Discrimination of ATP, ADP, and AMPPNP by Chaperonin GroEL
2003; Elsevier BV; Volume: 278; Issue: 29 Linguagem: Inglês
10.1074/jbc.m300806200
ISSN1083-351X
AutoresFumihiro Motojima, Masasuke Yoshida,
Tópico(s)Enzyme Structure and Function
ResumoThe double ring chaperonin GroEL binds unfolded protein, ATP, and GroES to the same ring, generating the cis ternary complex in which folding occurs within the cavity capped by GroES (cis folding). The functional role of ATP, however, remains unclear since several reports have indicated that ADP and AMPPNP (5′-adenylyl-β,γ-imidodiphosphate) are also able to support the formation of the cis ternary complex and the cis folding. To minimize the effect of contaminated ATP and adenylate kinase, we have included hexokinase plus glucose in the reaction mixtures and obtained new results. In ADP and AMPPNP, GroES bound quickly to GroEL but bound very slowly to the GroEL loaded with unfolded rhodanese or malate dehydrogenase. ADP was unable to support the formation of cis ternary complex and cis folding. AMPPNP supported cis folding of malate dehydrogenase to some extent but not cis folding of rhodanese. In the absence of hexokinase, apparent cis folding of rhodanese and malate dehydrogenase was observed in ADP and AMPPNP. Thus, the exclusive role of ATP in generation of the cis ternary complex is now evident. The double ring chaperonin GroEL binds unfolded protein, ATP, and GroES to the same ring, generating the cis ternary complex in which folding occurs within the cavity capped by GroES (cis folding). The functional role of ATP, however, remains unclear since several reports have indicated that ADP and AMPPNP (5′-adenylyl-β,γ-imidodiphosphate) are also able to support the formation of the cis ternary complex and the cis folding. To minimize the effect of contaminated ATP and adenylate kinase, we have included hexokinase plus glucose in the reaction mixtures and obtained new results. In ADP and AMPPNP, GroES bound quickly to GroEL but bound very slowly to the GroEL loaded with unfolded rhodanese or malate dehydrogenase. ADP was unable to support the formation of cis ternary complex and cis folding. AMPPNP supported cis folding of malate dehydrogenase to some extent but not cis folding of rhodanese. In the absence of hexokinase, apparent cis folding of rhodanese and malate dehydrogenase was observed in ADP and AMPPNP. Thus, the exclusive role of ATP in generation of the cis ternary complex is now evident. The bacterial chaperonin system consisting of GroEL and GroES facilitates folding of other proteins using the energy of ATP hydrolysis. GroEL is composed of 14 identical 57-kDa subunits, each containing a site for binding and hydrolysis of ATP. Seven GroEL subunits are arranged in a heptamer ring forming a central cavity, and two heptamer rings are stacked back to back. GroES is a dome-shaped, single heptamer ring of 10-kDa subunits. GroEL binds a wide range of unfolded proteins at the apical cavity surface and subsequently binds ATP and GroES to the same GroEL ring (the cis ring, a GroEL heptamer ring that binds to GroES), producing the complex consisting of GroEL, unfolded protein, and GroES (the cis ternary complex). Since the residues of the GroEL apical surface involved in GroES binding are mostly overlapped with substrate protein binding (1Fenton W.A. Kashi Y. Furtak K. Horwich A.L. Nature. 1994; 371: 614-619Crossref PubMed Scopus (575) Google Scholar), GroES binding results in encapsulating unfolded protein into the enlarged cavity of the cis GroEL ring capped by GroES (the cis cavity) (2Weissman J.S. Hohl C.M. Kovalenko O. Kashi Y. Chen S. Braig K. Saibil H.R. Fenton W.A. Horwich A.L. Cell. 1995; 83: 577-587Abstract Full Text PDF PubMed Scopus (391) Google Scholar). The unfolded protein initiates folding in the cis cavity without a risk of aggregation (the cis folding). ATP hydrolysis in the cis ring and subsequent ATP binding to the opposite side of GroEL ring (the trans ring) induce the release of GroES, ADP, and substrate protein (whether folded or not) from the cis ring (3Rye H.S. Burston S.G. Fenton W.A. Beechem J.M. Xu Z. Sigler P.B. Horwich A.L. Nature. 