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• W2782804945 abstract "•GroEL and GroES form a nano-cage for single protein molecules to fold in isolation•The two heptameric rings of GroEL separate and exchange between complexes•Ring separation is a consequence of inter-ring negative allostery upon ATP binding•Ring separation avoids formation of functionally impaired GroEL:GroES2 complexes The bacterial chaperonin GroEL and its cofactor, GroES, form a nano-cage for a single molecule of substrate protein (SP) to fold in isolation. GroEL and GroES undergo an ATP-regulated interaction cycle to close and open the folding cage. GroEL consists of two heptameric rings stacked back to back. Here, we show that GroEL undergoes transient ring separation, resulting in ring exchange between complexes. Ring separation occurs upon ATP-binding to the trans ring of the asymmetric GroEL:7ADP:GroES complex in the presence or absence of SP and is a consequence of inter-ring negative allostery. We find that a GroEL mutant unable to perform ring separation is folding active but populates symmetric GroEL:GroES2 complexes, where both GroEL rings function simultaneously rather than sequentially. As a consequence, SP binding and release from the folding chamber is inefficient, and E. coli growth is impaired. We suggest that transient ring separation is an integral part of the chaperonin mechanism. The bacterial chaperonin GroEL and its cofactor, GroES, form a nano-cage for a single molecule of substrate protein (SP) to fold in isolation. GroEL and GroES undergo an ATP-regulated interaction cycle to close and open the folding cage. GroEL consists of two heptameric rings stacked back to back. Here, we show that GroEL undergoes transient ring separation, resulting in ring exchange between complexes. Ring separation occurs upon ATP-binding to the trans ring of the asymmetric GroEL:7ADP:GroES complex in the presence or absence of SP and is a consequence of inter-ring negative allostery. We find that a GroEL mutant unable to perform ring separation is folding active but populates symmetric GroEL:GroES2 complexes, where both GroEL rings function simultaneously rather than sequentially. As a consequence, SP binding and release from the folding chamber is inefficient, and E. coli growth is impaired. We suggest that transient ring separation is an integral part of the chaperonin mechanism. The chaperonins are essential ATP-driven macromolecular machines of protein folding in bacteria, archaea, and eukarya (Hayer-Hartl et al., 2016Hayer-Hartl M. Bracher A. Hartl F.U. The GroEL-GroES chaperonin machine: A nano-cage for protein folding.Trends Biochem. Sci. 2016; 41: 62-76Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar, Lopez et al., 2015Lopez T. Dalton K. Frydman J. The mechanism and function of Group II chaperonins.J. Mol. Biol. 2015; 427: 2919-2930Crossref PubMed Scopus (117) Google Scholar). They form 800- to 1000-kDa double-ring complexes, with each ring enclosing a central cavity that functions as a nano-compartment for a single molecule of substrate protein (SP) to fold unimpaired by aggregation. In the case of the bacterial chaperonin, GroEL, and its homologs in mitochondria and chloroplasts, a lid-shaped cofactor, GroES, is required to close the folding chamber in an ATP-regulated reaction cycle. This process involves a network of nested cooperativity, with positive cooperativity of ATP-binding and hydrolysis within GroEL rings and negative cooperativity between rings (Gruber and Horovitz, 2016Gruber R. Horovitz A. Allosteric mechanisms in chaperonin machines.Chem. Rev. 2016; 116: 6588-6606Crossref PubMed Scopus (61) Google Scholar, Saibil et al., 2013Saibil H.R. Fenton W.A. Clare D.K. Horwich A.L. Structure and allostery