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• W2000673732 abstract "The C-terminal, polypeptide binding domain of the 70-kDa molecular chaperone DnaK is composed of a unique lidlike subdomain that appears to hinder steric access to the peptide binding site. We have expressed, purified, and characterized a lidless form of DnaK to test the influence of the lid on the ATPase activity, on interdomain communication, and on the kinetics of peptide binding. The principal findings are that loss of the lid creates an activated form of DnaK which is not equivalent to ATP-bound DnaK. For example, at 25 °C the NR peptide (NRLLLTG) dissociates from the ADP and ATP states of DnaK with observed off-rate constants of 0.001 and 4.8 s−1, respectively. In contrast, for DnaK that lacks most of the helical lid, residues 518–638, the NR peptide dissociates with observed off-rate constants of 0.1 and 188 s−1. These results show that the loss of the lid does not interfere with interdomain communication, that the β-sandwich peptide binding domain can exist in two discrete conformations, and that the lid functions to increase the lifetime of a DnaK·peptide complex. We discuss several mechanisms to explain how the lid affects the lifetime of a DnaK·peptide complex. The C-terminal, polypeptide binding domain of the 70-kDa molecular chaperone DnaK is composed of a unique lidlike subdomain that appears to hinder steric access to the peptide binding site. We have expressed, purified, and characterized a lidless form of DnaK to test the influence of the lid on the ATPase activity, on interdomain communication, and on the kinetics of peptide binding. The principal findings are that loss of the lid creates an activated form of DnaK which is not equivalent to ATP-bound DnaK. For example, at 25 °C the NR peptide (NRLLLTG) dissociates from the ADP and ATP states of DnaK with observed off-rate constants of 0.001 and 4.8 s−1, respectively. In contrast, for DnaK that lacks most of the helical lid, residues 518–638, the NR peptide dissociates with observed off-rate constants of 0.1 and 188 s−1. These results show that the loss of the lid does not interfere with interdomain communication, that the β-sandwich peptide binding domain can exist in two discrete conformations, and that the lid functions to increase the lifetime of a DnaK·peptide complex. We discuss several mechanisms to explain how the lid affects the lifetime of a DnaK·peptide complex. glutathioneS-transferase GST-cleaved DnaK high performance liquid chromatography a synthetic peptide (NRLLLTG) synthetic peptide representing residues 1–12 of the Cro repressor protein (MQERITLKDYAM) wild type α-N, dansyl-NR peptide DnaK, the 70-kDa molecular chaperone expressed byEscherichia coli, functions in protein folding, assembly, transport, and proteolysis in an ATP-dependent activity cycle that is regulated by the two cochaperones, GrpE and DnaJ (1Szabo A. Langer T. Schroder H. Flanagan J. Bukau B. Hartl F.-U. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10345-10349Crossref PubMed Scopus (443) Google Scholar, 2Hartl F.U. Nature. 1996; 381: 571-580Crossref PubMed Scopus (3090) Google Scholar, 3Bukau B. Horwich A.L. Cell. 1998; 92: 351-366Abstract Full Text Full Text PDF PubMed Scopus (2404) Google Scholar, 4Welch W.J. Eggers D.K. Hansen W.J. Nagata H. Fink A.L. Goto U. Molecular Chaperones in the Life Cycle of Proteins. Marcel Dekker, New York1998: 71-93Google Scholar). DnaK also functions in more specialized activities such as bacteriophage λ DNA replication (5Saito H. Uchida H. J. Mol. Biol. 1977; 113: 1-25Crossref PubMed Scopus (92) Google Scholar, 6Yochem J. Uchida H. Sunshine M. Saito H. Georgopoulos C.P. Feiss M. Mol. Gen. Genet. 