1997; 388: 792-798Crossref PubMed Scopus (357) Google Scholar, 4Rye H.S. Roseman A.M. Chen S. Furtak K. Fenton W.A. Saibil H.R. Horwich A.L. Cell. 1999; 97: 325-338Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar). When unfolded protein is added to the GroEL-GroES complex, it binds to the trans ring, and its folding is arrested (2Weissman J.S. Hohl C.M. Kovalenko O. Kashi Y. Chen S. Braig K. Saibil H.R. Fenton W.A. Horwich A.L. Cell. 1995; 83: 577-587Abstract Full Text PDF PubMed Scopus (391) Google Scholar). Binding and release from the trans ring enable the folding for some proteins (2Weissman J.S. Hohl C.M. Kovalenko O. Kashi Y. Chen S. Braig K. Saibil H.R. Fenton W.A. Horwich A.L. Cell. 1995; 83: 577-587Abstract Full Text PDF PubMed Scopus (391) Google Scholar), especially large ones that are too large to be encapsulated in the cis cavity, by lowering the concentration of aggregation-prone folding intermediates in bulk solution (5Chaudhuri T.K. Farr G.W. Fenton W.A. Rospert S. Horwich A.L. Cell. 2001; 107: 235-246Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). In contrast, the stringent substrate proteins for chaperonin fold efficiently by the cis folding in the presence of ATP (3Rye H.S. Burston S.G. Fenton W.A. Beechem J.M. Xu Z. Sigler P.B. Horwich A.L. Nature. 1997; 388: 792-798Crossref PubMed Scopus (357) Google Scholar, 6Burston S.G. Weissman J.S. Farr G.W. Fenton W.A. Horwich A.L. Nature. 1996; 383: 96-99Crossref PubMed Scopus (82) Google Scholar). However, it should be noted that the functional significance of ATP for the cis ternary complex formation is unclear yet. It was reported that slow cis folding of rhodanese, a stringent substrate protein, is observed when GroES and ADP or AMPPNP 1The abbreviations used are: AMPPNP, 5′-adenylyl-β,γ-imidodiphosphate; AMPPNPhex, AMPPNP that is always exposed to hexokinase and glucose; AMPPNPraw and ADPraw, commercial AMPPNP and ADP that have not been exposed to hexokinase; ADPhex, ADP that is always exposed to hexokinase and glucose; ATPsingle, ATP exposed to hexokinase at 3 s after initiation of the reaction, by which time only a single turnover of ATP hydrolysis of GroEL can occur; MDH, malate dehydrogenase; GroESN, GroES(T19C) labeled by 5(2-iodoacetylaminoethyl) aminonaphthalene-1-sulfonic acid; GroESC, GroES(98C) labeled by 5(2-iodoacetylaminoethyl) aminonaphthalene-1-sulfonic acid; Ap5A, diadenosine pentaphosphate; DTT, dithiothreitol; HPLC, high pressure liquid chromatography. were added to GroEL-unfolded rhodanese complex (2Weissman J.S. Hohl C.M. Kovalenko O. Kashi Y. Chen S. Braig K. Saibil H.R. Fenton W.A. Horwich A.L. Cell. 1995; 83: 577-587Abstract Full Text PDF PubMed Scopus (391) Google Scholar, 7Weissman J.S. Rye H.S. Fenton W.A. Beechem J.M. Horwich A.L. Cell. 1996; 84: 481-490Abstract Full Text Full Text PDF PubMed Scopus (360) Google Scholar, 8Hayer-Hartl M.K. Weber F. Hartl F.U. EMBO J. 1996; 15: 6111-6121Crossref PubMed Scopus (133) Google Scholar). The cis folding of dehydrofolate reductase in the presence of ADP was also reported (9Mayhew M. da Silva A.C.R. Martin J. Erdjument-Bromage H. Tempst P. Hartl F.U. Nature. 1996; 379: 420-426Crossref PubMed Scopus (345) Google Scholar). Malate dehydrogenase (MDH) and Rubisco were shown to form the cis ternary complex in ADP. However, these complexes did not promote folding (3Rye H.S. Burston S.G. Fenton W.A. Beechem J.M. Xu Z. Sigler P.B. Horwich A.L. Nature. 