of the chaperonin GroEL.J. Mol. Biol. 2013; 425: 1476-1487Crossref PubMed Scopus (133) Google Scholar). Despite intensive research, how exactly the two rings function in a coordinated manner is only partially understood. In one model, the two rings alternate between folding active and inactive states in a sequential mechanism, while in another, the two rings are proposed to function independently and to be folding active simultaneously (Hayer-Hartl et al., 2016Hayer-Hartl M. Bracher A. Hartl F.U. The GroEL-GroES chaperonin machine: A nano-cage for protein folding.Trends Biochem. Sci. 2016; 41: 62-76Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar, Taguchi, 2015Taguchi H. Reaction cycle of chaperonin GroEL via symmetric “football” intermediate.J. Mol. Biol. 2015; 427: 2912-2918Crossref PubMed Scopus (29) Google Scholar) (Figures 1A and 1B). GroEL consists of two heptameric rings of ∼57-kDa subunits that are stacked back to back such that each subunit in one ring contacts two subunits in the opposite ring (Hayer-Hartl et al., 2016Hayer-Hartl M. Bracher A. Hartl F.U. The GroEL-GroES chaperonin machine: A nano-cage for protein folding.Trends Biochem. Sci. 2016; 41: 62-76Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar, Saibil et al., 2013Saibil H.R. Fenton W.A. Clare D.K. Horwich A.L. Structure and allostery of the chaperonin GroEL.J. Mol. Biol. 2013; 425: 1476-1487Crossref PubMed Scopus (133) Google Scholar). The GroEL subunit is composed of an equatorial ATPase domain, an intermediate hinge domain, and an apical domain (Figures 1A and 1B). The apical domains form the flexible ring opening and expose hydrophobic amino acid residues toward the central cavity for the binding of non-native SP. GroES is a heptameric ring of ∼10-kDa subunits that caps the SP-bound GroEL ring, forming the so-called cis complex. This step is preceded by the binding of seven ATP to the cis ring and results in the displacement of SP into an enlarged hydrophilic chamber for folding (Xu et al., 1997Xu Z. Horwich A.L. Sigler P.B. The crystal structure of the asymmetric GroEL-GroES-(ADP)7 chaperonin complex.Nature. 1997; 388: 741-750Crossref PubMed Scopus (1039) Google Scholar). In the sequential model (also referred to as the asymmetric cycle), SP is free to fold inside this cage for the time needed for ATP hydrolysis (∼2–7 s, dependent on temperature) (Figure 1A). During this time, the open trans ring can bind a new non-native SP. After ATP hydrolysis in the cis ring, ATP binding to the trans ring causes the dissociation of ADP and GroES, allowing SP release. GroES binding to the trans ring forms a new, folding-active cis chamber (Rye et al., 1999Rye H.S. Roseman A.M. Chen S. Furtak K. Fenton W.A. Saibil H.R. Horwich A.L. GroEL-GroES cycling: ATP and nonnative polypeptide direct alternation of folding-active rings.Cell. 1999; 97: 325-338Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar). Asymmetric GroEL:GroES complexes predominate, due to the negative allosteric coupling of the GroEL rings, which is mediated by critical contacts at the inter-ring interface of the equatorial domains (Gruber and Horovitz, 2016Gruber R. Horovitz A. Allosteric mechanisms in chaperonin machines.Chem. Rev. 2016; 116: 6588-6606Crossref PubMed Scopus (61) Google Scholar, Saibil et al., 2013Saibil H.R. Fenton W.A. Clare D.K. Horwich A.L. Structure and allostery of the chaperonin GroEL.J. Mol. Biol. 2013; 425: 1476-1487Crossref PubMed Scopus (133) Google Scholar). In contrast, in the non-sequential model (also referred to as the symmetric cycle; Ye and Lorimer, 2013Ye X. Lorimer G.H. Substrate protein switches GroE chaperonins from asymmetric to symmetric cycling by catalyzing nucleotide exchange.Proc. Natl. Acad. Sci. USA. 2013; 110: E4289-E4297Crossref PubMed Scopus (49) Google Scholar) (Figure 1B), GroES binds simultaneously to both GroEL rings and dissociates stochastically upon ATP hydrolysis, with SP catalyzing nucleotide exchange (Hayer-Hartl et al., 2016Hayer-Hartl M. Bracher A. Hartl F.U. The GroEL-GroES chaperonin machine: A nano-cage for protein folding.Trends Biochem. Sci. 2016; 41: 62-76Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar, Taguchi, 2015Taguchi H. Reaction cycle of chaperonin GroEL via symmetric “football” intermediate.J. Mol. Biol. 2015; 427: 2912-2918Crossref PubMed Scopus (29) Google Scholar). Symmetric GroEL:GroES2 complexes are thought to be the major populated species (Yang et al., 2013Yang D. Ye X. Lorimer G.H. Symmetric GroEL:GroES2 complexes are the protein-folding functional form of the chaperonin nanomachine.Proc. Natl. Acad. Sci. USA. 2013; 110: E4298-E4305Crossref PubMed Scopus (53) Google Scholar). Due to the stochastic nature of GroES binding and release, the residence time of SP in the folding cage would vary in this model. Here, we performed a series of biochemical and biophysical experiments to investigate the GroEL/GroES reaction and distinguish between the different models. We find that transient ring separation permits the GroEL rings to function sequentially in SP folding. Ring separation is triggered by ATP binding to the trans ring of the asymmetric GroEL:GroES complex and is concomitant with the dissociation of GroES from the cis ring for SP release. At this point, ring exchange between complexes can occur. A folding-active GroEL mutant with covalently linked rings populated symmetric GroEL:GroES2 complexes. In this mutant, both GroEL chambers function simultaneously. Failure of ring separation in the reaction cycle resulted in delayed release of folded SP and inefficient binding of non-native SP, consistent with the reduced activity of this mutant in vivo. Together, our findings define transient ring separation as an integral step of the sequential GroEL/GroES chaperonin cycle. To distinguish between the two rings of GroEL in the chaperonin cycle, we took advantage of previous reports that GroEL ring hybrids can be produced by incubating wild-type GroEL (EL-WT) with GroEL mutant EL-379 or with GroEL from Thermus thermophilus in the presence of ATP (Burston et al., 1996Burston S.G. Weissman J.S. Farr G.W. Fenton W.A. Horwich A.L. Release of both native and non-native proteins from a cis-only GroEL ternary complex.Nature. 1996; 383: 96-99Crossref PubMed Scopus (82) Google Scholar, Taguchi et al., 1997Taguchi H. Amada K. Murai N. Yamakoshi M. Yoshida M. ATP-, K+-dependent heptamer exchange reaction produces hybrids between GroEL and chaperonin from Thermus thermophilus.J. Biol. Chem. 1997; 272: 18155-18160Crossref PubMed Scopus (15) Google Scholar). EL-379 carries the mutations Y203E, G337S, and I349E in the apical domain. It is ATPase active but was reported to be unable to bind SP and GroES (Burston et al., 1996Burston S.G. Weissman J.S. Farr G.W. Fenton W.A. Horwich A.L. Release of both native and non-native proteins from a cis-only GroEL ternary complex.Nature. 1996; 383: 96-99Crossref PubMed Scopus (82) Google Scholar). On native-PAGE, the EL-379 double ring migrates more slowly than GroEL, and the mixed ring (MR) complex migrates to an intermediate position (Figure 1C, lanes 1 and 2). We found that MR could not only be generated at 42°C (Burston et al., 1996Burston S.G. Weissman J.S. Farr G.W. Fenton W.A. Horwich A.L. Release of both native and non-native proteins from a cis-only GroEL ternary complex.Nature. 