1978; 164: 9-14Crossref PubMed Scopus (102) Google Scholar). In all of these processes, DnaK is thought to interact transiently with unraveled segments of partially unfolded or denatured protein substrates. Studies have shown that the reversible switching from a high affinity conformation, which binds substrate tightly, to a low affinity conformation, which binds substrate weakly, is the hallmark of DnaK activity (7Palleros D.R. Reid K.L. Shi L. Welch W.J. Fink A.L. Nature. 1993; 365: 664-666Crossref PubMed Scopus (347) Google Scholar, 8Schmid D. Baici A. Gehring H. Christen P. Science. 1994; 263: 971-973Crossref PubMed Scopus (420) Google Scholar, 9Buchberger A. Theyssen H. Schroder H. McCarty J.S. Virgallita G. Milkereit P. Reinstein J. Bukau B. J. Biol. Chem. 1995; 270: 16903-16910Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar, 10Theyssen H. Schuster H.-P. Packschies L. Bukau B. Reinstein J. J. Mol. Biol. 1996; 263: 657-670Crossref PubMed Scopus (199) Google Scholar, 11Slepenkov S.V. Witt S.N. Biochemistry. 1998; 37: 16749-16756Crossref PubMed Scopus (47) Google Scholar). Such a mechanism is shown in Scheme FS1, where ADP·DnaK·P and ATP·DnaK* are the high and low affinity states, respectively, and E and J are GrpE and DnaJ, respectively. In this report, we examine the role of a specific subdomain of DnaK on this conformational switch.DnaK is composed of two functional domains: residues 1–387 comprise the N-terminal ATPase domain, and residues 388–638 comprise the C-terminal polypeptide binding domain. The three-dimensional structure of the ATPase domain of bovine brain Hsc70, a 70-kDa chaperone, is a bilobed structure in which a nucleotide molecule sits at the base of a cleft formed by the two lobes (12Flaherty K.M. DeLuca-Flaherty C. McKay D.B. Nature. 1990; 346: 623-628Crossref PubMed Scopus (823) Google Scholar). The three-dimensional structure of a fragment of the C-terminal polypeptide binding domain comprising residues 387–601 consists of a β-sandwich subdomain that is followed by an α-helical subdomain that acts like a lid over the β-sandwich subdomain (13Morshauser R.C. Wang H. Glynn G.C. Zuiderweg R.P. Biochemistry. 1995; 34: 6261-6266Crossref PubMed Scopus (94) Google Scholar, 14Zhu X. Zhao X. Burkholder W.F. Gragerov A. Ogata C.M. Gottesman M.E. Hendrickson W.A. Science. 1996; 272: 1606-1614Crossref PubMed Scopus (1049) Google Scholar) (Fig. 1). The bound peptide contacts the β-sandwich but not the lid.Figure 1Structure of the polypeptide binding domain (residues 394–607) of DnaK. The bound NR peptide (NRLLLTG) is depicted in black. The arrow indicates residue 517. The image was constructed from PDB file 1DKX.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The molecular structure depicted in Fig. 1, with the lid subdomain blocking entry to and exit from the peptide binding site, has been suggested to be the high affinity conformation of the C-terminal domain of DnaK (14Zhu X. Zhao X. Burkholder W.F. Gragerov A. Ogata C.M. Gottesman M.E. Hendrickson W.A. Science. 1996; 272: 1606-1614Crossref PubMed Scopus (1049) Google Scholar). ATP binding to the N-terminal domain induces a global conformational change in DnaK (7Palleros D.R. Reid K.L. Shi L. Welch W.J. Fink A.L. Nature. 