1997; 388: 792-798Crossref PubMed Scopus (357) Google Scholar). These observations have raised an intriguing question: what is the difference between the non-productive ADP-induced cis ternary complex and the productive ATP-induced cis ternary complex? As reported previously (10Makino Y. Amada K. Taguchi H. Yoshida M. J. Biol. Chem. 1997; 272: 12468-12474Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar), we have noticed that inclusion of hexokinase and glucose in the reaction mixtures diminished ADP-dependent cis folding of green fluorescent protein. Commercially prepared ADP and AMPPNP usually contain a trace amount of ATP. Also, if a trace amount of adenylate kinase is contaminated in purified protein, it can produce ATP from ADP constantly. Hexokinase would eliminate these unwanted ATPs during the reaction period. Another potential factor that can affect the results is heterogeneity of GroEL complexes in the reaction mixtures. For example, if two GroEL rings are not saturated by unfolded proteins and if GroES can bind preferably to free GroEL rings rather than the unfolded protein-loaded GroEL rings, two kinds of complexes would be generated: GroEL-unfolded protein complex without GroES or GroEL-GroES complex without unfolded protein in the cis cavity. Since these two complexes cannot be separated, the results may be taken as an evidence for the existence of the GroEL-GroES-unfolded protein ternary complex. Here, we reexamined the nucleotide requirement for the cis ternary complex by taking the precautions described above. The results showed that ADP is incompetent to generate the cis ternary complex, that is, ATP is stringent for the fast GroES binding to the unfolded protein-loaded GroEL rings and subsequent cis folding of substrate protein. Materials—Single-stranded DNAs of the plasmid pET-EL and pET-ES2 were obtained by infecting Escherichia coli CJ236 cells with helper phage M13KO7 (Amersham Biosciences). Mutant GroES(T19C) was made by Kunkel methods using an oligonucleotide 5′-CAGCAGATTTGCATTCAACTTCTTTACG-3′. GroEL, GroES mutants, and rhodanese were expressed and purified as described (11Motojima F. Makio T. Aoki K. Makino Y. Kuwajima K. Yoshida M. Biochem. Biophys. Res. Commun. 2000; 267: 842-849Crossref PubMed Scopus (39) Google Scholar). GroEL purified by the procedures including gel permeation column chromatography in the presence of 30% methanol (11Motojima F. Makio T. Aoki K. Makino Y. Kuwajima K. Yoshida M. Biochem. Biophys. Res. Commun. 2000; 267: 842-849Crossref PubMed Scopus (39) Google Scholar) contained only a very small amount of contaminated proteins ( 107m–1 s–1) (Fig. 2B). In AMPPNPraw, the rates were slowed down by ∼102-fold (Fig. 2C). In AMPPNPhex, the rates were slowed down further; GroES binding to the MDH-loaded GroEL and the rhodanese-loaded GroEL were reached only 40 and 5% after 5 min, respectively. Similarly, the binding of GroESN to the loaded GroEL was slow in ADPraw and even slower in ADPhex (Fig. 2D). Although these slow GroES binding to the loaded GroEL in ADP and AMPPNP have not been known previously, this can be expected because GroES and unfolded protein compete for the overlapping binding sites on the GroEL. Rather, a new mechanism is required to understand this rapid GroES binding in ATP to the loaded GroEL.Fig. 2Binding of GroES to free GroEL and the loaded GroEL in various nucleotides. Binding was monitored with the fluorescence change of GroESN. The reactions were initiated by mixing the solution containing GroESN and nucleotide with the solution containing free GroEL or the loaded GroEL. Final concentrations of GroEL, GroESN, and nucleotides were 0.1 μm, 0.02 μm, and 1 mm, respectively. Single exponential fitting curves that were used to obtain association rate constants in Table I are shown in white lines. A, GroESN binding to free GroEL in ATP, AMPPNPhex, and ADPhex. A.U., arbitrary units. B, GroESN binding to the rhodanese-loaded GroEL and the MDH-loaded GroEL in ATP. C, GroESN binding to the rhodanese-loaded GroEL and the MDH-loaded GroEL in AMPPNPraw and AMPPNPhex. D, GroESN binding to the rhodanese-loaded GroEL and the MDH-loaded GroEL in ADPraw and ADPhex.