1996; 383: 96-99Crossref PubMed Scopus (82) Google Scholar), but also formed efficiently at 25°C (Figure 1C, lanes 1–3). MR formation was ATP-dependent and was not observed when EL-379 was incubated with the GroEL mutant EL-D87K, which does not bind nucleotide (Fenton et al., 1994Fenton W.A. Kashi Y. Furtak K. Horwich A.L. Residues in chaperonin GroEL required for polypeptide binding and release.Nature. 1994; 371: 614-619Crossref PubMed Scopus (574) Google Scholar) (Figure 1C, lane 4). To verify that MR is composed of distinct rings from GroEL and EL-379, and not of scrambled subunits, we dissociated EL-WT and EL-379 into subunits with 3.5 M urea and then reassembled the subunits in the presence of ATP (Ybarra and Horowitz, 1995Ybarra J. Horowitz P.M. Inactive GroEL monomers can be isolated and reassembled to functional tetradecamers that contain few bound peptides.J. Biol. Chem. 1995; 270: 22962-22967Crossref PubMed Scopus (31) Google Scholar) (Figure S1A). This resulted in scrambled complexes migrating as multiple bands on native-PAGE between EL-379 and EL-WT (Figure S1B), but not as a distinct band as observed for MR (Figure 1C). Interestingly, when MR was first purified by anion-exchange chromatography (Figure S1C), incubation with ATP resulted in redistribution of the rings to the typical three-band pattern at a molar ratio of EL-WT to MR to EL-379 of 1:2:1 (Figures 1C, lanes 5-6, and 1D). This indicated that the MR complex, once formed, is not a stable entity. Time course experiments showed that MR formation using 1 μM EL-379 and 1 μM EL-WT occurred with an apparent t1/2 of ∼50 s at 25°C (Figures 1E and S1D), i.e., with kinetics comparable to the ATPase rate of GroEL (∼3 rounds of ATP hydrolysis per tetradecamer; Figure S1E). MR formation also occurred at a similar rate in the presence of 30% Ficoll 70 (Figures 1E and S1D), a crowding agent used to mimic the excluded volume effects of the cell cytosol (Zimmerman and Minton, 1993Zimmerman S.B. Minton A.P. Macromolecular crowding: biochemical, biophysical, and physiological consequences.Annu. Rev. Biophys. Biomol. Struct. 1993; 22: 27-65Crossref PubMed Scopus (1265) Google Scholar). These findings indicated that ring separation and exchange can occur during the GroEL ATPase cycle. Next, we tested the nucleotide requirement of ring separation and also analyzed the effect of GroES. MR formation occurred in the presence of ATP but was inefficient with ADP (Figure 1F, lanes 1–3). Note that ADP preparations may contain small amounts of ATP (Hayer-Hartl et al., 1996Hayer-Hartl M.K. Weber F. Hartl F.U. Mechanism of chaperonin action: GroES binding and release can drive GroEL-mediated protein folding in the absence of ATP hydrolysis.EMBO J. 1996; 15: 6111-6121Crossref PubMed Scopus (133) Google Scholar). In the additional presence of GroES, ring separation and exchange was observed again only with ATP (Figure 1F, lanes 4 and 5). These reactions were stopped by addition of CDTA, which results in dissociation of GroES from EL-WT (Hayer-Hartl et al., 1995Hayer-Hartl M.K. Martin J. Hartl F.U. Asymmetrical interaction of GroEL and GroES in the ATPase cycle of assisted protein folding.Science. 1995; 269: 836-841Crossref PubMed Scopus (138) Google Scholar). The upshift of EL-379 observed in the presence of GroES (Figure 1F, lanes 4 and 5) was due to inefficient GroES dissociation, as confirmed by excision of the band and reanalysis by SDS-PAGE (Figure S1F). This observation, together with the finding that GroES inhibits the EL-379 ATPase by ∼30% (Figure S1E), indicated that the reported defect of EL-379 in GroES binding is only partial. To determine whether ATP binding or hydrolysis is required for ring separation, we used the GroEL mutant EL-D398A, which binds ATP but hydrolyzes it at a very slow rate of less than 2% of EL-WT (Rye et al., 1997Rye H.S. Burston S.G. Fenton W.A. Beechem J.M. Xu Z. Sigler P.B. Horwich A.L. Distinct actions of cis and trans ATP within the double ring of the chaperonin GroEL.Nature. 