1993; 365: 664-666Crossref PubMed Scopus (347) Google Scholar) which is thought to displace the lid from the top of the β-sandwich subdomain, creating the low affinity form of the protein. To gain insight into the function of the lid, slightly different lidless forms of DnaK and eukaryotic Hsp70s have been engineered (9Buchberger A. Theyssen H. Schroder H. McCarty J.S. Virgallita G. Milkereit P. Reinstein J. Bukau B. J. Biol. Chem. 1995; 270: 16903-16910Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar, 15Misselwitz B. Staeck O. Rapoport T.A. Mol. Cell. 1998; 2: 593-603Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar, 16Fouchaq B. Benaroudj N. Ebel C. Ladjimi M.M. Eur. J. Biochem. 1999; 259: 379-384Crossref PubMed Scopus (33) Google Scholar, 17Pellecchia M. Montgomery D.L. Stevens S.Y. Vander Kooi C.W. Feng H.-P. Gierasch L.M. Zuiderweg E.R.P. Nat. Struct. Biol. 2000; 7: 298-303Crossref PubMed Scopus (179) Google Scholar, 18Mayer M. Schroder H. Rudiger S. Paal K. Laufen T. Bukau B. Nat. Struct. Biol. 2000; 7: 586-593Crossref PubMed Scopus (305) Google Scholar). What has emerged from these studies is that loss of the lid does not interfere with interdomain coupling (15Misselwitz B. Staeck O. Rapoport T.A. Mol. Cell. 1998; 2: 593-603Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar), and loss of the lid results in a 2–20-fold increase inKd values for peptide interaction with the ADP-bound state of lidless DnaK (17Pellecchia M. Montgomery D.L. Stevens S.Y. Vander Kooi C.W. Feng H.-P. Gierasch L.M. Zuiderweg E.R.P. Nat. Struct. Biol. 2000; 7: 298-303Crossref PubMed Scopus (179) Google Scholar, 18Mayer M. Schroder H. Rudiger S. Paal K. Laufen T. Bukau B. Nat. Struct. Biol. 2000; 7: 586-593Crossref PubMed Scopus (305) Google Scholar). The lidless protein reversibly oligomerizes (16Fouchaq B. Benaroudj N. Ebel C. Ladjimi M.M. Eur. J. Biochem. 1999; 259: 379-384Crossref PubMed Scopus (33) Google Scholar). The β-sandwich subdomain can exist in a closed conformation (17Pellecchia M. Montgomery D.L. Stevens S.Y. Vander Kooi C.W. Feng H.-P. Gierasch L.M. Zuiderweg E.R.P. Nat. Struct. Biol. 2000; 7: 298-303Crossref PubMed Scopus (179) Google Scholar). Lidless DnaK(2–538) does not function in anin vitro folding assay (18Mayer M. Schroder H. Rudiger S. Paal K. Laufen T. Bukau B. Nat. Struct. Biol. 2000; 7: 586-593Crossref PubMed Scopus (305) Google Scholar), whereas DnaK(1–507) supports λ phage DNA replication in vivo, albeit at reduced activity relative to the wild type protein (17Pellecchia M. Montgomery D.L. Stevens S.Y. Vander Kooi C.W. Feng H.-P. Gierasch L.M. Zuiderweg E.R.P. Nat. Struct. Biol. 2000; 7: 298-303Crossref PubMed Scopus (179) Google Scholar).The hypothesis tested in this study was that removal of the DnaK lid should produce a constitutive low affinity state of the protein. In other words, if the lid enables high affinity peptide binding to DnaK, then deletion of the lid should produce a state that rapidly binds and rapidly releases peptide, even in the presence of ADP. Such experiments are important because the kinetics of peptide interactions with lidless DnaK is unexplored. To this end, we have expressed, purified, and characterized a lidless variant of DnaK which contains an N-terminal glutathione S-transferase (GST)1tag. Deletion of these residues (see the arrow in Fig. 1) completely eliminates four of the five helices that make up the lid. Because GST domains dimerize (19Maru Y. Afar D.E. Witte O. Shibuya M. J. Biol. Chem. 1996; 271: 15353-15357Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 20Tudyka T. Skerra A. Prot. Sci. 1997; 6: 2180-2187Crossref PubMed Scopus (65) Google Scholar), the GST tag was removed by thrombin cleavage prior to characterization. In agreement with two earlier studies (17Pellecchia M. Montgomery D.L. Stevens S.Y. Vander Kooi C.W. Feng H.