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table IKinetic constants of GroES binding and releaseLoaded proteinATPAMP-PNPhexADPhexAMP-PNPrawADPrawk on (M-1 S-1)None7.5 × 1072.2 × 1073.6 × 1073.5 × 1075.0 × 107Rhodanese3.0 × 1071.8 × 1035.8 × 1032.6 × 1051.0 × 106MDH4.3 × 1077.1 × 1044.9 × 1051.1 × 1062.1 × 106ATPsingleAMP-PNPhexADPhexk off (S)None2.0 × 10-72.4 × 10-51.7 × 10-7Rhodanese5.6 × 10-7K d (fM)None2.71.1 × 1034.7Rhodanese1.9 × 101 Open table in a new tab When the substrate binding sites of GroEL are not saturated with unfolded proteins, it is predicted that rapid GroES binding in ADPhex to free GroEL rings in partially loaded GroEL can be observed. Indeed, in ADPhex, GroESN bound rapidly to two-thirds of the 1:1 (molar ratio of rhodanese and GroEL) rhodanese-loaded GroEL and to a quarter of the 2:1 rhodanese-loaded GroEL (Fig. 3). The rapid binding was no longer observed for the 2.5:1 rhodanese-loaded GroEL, indicating that all the substrate binding sites of GroEL were occupied by unfolded rhodanese. These results showed that 2.5 rhodanese and MDH can bind to GroEL even if GroEL has only two rings for substrate protein binding. This discrepancy may be due to the error in estimation of protein concentration or the occasional binding of two substrate proteins to one GroEL ring as reported using citrate synthase as substrate protein (14Grallert H. Rutkat K. Buchner J. J. Biol. Chem. 1998; 273: 33305-33310Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). Dissociation of GroES from GroEL—Dissociation of GroES from GroEL was measured by the exchange of fluorescently labeled GroESC bound on GroEL with an excess amount of unlabeled GroES. As shown in Fig. 4, the GroEL-GroESC complexes formed in ATPsingle and ADPhex without substrate protein are surprisingly stable; only 10% of the GroEL-GroESC complexes released GroESC after 1 week. The GroEL-GroESC complex formed in ATPsingle becomes identical to that in ADPhex since the bound ATP in the complex is hydrolyzed to ADP. This result shows the extraordinary stability of the GroEL-GroESC complexes in ADP. The complex without substrate protein formed in AMPPNPhex was relatively unstable and decayed in 2 days. The GroEL-GroESC-rhodanese complex formed in ATPsingle was slightly less stable than the GroEL-GroESC complex in ATPsingle in the absence of unfolded protein. This instability in the presence of rhodanese may be due to the stimulation of GroES release by substrate protein binding to trans ring (4Rye H.S. Roseman A.M. Chen S. Furtak K. Fenton W.A. Saibil H.R. Horwich A.L. Cell. 1999; 97: 325-338Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar). Calculated dissociation rate constants (k off) (Table I) are smaller than the previously reported value (3.8 × 10–5 s) estimated from the dissociation of ADP moiety from the GroEL-GroES complex (15Todd M.J. Viitanen P.V. Lorimer G.H. Science. 1994; 265: 659-666Crossref PubMed Scopus (429) Google Scholar). The reason of this discrepancy is not known, but there is a possibility that a trace amount of contaminated ATP would stimulate the exchange of bound ADP. The estimated dissociation constants of GroES binding to GroEL are extremely small (∼10 fM) and comparable with the dissociation constant of the biotin-avidin binding. This strong GroES binding may enable GroES to bind the loaded GroEL, the GroES binding site of which is covered by substrate protein. cis Folding of Rhodanese—From the slow binding of GroES to the rhodanese-loaded GroEL in ADPhex and AMPPNPhex, it can be predicted that formation of the cis ternary complex and the cis folding cannot occur in these nucleotides. To examine this, we compared the effect of these nucleotides on the chaperonin-assisted folding of rhodanese. Since it has been known that rhodanese folds in the cis cavity but does not fold spontaneously (16Martin J. Langer T. Boteva R. Schramel A. Horwich A.L. Hartl F.-U. Nature. 