1997; 388: 792-798Crossref PubMed Scopus (356) Google Scholar). ATP-dependent MR formation of EL-D398A with EL-379 (Figure 1G) occurred with the same efficiency as for EL-WT (Figure 1F), suggesting that ATP binding, but not hydrolysis, causes ring separation. Ring separation and exchange was next analyzed in the presence of unfolded SP and GroES. We used SPs varying in size and folding properties: double mutant of maltose-binding protein (DM-MBP; ∼41 kDa) refolds spontaneously with slow kinetics (t1/2 ∼30 min), but the folding intermediate has high affinity for GroEL (Tang et al., 2006Tang Y.C. Chang H.C. Roeben A. Wischnewski D. Wischnewski N. Kerner M.J. Hartl F.U. Hayer-Hartl M. Structural features of the GroEL-GroES nano-cage required for rapid folding of encapsulated protein.Cell. 2006; 125: 903-914Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar), thus allowing chaperonin function to be analyzed at SP saturation (Haldar et al., 2015Haldar S. Gupta A.J. Yan X. Miličić G. Hartl F.U. Hayer-Hartl M. Chaperonin-assisted protein folding: Relative population of asymmetric and symmetric GroEL:GroES complexes.J. Mol. Biol. 2015; 427: 2244-2255Crossref PubMed Scopus (28) Google Scholar), and mitochondrial rhodanese (Rho; ∼33 kDa) and mitochondrial malate dehydrogenase (mMDH; ∼35 kDa) aggregate upon dilution from denaturant in the absence of GroEL (Martin et al., 1991Martin J. Langer T. Boteva R. Schramel A. Horwich A.L. Hartl F.U. Chaperonin-mediated protein folding at the surface of groEL through a ‘molten globule’-like intermediate.Nature. 1991; 352: 36-42Crossref PubMed Scopus (726) Google Scholar, Ranson et al., 1995Ranson N.A. Dunster N.J. Burston S.G. Clarke A.R. Chaperonins can catalyse the reversal of early aggregation steps when a protein misfolds.J. Mol. Biol. 1995; 250: 581-586Crossref PubMed Scopus (134) Google Scholar). Efficient MR formation was observed in all cases (Figure 1H). To determine the step in the GroEL/GroES cycle at which the GroEL rings separate, we used complexes of ADP with metal fluoride or vanadate ion to mimic different states of the γ phosphate along the reaction coordinate of ATP hydrolysis. ADP·BeFx mimics the ATP-bound state, ADP·AlFx mimics the transition state of ATP hydrolysis, and ADP·VO4 mimics the post-hydrolysis state (Chaudhry et al., 2003Chaudhry C. Farr G.W. Todd M.J. Rye H.S. Brunger A.T. Adams P.D. Horwich A.L. Sigler P.B. Role of the gamma-phosphate of ATP in triggering protein folding by GroEL-GroES: function, structure and energetics.EMBO J. 2003; 22: 4877-4887Crossref PubMed Scopus (113) Google Scholar, Meyer et al., 2003Meyer A.S. Gillespie J.R. Walther D. Millet I.S. Doniach S. Frydman J. Closing the folding chamber of the eukaryotic chaperonin requires the transition state of ATP hydrolysis.Cell. 2003; 113: 369-381Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). In the presence of these nucleotide analogs, asymmetric GroEL:GroES complexes form with nucleotides bound in the cis ring (Haldar et al., 2015Haldar S. Gupta A.J. Yan X. Miličić G. Hartl F.U. Hayer-Hartl M. Chaperonin-assisted protein folding: Relative population of asymmetric and symmetric GroEL:GroES complexes.J. Mol. Biol. 2015; 427: 2244-2255Crossref PubMed Scopus (28) Google Scholar, Yang et al., 2013Yang D. Ye X. Lorimer G.H. Symmetric GroEL:GroES2 complexes are the protein-folding functional form of the chaperonin nanomachine.Proc. Natl. Acad. Sci. USA. 2013; 110: E4298-E4305Crossref PubMed Scopus (53) Google Scholar). No MR formation was observed in the presence of these nucleotides (Figure S1G). Thus, the action of ATP solely in the cis ring of EL-WT apparently does not cause ring