-P. Gierasch L.M. Zuiderweg E.R.P. Nat. Struct. Biol. 2000; 7: 298-303Crossref PubMed Scopus (179) Google Scholar, 18Mayer M. Schroder H. Rudiger S. Paal K. Laufen T. Bukau B. Nat. Struct. Biol. 2000; 7: 586-593Crossref PubMed Scopus (305) Google Scholar), we show here that our GST-cleaved DnaK(1–517) is not comparable to the ATP-bound low affinity state of DnaK. Instead, clDnaK(1–517) is an activated state in that the rates of peptide binding and release in the presence of ADP or ATP are increased relative to the wild type protein.RESULTSWild type DnaK and two variants were studied. (i) The primary form of lidless DnaK used in this report was expressed with an N-terminal GST tag that was removed by treatment with thrombin prior to characterization. N-terminal sequence analysis revealed that 10 residues, GSPEFPGRLE, remain attached to the N terminus of DnaK after cleavage of the GST tag. GSPEFPGRLE-DnaK(1–517) is referred to below as clDnaK(1–517). (ii) To control for the effect, if any, of this 10-amino acid prepiece a GST fusion of wtDnaK was also expressed; cleavage of the tag leads to GSPEFPGRLE-DnaK, and this form of the protein is referred to as clDnaKwt.ATPase ActivityThe steady-state ATPase activities of wild type, clDnaK(1–517), and clDnaKwt are compared in Fig.2. In the absence of peptide, the steady-state ATPase activity of wtDnaK was 0.02 ± 0.01 min–1, and added peptide increased this value ∼5-fold to 0.11 ± 0.01 min–1. In contrast, in the absence of peptide clDnaK(1–517) hydrolyzes ATP (0.05 ± 0.015 min–1) about two times faster than wtDnaK, and, in agreement with Pellecchia and co-workers (17Pellecchia M. Montgomery D.L. Stevens S.Y. Vander Kooi C.W. Feng H.-P. Gierasch L.M. Zuiderweg E.R.P. Nat. Struct. Biol. 2000; 7: 298-303Crossref PubMed Scopus (179) Google Scholar), added peptide produces a ∼2-fold stimulation of the ATPase activity of lidless DnaK. As a control, we determined the steady-state ATPase activity of clDnaKwt. The ATPase values with and without peptide are almost identical to those found for clDnaK(1–517). On the basis of these results we conclude that clDnaK(1–517) is fully capable of binding peptide because added peptide stimulates its ATPase activity, and the 10-amino acid prepiece stimulates the endogenous ATPase activity of DnaK by a factor of 2.Figure 2ATPase assay. The ATPase activity of DnaK (wtDnaK, clDnaK(1–517), and clDnaKwt) was measured in the absence or presence of excess peptide. For details on the ATPase assay, see “Experimental Procedures.” Conditions: [protein] = 1–3 µm; [NR peptide] = 200–400 µm; temperature = 25 °C.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Peptide BindingThe effect of nucleotide on equilibrium peptide binding to wild type, clDnaK(1–517), and clDnaKwt is shown in Fig. 3. A spin column assay was used instead of size exclusion column because the former assay enables the separation of relatively short lived DnaK·peptide complexes from free peptide, whereas the latter method requires relatively long lived complexes (t 1/2 > 15 min). The assay was conducted as described under “Experimental Procedures.” A tritiated Cro peptide ([3H]MQERITLKDYAM) was used.Figure 3Equilibrium peptide binding to DnaK variants. A spin column assay was used to measure equilibrium peptide binding to DnaK and its various forms (wtDnaK, clDnaK(1–517), and clDnaKwt). For details regarding the spin column assay, see “Experimental Procedures.” Conditions: [protein] = 4 µm; [[3H]Cro] = 20 µm; temperature = 25 °C.