1991; 352: 36-42Crossref PubMed Scopus (726) Google Scholar), all the recovered rhodanese activity can be attributed to the result of the cis folding. The reaction was started by mixing the rhodanese-loaded GroEL, GroES, and nucleotides (Fig. 5A). In ATP, the yield of recovered rhodanese reached to more than 80% of the total bound rhodanese in 60 min. In ATPsingle, the recovered rhodanese was nearly 50%, showing that unfolded rhodanese bound in one GroEL ring is folded in the cis cavity after single ATP hydrolytic cycle was terminated. We observed significant recovery of rhodanese activity also in ADPraw (∼50%) and AMPPNPraw (∼20%). The same results were reported previously by others (7Weissman J.S. Rye H.S. Fenton W.A. Beechem J.M. Horwich A.L. Cell. 1996; 84: 481-490Abstract Full Text Full Text PDF PubMed Scopus (360) Google Scholar, 8Hayer-Hartl M.K. Weber F. Hartl F.U. EMBO J. 1996; 15: 6111-6121Crossref PubMed Scopus (133) Google Scholar, 9Mayhew M. da Silva A.C.R. Martin J. Erdjument-Bromage H. Tempst P. Hartl F.U. Nature. 1996; 379: 420-426Crossref PubMed Scopus (345) Google Scholar), and it has been thought that ADP and AMPPNP can substitute ATP to some extent for the chaperonin function. However, hexokinase treatment diminished rhodanese activity in ADPhex and AMPPNPhex (Fig. 5A). To determine which is the cause of ATP contamination, contaminated ATP or ATP production by contaminated adenylate kinase, rhodanese activity was measured using pre-hexokinase-treated ADP and AMPPNP (ADPpre-hex and AMPPNPpre-hex) that were treated with hexokinase and separated from hexokinase by ultrafiltration (Fig. 5B). Approximately 50% of rhodanese was recovered in ADPpre-hex. The initial lag period in the rhodanese recovery in ADPpre-hex can be interpreted as the time required for the accumulation of ATP by adenylate kinase. Indeed, in the presence of Ap5A, a potent inhibitor of adenylate kinase (17Reinstein J. Vetter I.R. Schlichting I. Rosch P. Wittinghofer A. Goody R.S. Biochemistry. 1990; 29: 7440-7450Crossref PubMed Scopus (89) Google Scholar), rhodanese reactivation disappeared in ADPraw, whereas it was not affected in ATP. The reason for no rhodanese recovery in AMPPNPpre-hex may be explained by the low concentration of contaminated ADP that is not enough for adenyate kinase to produce ATP. These results lead to conclusion that the causes for rhodanese recovery in ADPraw and AMPPNPraw are trace amounts of contaminated adenylate kinase and contaminated ATP, respectively. Formation of the cis Ternary Complex—We assessed the formation of the cis ternary complexes by using protease treatment; the substrate proteins entrapped in the cis cavity are protected from the protease digestion, whereas those bound to the trans ring are readily digested as well as free unfolded proteins (2Weissman J.S. Hohl C.M. Kovalenko O. Kashi Y. Chen S. Braig K. Saibil H.R. Fenton W.A. Horwich A.L. Cell. 1995; 83: 577-587Abstract Full Text PDF PubMed Scopus (391) Google Scholar, 16Martin J. Langer T. Boteva R. Schramel A. Horwich A.L. Hartl F.-U. Nature. 1991; 352: 36-42Crossref PubMed Scopus (726) Google Scholar). The samples incubated for 120 min as in Fig. 5A were ultrafiltrated to remove free GroES, digested by proteinase K, and ultrafiltrated to remove proteinase K and digested polypeptide. Three kinds of GroEL complexes, GroEL, GroEL-GroES, and the GroEL-GroES-rhodanese cis ternary complex, should exist after this procedure. The relative populations of GroEL, GroES, and rhodanese were estimated from the band intensities in the CBB-stained SDS-PAGE (Fig. 6A). The rhodanese-loaded GroEL contained 2.4 mol rhodanese/mol of GroEL (lane 1), and all rhodanese molecules were digested by proteinase K (lane 2). In ATP, the folded rhodanese molecules had been removed by ultrafiltration, and GroEL-GroES complexes remained (lane 3). In AMPPNPraw, ADPraw, and ATPsingle, significant amount of proteinase K-resistant rhodanese were observed (lanes 4–6). Molar ratios among GroEL, rhodanese, and GroES in these three lanes were 1.0:0.8–1.4: 0.7–1.1, indicating that the GroEL-GroES-rhodanese