separation, suggesting that ATP binding to the trans ring triggers ring separation after ATP hydrolysis in the cis ring. To test this possibility directly, we first generated the asymmetric GroEL:7ADP:GroES complex. Addition of ATP, resulting in ATP binding to the trans ring, triggered ring separation and efficient formation of MR complexes within 10 s (Figure 1I, lanes 1–3). Importantly, MR formation within seconds was also observed upon ATP binding to the ATPase-defective EL-D398A:7ADP:GroES complex (Figure 1I, lanes 4–6). Note that EL-D398A requires ∼40 min to complete one round of ATP hydrolysis (Rye et al., 1997Rye H.S. Burston S.G. Fenton W.A. Beechem J.M. Xu Z. Sigler P.B. Horwich A.L. Distinct actions of cis and trans ATP within the double ring of the chaperonin GroEL.Nature. 1997; 388: 792-798Crossref PubMed Scopus (356) Google Scholar, Yang et al., 2013Yang D. Ye X. Lorimer G.H. Symmetric GroEL:GroES2 complexes are the protein-folding functional form of the chaperonin nanomachine.Proc. Natl. Acad. Sci. USA. 2013; 110: E4298-E4305Crossref PubMed Scopus (53) Google Scholar). Thus, ATP binding to the trans ring of the GroEL:7ADP:GroES complex is the step at which ring separation is triggered (Figure 1J), concomitant with the dissociation of ADP and GroES from the cis ring. The GroEL/GroES ATPase cycle is regulated by a system of nested cooperativity (Gruber and Horovitz, 2016Gruber R. Horovitz A. Allosteric mechanisms in chaperonin machines.Chem. Rev. 2016; 116: 6588-6606Crossref PubMed Scopus (61) Google Scholar). ATP binds to a GroEL ring with positive cooperativity (Hill coefficient, n = ∼2.8), and there is negative allostery between rings (Figure S2A). Considering that ring separation is triggered by ATP binding to the trans ring, it seemed plausible that negative inter-ring cooperativity was required for this effect. Negative cooperativity is reflected by a small decrease in the ATPase rate from its maximum as the ATP concentration is raised above ∼20 μM (Gruber and Horovitz, 2016Gruber R. Horovitz A. Allosteric mechanisms in chaperonin machines.Chem. Rev. 2016; 116: 6588-6606Crossref PubMed Scopus (61) Google Scholar) (Figure S2A). We tested the efficiency of MR formation with GroEL mutants displaying defects in either positive or negative cooperativity. Mutation D155A converts the ATP-induced intra-ring allosteric transitions from concerted to sequential, reducing positive cooperativity (n = ∼1.5) while preserving negative inter-ring allostery (Danziger et al., 2003Danziger O. Rivenzon-Segal D. Wolf S.G. Horovitz A. Conversion of the allosteric transition of GroEL from concerted to sequential by the single mutation Asp-155 -> Ala.Proc. Natl. Acad. Sci. USA. 2003; 100: 13797-13802Crossref PubMed Scopus (34) Google Scholar) (Figure S2B). We generated a GroEL mutant essentially lacking positive cooperativity by combining mutations D155A and R197A (Figure S2C). The latter destabilizes one of the inter-subunit salt bridges (R197-E386), reducing positive cooperativity (White et al., 1997White H.E. Chen S. Roseman A.M. Yifrach O. Horovitz A. Saibil H.R. Structural basis of allosteric changes in the GroEL mutant Arg197-->Ala.Nat. Struct. Biol. 1997; 4: 690-694Crossref PubMed Scopus (52) Google Scholar). The EL-D155A/R197A double mutant nevertheless preserved negative inter-ring cooperativity (Figure S2C). Both EL-D155A and EL-D155A/R197A formed MR complexes in the presence of GroES/ATP with an efficiency similar to EL-WT (Figure 1K), indicating that intra-ring cooperativity is not critical for ring separation. In contrast, mutant EL-E461K had lost the negative inter-ring cooperativity but largely preserved positive intra-ring cooperativity (n = ∼2.3) (Figure S2D). In this mutant, the critical inter-ring