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Wild type DnaK binds the [3H]Cro peptide in a nucleotide-dependent fashion, with high affinity binding and reduced or low affinity binding in the presence of ADP and ATP (Fig. 3), respectively. By comparison, clDnaK(1–517) binds the [3H]Cro peptide with about one-half to one-fourth the affinity as wtDnaK in the presence of ADP. However, similar to wtDnaK, added ATP reduces the amount of peptide binding to the lidless protein. To control for the presence of the prepiece, [3H]Cro peptide binding to clDnaKwt was also examined. This form of DnaK displays high and low affinity binding in the presence of ADP and ATP, respectively. One difference between clDnaK and wtDnaK is that there is about twice the amount of peptide binding in the presence of ATP to the cleaved form of wild type. The reduced amount of [3H]Cro peptide binding to clDnaK(1–517), coupled with the ability of ATP to reduce further the amount of binding, agrees with earlier studies (17Pellecchia M. Montgomery D.L. Stevens S.Y. Vander Kooi C.W. Feng H.-P. Gierasch L.M. Zuiderweg E.R.P. Nat. Struct. Biol. 2000; 7: 298-303Crossref PubMed Scopus (179) Google Scholar, 18Mayer M. Schroder H. Rudiger S. Paal K. Laufen T. Bukau B. Nat. Struct. Biol. 2000; 7: 586-593Crossref PubMed Scopus (305) Google Scholar).Kinetics of DnaK·Peptide Complex Formation/DissociationThe effect of the loss of the lid on the kinetics of DnaK·peptide complex formation and dissociation was determined using a dansyl-labeled form of the NR peptide. A fluorescence spectrometer in time-based mode was used for experiments conducted on wtDnaK and clDnaKwt, and a stopped-flow instrument was used for experiments conducted on clDnaK(1–517).Fig. 4 A compares fNR dissociation from wild type, clDnaKwt, and clDnaK(1–517) in the presence of ADP. Over a time base of 2,000–3,000 s for dissociation from wtDnaK and clDnaKwt and a time base of 100 s for clDnaK(1–517), fNR dissociation follows single exponential kinetics. The dissociation of the fNR peptide from preformed wtDnaK·fNR complexes occurs with an apparent first-order rate constant,k off, equal to 1.0 (± 0.1) × 10–3 s–1. In contrast, the dissociation of the fNR peptide from preformed clDnaK(1–517)·fNR complexes is 100 times faster, occurring with an apparent k offequal to 1.1 (± 0.2) × 10–1 s–1. (An expanded scale version of this dissociation trace is shown in theinset.) As a control, the dissociation of the fNR peptide from clDnaKwt, which, like clDnaK(1–517), possesses a 10-amino acid, N-terminal prepiece, was examined. The fNR peptide dissociates from clDnaKwt with k off = 8.7 (± 0.5) × 10–4 s–1, which demonstrates that the 10-amino acid prepiece does not affect the rate of fNR dissociation from DnaK. The dissociation experiments show that loss of the lid increases the rate of peptide release from ADP-DnaK by 2 orders of magnitude.Figure 4Kinetics of DnaK·peptide complex dissociation. Panel A, comparison of fNR dissociation from wild type, clDnaKwt, and clDnaK(1–517) in the presence of ADP. Experiments were initiated by mixing preformed ADP·wtDnaK·fNR complexes with excess unlabeled NR plus excess ADP. Each dissociation trace was fit to a single exponential function (solid line). wtDnaK·fNR → wtDnaK + fNR, k off = 0.00099 s–1; clDnaKwt·fNR → clDnaKwt + fNR,k off = 0.00093 s–1; and clDnaK(1–517)·fNR → clDnaK(1–517) + fNR,k off = 0.073 s–1. Theinset shows an expanded scale trace for dissociation from clDnaK(1–517). Concentrations after mixing: [protein] = 1–2 µm; [fNR] = 1–2 µm; [NR] = 30–60 µm; [ADP] = 1 mm. Temperature = 25 °C. Panel B, comparison of fNR dissociation from wild type, clDnaKwt, and clDnaK(1–517) in the presence of ATP. Each dissociation trace was fit to a single exponential function (solid line). wtDnaK·fNR → wtDnaK + fNR,k off = 4.8 s–1; clDnaKwt·fNR → clDnaKwt + fNR, k off = 4.1 s–1; and clDnaK(1–517)·fNR → clDnaK(1–517) + fNR,k off = 152 s–1. Theinset shows an expanded scale trace for fNR dissociation from clDnaK(1–517). Concentrations after mixing: [protein] = 1–2 µm; [fNR] = 1–2 µm; [NR] = 30–60 µm; [ATP] = 1 mm. Temperature = 25 °C.