cis ternary complex was formed in these nucleotides. The rhodanese molecules held in the cis cavity of these complexes had finished folding because rhodanese activity of these complexes is equivalent to the estimated amount of rhodanese from SDS-PAGE (Fig. 6A, lower panel). In contrast, in AMPPNPhex and ADPhex, no rhodanese and a faint amount of GroES were observed (lanes 7 and 8), suggesting that GroES cannot bind to the rhodanese-loaded GroEL or can form only unstable GroEL-GroES-rhodanese ternary complex. Slow GroES binding to rhodanese-loaded GroEL in AMPPNPhex and ADPhex (Fig. 2, C and D) can be due to the formation of such unstable complex. Next, we used MDH as a substrate protein (Fig. 6B). Molar ratios among GroEL, MDH, and GroES in AMPPNPraw, ADPraw, and ATPsingle (lanes 4–6) were 1.0:0.8–1.1:1.0–1.1, indicating the stoichiometric formation of the cis ternary complexes as well as rhodanese. The results of AMPPNPhex (lane 7) and ADPhex (lane 8) are different from those of rhodanese. In AMPPNPhex, smaller amounts of MDH than ATPsingle were encapsulated in the cis cavity, whereas rhodanese was not encapsulated. In ADPhex, no MDH band was observed as rhodanese; however, GroES was bound to GroEL in contrast to rhodanese. Probably, MDH was gradually released from GroEL, and then GroES bound to these newly available binding sites. Folding of MDH in the cis cavity was assessed by measuring MDH activity after the MDH dimer was formed by releasing the MDH monomer from the cis cavity using EDTA to open the GroES cap (Fig. 6B, lower panel). Recovered MDH activity of each sample roughly corresponds to the amount of MDH in SDS-PAGE. ADPhex did not support cis folding of MDH, but AMPPNPhex did so in some extent. This ATP analogue seems to mimic the action of ATP in this case by a yet unknown mechanism. 3In AMPPNPhex and ADPhex, MDH in the MDH-loaded GroEL appeared to be gradually released from GroEL, and then GroES bound tightly to the newly available binding sites, because recovery of some MDH activity was observed even before the addition of EDTA (data not shown). In this case, recovery occurred much more slowly in AMPPNPhex than in ADPhex, indicating higher affinity of MDH to GroEL in AMPPNPhex than in ADPhex, which is consistent with slower GroESN binding to MDH-loaded GroEL in AMPPNPhex than in ADPhex (Fig. 2, C and D). The small cis folding of MDH in AMPPNPhex detected in Fig. 6B may be related to this relatively strong affinity of MDH to GroEL in AMPPNPhex and accidental entrapping of MDH in the cis cavity, whereas GroES competes for its binding sites. Conclusions—The previously reported ability of ADP, less efficient than that of ATP in the formation of the cis ternary complex, has made the current conception of the ATP-dependent chaperonin function ambiguous. However, our results clearly show that the cis ternary complex is not formed and that the cis folding does not occur in ADP. Also, cis folding of substrate proteins in ADP has been caused by the contaminated adenylate kinase because its inhibitor, diadenosine pentaphosphate, suppressed the cis folding of rhodanese. It should be noted that the direct measurement of adenylate kinase activity contaminated in the purified GroEL is hard to detect due to the immediate hydrolysis of produced ATP by GroEL. In contrast, AMPPNP appears to be able to mimic the action of ATP to some extent; it supports cis folding for MDH but not for rhodanese. This work sheds light on the key question on the chaperonin function; how can ATP enable rapid binding of GroES to the substrate protein-loaded GroEL whose binding sites for GroES are occupied by the substrate protein? We are grateful to Drs. T. Hisabori, E. Muneyuki, and H. Taguchi for valuable discussion, Drs. K. Kinosita, T. Kawashima, and T. Masaike for the use of the stopped-flow apparatus, and S. Murayama, T. Inoue, and R. Suno for experimental assistance.
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