salt-bridges (E461-R452 and K105-E434) of the equatorial domains were disrupted, and the two rings were realigned in a 1:1 subunit interaction (Cabo-Bilbao et al., 2006Cabo-Bilbao A. Spinelli S. Sot B. Agirre J. Mechaly A.E. Muga A. Guérin D.M. Crystal structure of the temperature-sensitive and allosteric-defective chaperonin GroELE461K.J. Struct. Biol. 2006; 155: 482-492Crossref PubMed Scopus (13) Google Scholar, Sewell et al., 2004Sewell B.T. Best R.B. Chen S. Roseman A.M. Farr G.W. Horwich A.L. Saibil H.R. A mutant chaperonin with rearranged inter-ring electrostatic contacts and temperature-sensitive dissociation.Nat. Struct. Mol. Biol. 2004; 11: 1128-1133Crossref PubMed Scopus (36) Google Scholar). No MR formation was observed with EL-E461K. In conclusion, structural changes underlying negative inter-ring cooperativity of ATP-binding are critical in triggering ring separation. To determine the kinetics of GroEL ring exchange, we established a dual-color fluorescence cross-correlation spectroscopy (dcFCCS) assay using the apical domain cysteine mutant, EL-E315C, labeled either with the fluorophore Atto 532 (green) or Atto 655 (red) (Haldar et al., 2015Haldar S. Gupta A.J. Yan X. Miličić G. Hartl F.U. Hayer-Hartl M. Chaperonin-assisted protein folding: Relative population of asymmetric and symmetric GroEL:GroES complexes.J. Mol. Biol. 2015; 427: 2244-2255Crossref PubMed Scopus (28) Google Scholar). Equimolar amounts of the labeled proteins (0.5 μM each) were mixed and allowed to undergo ring exchange in the presence of GroES, unfolded DM-MBP, and ATP at 25°C (Figure 2A). MR formation was stopped at different times by addition of apyrase to rapidly hydrolyze ATP, followed by dcFCCS. MR formation occurred with an apparent t1/2 of ∼22 s (Figure 2A). Note that this experimental setup may underestimate the kinetics of ring separation due to the concentration dependence of ring re-association. To obtain the concentration-independent rate of ring dissociation, we incubated preformed MR complexes with excess unlabeled GroEL (plus GroES and unfolded DM-MBP) and monitored the decay of the dcFCCS signal (Figure 2B). ATP-dependent ring separation in ∼70% of the complexes occurred rapidly with a t1/2 of ∼12 s (Figure 2B). The remainder of the complexes showed a slow rate of ring separation, suggesting that a fraction of the labeled MR complexes were not fully functional. The fluorescent-labeled EL-315C hydrolyzed ATP in the presence of GroES and DM-MBP at a rate of ∼50 min−1 (Figure S3), equivalent to successive ATPase cycles in the two GroEL rings being completed in ∼17 s. Thus, ring separation allowing exchange occurs at the timescale of the ATPase reaction.Figure S3ATPase Activity of Fluorescently Labeled GroEL Mutant, Related to Figure 2Show full captionATPases activity of EL-E315C labeled with Atto532 or Atto655 alone, in the presence of GroES or in the presence of GroES and excess non-native DM-MBP. ATPase activities were measured photometrically at 25°C. Data represent the mean ± SEM of three independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) ATPases activity of EL-E315C labeled with Atto532 or Atto655 alone, in the presence of GroES or in the presence of GroES and excess non-native DM-MBP. ATPase activities were measured photometrically at 25°C. Data represent the mean ± SEM of three independent experiments. To understand the functional significance of ring separation, we designed a cysteine mutant of GroEL in which the two rings are covalently coupled via disulfide bonds. In the GroEL crystal structure (PDB: 1XCK), residue Ala109 of helix D in the equatorial domain of each subunit of one ring is in van der Waals contact with Ala109 in a subunit of the opposing ring, having a role in communicating