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 4 B compares fNR dissociation from wild type, clDnaKwt, and clDnaK(1–517) in the presence of ATP over a time base of 1.5 s. ATP accelerates the rate of fNR dissociation from all three forms of DnaK compared with ADP. For example, the apparent first-order rate constant, k off, for fNR dissociation from wtDnaK and clDnaKwt is 4.8 ± 0.3 s–1 and 4.1 ± 0.3 s–1, respectively. In contrast, fNR dissociates from preformed clDnaK(1–517)·fNR complexes with an apparentk off equal to 188 (± 36) s–1. ATP therefore produces a ∼1,700-fold increase in the rate of peptide dissociation from clDnaK(1–517). The combined results reveal that loss of the lid region of DnaK produces a significant increase in the peptide off-rates.The kinetics of DnaK·peptide complex formation was also investigated (Fig. 5). Representative formation traces for the reaction between fNR and ADP-bound, lidless DnaK are shown in Fig. 5 A. The reactions were carried out at a fixed concentration of the fNR peptide (1.0 µm) while the concentration of wtDnaK was varied (4–20 µm). The relatively large concentration of the fNR peptide was used to obtain an adequate fluorescence signal. The fNR peptide binds to ADP-bound clDnaK(1–517) with double exponential kinetics (F(t) =ΔF1exp[−k obs1 t] +ΔF2exp[-k obs2 t] +F ∞) (Fig. 5 A), consistent with the following sequential two-step mechanism P+ADP·lidless↔k−1k+1(ADP·lidless·P)i↔k−2k+2(ADP·lidless·P)t REACTION1where (ADP·lidless·P)i and (ADP·lidless·P)t denote intermediate and terminal complexes of clDnaK(1–517). For Reaction 1, when the bimolecular reaction P + ADP·lidless ⇔ (ADP·lidless·P)i is faster than the unimolecular reaction (ADP·lidless·P)i ⇔ (ADP·lidless·P)t,k obs1 exhibits a linear dependence on the concentration of lidless DnaK, whereas k obs2exhibits a hyperbolic dependence on the concentration of the lidless protein (29Hiromi K. Kinetics of Fast Enzyme Reactions Theory and Practice. John Wiley & Sons, New York1979Google Scholar). The plot of k obs1 versus [clDnaK(1–517)] (see Fig. 5 B) is linear; the slope and y intercept yield apparent on- and off-rate constants of 11,000 (± 2,000) m–1s–1 and 0.13 (± 0.04) s–1, respectively. This latter value agrees, within the experimental error, with the value of k off1 determined from the direct dissociation experiment (k off1 = 0.11 ± 0.02 s–1) (Table I). The plot ofk obs2 versus [clDnaK(1–517)] (see Fig. 5 B) shows a weak dependence ofk obs on [clDnaK(1–517)], and the asymptote of the plot, k +2 + k –2, equals 0.06–0.07 s–1.Figure 5Kinetics of DnaK·peptide complex formation. Panel A, kinetics of ADP·clDnaK(1–517)·fNR complex formation. The reagents in the two stopped-flow syringes are indicated in brackets: [clDnaK(1–517) + ADP] + [fNR + ADP]. Concentrations after mixing: [fNR] = 1.0 µm, and [clDnaK(1–517)] = 15 (curve a), 10 (curve b), and 5 (curve c) µm; 1 mm ADP. All curves followed double exponential kinetics. Residuals to the fit of curve a to a single and double exponential functions are shown above the panel containing the three curves. The double exponential function,F(t) = 0.104 exp(−0.326t) + 0.169 exp(−0.068t) + 0.342, yielded the best fit.Panel B, plots of k obs1,2 versus [clDnaK(1–517)]. ▪, k obs1 versus [clDnaK(1–517)]. The least squares fit to the equation k obs1 = k +1[clDnaK(1–517)] + k –1 (solid line) yields k +1 = 11,000 ± 2,000m–1 s–1 andk –1 = 0.13 + 0.04 s–1. Δ,k obs2 versus [clDnaK(1–517)]. The asymptote of the curve equals ≈ 0.06–0.07 s–1.Error bars represent the S.E. of duplicates. Panel C, kinetics of ADP·wtDnaK·fNR complex formation. The reagents in the two stopped-flow syringes are indicated in brackets: [wtDnaK + ADP] + [fNR + ADP]. Concentrations after mixing: [fNR] = 0.2 µm, and [wtDnaK] = 8 (curve a), 4 (curve b), and 1 (curve c) µm; 1 mm ADP. Curves follow single exponential kinetics (solid lines). Residuals from the fit of curve aare shown in the panel above the three curves. Panel D, plot of k obs versus [wtDnaK]. Thesolid line is the least squares fit of the data sets to the equation k obs = k +1[wtDnaK] + k –1. ●,k +1 = 910 ± 40m–1 s–1 andk –1 = 0.0019 + 0.0002 s–1. Temperature = 25 °C.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table IKinetic constants for DnaK · peptide complex formation and dissociation in the presence of ADPSpeciesFormation experimentsDirect dissociation experimentsk +1k −1k offm−1s−1s−1s−1wtDnaK910 (±40)0.0019 (±0.0002)0.0010 (±0.0001)clDnaK(1–517)11,000 (±2,000)0.13 (±0.04)0.11 (±0.02)clDnaKwtND1-aND, not determined.ND0.00087 (±0.00005)1-a ND, not determined. Open table in a new tab For comparison, the kinetics of complex formation between the fNR peptide and wtDnaK in the presence of excess ADP was investigated. The reactions were carried out at a fixed concentration of the fNR peptide (0.2 µm) while the concentration of wtDnaK was varied (2–10 µm). Over this range of protein concentrations, the fNR peptide binds to ADP·wtDnaK in one phase (F(t) = ΔFexp[−k obs t] +F ∞) (Fig. 5 C), consistent with complex formation according to Reaction 2. P+ADP·wtDnaK⇔k−1k+1ADP·wtDnaK·P REACTION2Assuming complex formation according to Reaction 2, a plot ofk obs versus [wtDnaK] should be linear, with slope and intercept equal to the apparent on-rate (k +1) and off-rate (k –1) constants, respectively. The plot of k obs versus [wtDnaK] in Fig. 5 D is linear and yields apparent on- and off-rate constants of 910 (± 40)m–1 s–1 and 0.0019 (± 0.0002) s–1, respectively. This latter value is similar to the apparent k off value determined from the direct dissociation experiment (k off = 0.0010 ± 0.0001 s–1) (Table I). These combined kinetic experiments show that deletion of the lid increases the magnitudes of the apparent on- and off-rate constants (k +1 andk –1) by 12- and ∼100-fold, respectively.When complex formation reactions were carried out at a larger fixed concentration of the fNR peptide (1.0 µm) while the concentration of wtDnaK was varied over a larger range (4–20 µm) there was a rapid (k obs ∼ 2 s–1) initial increase in fluorescence which constituted 5% of the total signal change. Based on this observation, it is likely that the reaction between wtDnaK and the fNR peptide is also biphasic, but because of the small amplitude we could not characterize this phase.DISCUSSIONStriking differences were found in the kinetic constants for the interactions of the NR peptide with wtDnaK and lidless DnaK (Table I). Differences in off-rate constants between wild type and lidless DnaK are illustrated in Reactions 3–R6. Reactions 3 and 4 compare peptide dissociation from wild type and clDnaK(1–517) in the presence of ADP, respectively; Reactions 5 and 6 compare peptide dissociation from wild type and clDnaK(1–517) in the presence of ATP, respectively. ADP·wtDnaK·fNR→0.001s−1ADP·wtDnaK+fNR REACTION3 ADP·clDnaK(1–517)·fNR→0.1s−1ADP·clDnaK(1–517)+fNR REACTION4 ATP+wtDnaK·fNR→5s−1ATP·wtDnaK*+fNR REACTION5 ATP+clDnaK(1–517)·fNR→188s−1ATP·clDnaK(1–517)*+fNR REACTION6The above results show