terminus of GrpE is unstructured and could an The published for the of does not a high within the N-terminal of GrpE, in because of the protein is in S. L. Schneider-Mergener J. Bukau B. EMBO J. 1997; 16: PubMed Scopus Google Scholar). However, a high might not be because GrpE with a of to the ATPase domain, and when the N terminus in of the substrate-binding even a the substrate-binding because of the high of peptide with hypothesis are the of a by Witt and Witt S.N. Biochemistry. 2003; 42: PubMed Scopus Google Scholar) in which they demonstrate that GrpE not with with a substrate-binding domain of DnaK This interaction with the substrate-binding domain, does not to the of the interaction because with to DnaK as GrpE and of an in the ATPase domain strongly the binding of GrpE to DnaK (25Buchberger A. Schröder M. A. Bukau B. Nat. Struct. Biol. 1994; PubMed Scopus Google Scholar, Witt S.N. Biochemistry. 2003; 42: PubMed Scopus Google Scholar). nucleotide exchange factors of Hsp70s also interact with the substrate-binding domain of their protein has not been and others M.P. T. K. J.S. Bukau B. J. Mol. Biol. 1999; PubMed Scopus Google Scholar, T. Mayer M.P. C. Klostermeier D. Reinstein J. Bukau B. U. S. A. 1999; PubMed Scopus Google Scholar, J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar) show that DnaJ also with the substrate-binding of DnaK, with the interaction occurs in the ATPase domain C.S. Buchberger A. T. Mayer M.P. Schröder A. Bukau B. U. S. A. 1998; PubMed Scopus Google Scholar, W.F. Gottesman M.E. U. S. A. 1998; PubMed Scopus Google Scholar, K. S.J. U. S. A. 1998; PubMed Scopus Google Scholar, S.J. Biochemistry. 2003; 42: PubMed Scopus Google Scholar, P. J. S.J. Biochemistry. 2003; 42: PubMed Scopus Google Scholar). It is that co-chaperones that the ATPase of DnaK also interact with the substrate-binding ATP binding and hydrolysis not only in the ATPase domain of DnaK also in the substrate-binding domain, an interaction of with of DnaK to the of the of the substrate-binding domain of DnaK association of ATP to its ATPase domain to the release of This might be the that GrpE with high to the ATPase domain in the ADP has a dissociation rate Witt S.N. Biochemistry. 2003; 42: PubMed Scopus Google Scholar). The dissociation rate of GrpE from DnaK the of ATP has not been A of study was that the dissociation rate of the complex of DnaK with a protein substrate was down in the presence of This under the of high ATP the release of the substrate and the release of GrpE are and when the ATP might substrate dissociation is down further through the of under the ATP chaperone is not the release of a protein substrate would to its with high it was shown recently that substrate release from DnaK a high above 45 °C, when for to the are also is down because of a of GrpE, which the to trigger nucleotide exchange (16Grimshaw J.P. Jelesarov I. Schönfeld H.J. Christen P. J. Biol. Chem. 2001; 276: 6098-6104Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 17Grimshaw J.P. Jelesarov I. Siegenthaler R.K. Christen P. J. Biol. Chem. 2003; 278: 19048-19053Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 18Groemping Y. Reinstein J. J. Mol. Biol. 2001; 314: 167-178Crossref PubMed Scopus (56) Google Scholar). findings that the N-terminal 33 amino acids are for the folding of GrpE are with which is essential for the nucleotide exchange of GrpE, with an interaction of the N-terminal 33 amino acid of with each and through the long in a fashion. independent claim that GrpE accelerates substrate binding to and the release from DnaK independent of its nucleotide exchange (14Mally A. Witt S.N. Nat. Struct. Biol. 2001; 8: 254-257Crossref PubMed Scopus (35) Google Scholar, W. Christen P. Biochem. J. 2003; 369: 627-634Crossref PubMed Scopus (29) Google Scholar). findings are in to first that GrpE might have a of Therefore, we the of protein the was using the of and the nucleotide exchange activity was in of the ADP and ATP dissociation rates with GrpE as L. Buchberger A. Bukau B. Reinstein J. Biochemistry. 1997; PubMed Scopus Google Scholar) or by the ATPase rates of DnaK in the presence of DnaJ and the of GrpE also was shown in chaperone by the and and chemically Finally, for we used independent protein purified GrpE from to with DnaK, we also purified it from a in that DnaK might be for the folding of DnaK was in removed by with an To that GrpE is not with ATP, we ATP by the luciferase and found that the is of the GrpE GrpE was purified in a of H.-J. which also the GrpE that was used in of the above (15Han W. Christen P. Biochem. J. 2003; 369: 627-634Crossref PubMed Scopus (29) Google Scholar). could not that GrpE are and that be a for The (14Mally A. Witt S.N. Nat. Struct. Biol. 2001; 8: 254-257Crossref PubMed Scopus (35) Google Scholar, W. Christen P. Biochem. J. 2003; 369: 627-634Crossref PubMed Scopus (29) Google Scholar) the substrate release of GrpE are not Whereas Mally and Witt (14Mally A. Witt S.N. Nat. Struct. Biol. 2001; 8: 254-257Crossref PubMed Scopus (35) Google Scholar) show that GrpE peptide release in the absence of nucleotide as well as in the presence of Han and Christen (15Han W. Christen P. Biochem. J. 2003; 369: 627-634Crossref PubMed Scopus (29) Google Scholar) could only the GrpE of substrate release in the presence of DnaK has a for ATP for ADP Bukau B. Reinstein J. J. Mol. Biol. 1996; PubMed Scopus Google Scholar, R. Jordan R. McMacken R. Biochemistry. 1998; PubMed Scopus Google Scholar), of the ADP with ATP could the because GrpE would accelerate ADP release and ATP would to DnaK substrate release. found that ADP is with ATP and ADP by high liquid chromatography. a of ADP with ATP, could not the of Mally and Witt (14Mally A. Witt S.N. Nat. Struct. Biol. 2001; 8: 254-257Crossref PubMed Scopus (35) Google Scholar), that GrpE the substrate release from DnaK in the absence of nucleotide. Mally and Witt (14Mally A. Witt S.N. Nat. Struct. Biol. 2001; 8: 254-257Crossref PubMed Scopus (35) Google Scholar) purified their GrpE by on DnaK according to Zylicz M. Ang D. Georgopoulos C. J. Biol. Chem. Full Text PDF PubMed Google Scholar). In GrpE is with a ATP and is not as with of amounts of ATP can in the GrpE which would be able to trigger substrate release by on and we that GrpE does not have the claimed to trigger substrate release from DnaK even in the absence of nucleotide. GrpE the dissociation of protein substrates in the absence of ATP, and it is that a dissociation of GrpE and substrate occurs when ATP to the ATPase domain of K. for and H.-J. Schönfeld for the of