that loss of the lid produces an activated form of DnaK in which the rate constants for peptide dissociation are increased substantially compared with wild type. For example, in the presence of ADP the deletion of the lid increasesk off by a factor of 100 (0.001 → 0.1 s–1), whereas in the presence of ATP the deletion of the lid increases k off by a factor of 37.6 (5 → 188 s–1). Further, that the k offvalue for NR peptide dissociation from clDnaK(1–517) is so nucleotide-dependent, increasing from 0.1 to 188 s–1 when ADP is replaced with ATP shows that the β-sandwich subdomain can exist in two distinct conformations, in agreement with earlier studies (17Pellecchia M. Montgomery D.L. Stevens S.Y. Vander Kooi C.W. Feng H.-P. Gierasch L.M. Zuiderweg E.R.P. Nat. Struct. Biol. 2000; 7: 298-303Crossref PubMed Scopus (179) Google Scholar, 18Mayer M. Schroder H. Rudiger S. Paal K. Laufen T. Bukau B. Nat. Struct. Biol. 2000; 7: 586-593Crossref PubMed Scopus (305) Google Scholar). The combined results show that the lid acts like a brake on the rate of ATP-induced peptide dissociation and that the lid and the β-sandwich facilitate peptide binding to DnaK.One of the findings in this report is that the presence of DnaK lid increases the lifetime of a DnaK·peptide complex. For example, in the presence of ATP the lifetime, τ, of an ATP·DnaK*·fNR complex is 140 ms (ln 2/5 s–1), whereas deletion of the lid decreases the lifetime to 3.7 ms (ln 2/188 s–1). To explain the effect of the lid on the lifetime of the ATP·DnaK·peptide complex, below we discuss several variations of a two-step reaction mechanism that involves sequential conformational changes in the lid and β-sandwich subdomains. For simplicity, only forward reactions, i.e. closed-to-open transitions in the β-sandwich (βc→o) and lid (lc→o), are depicted.If the β-sandwich and lid subdomains are uncoupled in that deletion of the lid does not affect the rate constants for the βc→o or βo→ctransitions, then our results are consistent with Reaction 7, where ATP binding triggers conformational changes first in the β-sandwich and then in the lid ATP+Eβclc·P→188s−1ATP·Eβolc·P→5s−1ATP·Eβolo+P REACTION7where Eβclc·P is the high affinity wtDnaK·peptide complex in which both the β-sandwich and lid are closed; ATP·Eβolc·P is an intermediate in which the β-sandwich and lid are open and closed, respectively; and ATP·Eβolo is the low affinity state in which both the β-sandwich and lid subdomains are open. Note that the second step in Reaction 7, lid opening (k lc→o = 5 s–1), is the rate-limiting step for peptide dissociation peptide. Upon deletion of the lid peptide dissociation is governed by the βc→o transition, therefore the maximal rate of peptide dissociation jumps from 5 to 188 s–1.Because at present we cannot determine the sequence of the conformational changes upon ATP binding to DnaK, it must be noted that our results are also consistent with Reaction 8, where ATP binding triggers conformational changes first in the lid and then in the β-sandwich. ATP+Eβclc·P→5s−1ATP·Eβclo·P→188s−1ATP·Eβolo+P REACTION8In the above reaction, the first step, lid opening (k lc→o = 5 s–1), is the rate-limiting step for peptide dissociation peptide from wtDnaK. Upon deletion of the lid peptide dissociation becomes governed by the βc→o transition, therefore the maximal rate of peptide dissociation jumps from 5 to 188 s–1.Alternatively, suppose the β-sandwich and lid" @default.
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• W2000673732 title "Characterization of a Lidless Form of the Molecular Chaperone DnaK" @default.
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