negative inter-ring allostery (Gruber and Horovitz, 2016Gruber R. Horovitz A. Allosteric mechanisms in chaperonin machines.Chem. Rev. 2016; 116: 6588-6606Crossref PubMed Scopus (61) Google Scholar, Saibil et al., 2013Saibil H.R. Fenton W.A. Clare D.K. Horwich A.L. Structure and allostery of the chaperonin GroEL.J. Mol. Biol. 2013; 425: 1476-1487Crossref PubMed Scopus (133) Google Scholar). We mutated Ala109 to Cys. The resulting mutant, EL-A109C, readily formed inter-ring disulfide bonds upon expression in E. coli, indicating that the Cys residues are properly positioned for disulfide-bond formation. Disulfide-bonded GroEL-subunit dimers were confirmed by mass spectrometry and SDS-PAGE (Figures S4A and S4B). We solved the crystal structure of EL-A109C by molecular replacement at 3.2 Å resolution (Figure 3A and Table S1). The inter-ring disulfide bonds between opposite A109C residues are clearly observed (Figures 3A and 3B). The structure of the disulfide-bonded EL-A109C (henceforth EL-SS) was otherwise essentially identical to EL-WT with an overall root mean square deviation (rmsd) between Cα atoms of 1.128 Å.Figure 3Characterization of EL-A109C Defective in Ring Separation and ExchangeShow full caption(A) Overlay of the crystal structures of disulfide-bonded EL-A109C (EL-SS) and EL-WT (PDB: 1XCK) in orange and aquamarine, respectively. The green meshwork at the equator shows the omit electron density for the disulfide bonds at 5 sigma.(B) A close-up of one inter-ring contact. The cysteine moiety (pink) is shown in stick representation.(C) MR formation of EL-379 with EL-SS or EL-A109S in the presence of GroES and ATP at 25°C monitored by native-PAGE. MR formation of EL-379 and EL-WT is shown as control.(D) ATPase rates of EL-SS as a function of ATP concentration. ATPase activities were measured photometrically at 25°C and fitted to Equation 1. Insert shows the ATPase rate at low ATP concentrations up to 20 μM and fitted to Hill Equation 2 (see STAR Methods). ATPase activities of EL-WT under the same conditions are shown in Figure S2A. Data represent the mean ± SEM of three independent experiments.See also Figure S4 and Table S1.View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A) Overlay of the crystal structures of disulfide-bonded EL-A109C (EL-SS) and EL-WT (PDB: 1XCK) in orange and aquamarine, respectively. The green meshwork at the equator shows the omit electron density for the disulfide bonds at 5 sigma. (B) A close-up of one inter-ring contact. The cysteine moiety (pink) is shown in stick representation. (C) MR formation of EL-379 with EL-SS or EL-A109S in the presence of GroES and ATP at 25°C monitored by native-PAGE. MR formation of EL-379 and EL-WT is shown as control. (D) ATPase rates of EL-SS as a function of ATP concentration. ATPase activities were measured photometrically at 25°C and fitted to Equation 1. Insert shows the ATPase rate at low ATP concentrations up to 20 μM and fitted to Hill Equation 2 (see STAR Methods). ATPase activities of EL-WT under the same conditions are shown in Figure S2A. Data represent the mean ± SEM of three independent experiments. See also Figure S4 and Table S1. As expect" @default.
• W2782804945 created "2018-01-26" @default.
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• W2782804945 creator A5036116391 @default.
• W2782804945 creator A5053437073 @default.
• W2782804945 creator A5060999263 @default.
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• W2782804945 date "2018-01-01" @default.
• W2782804945 modified "2023-10-17" @default.
• W2782804945 title "GroEL Ring Separation and Exchange in the Chaperonin Reaction" @default.
• W2782804945 cites W1591074156 @default.
• W2782804945 cites W1646791064 @default.
• W2782804945 cites W1928288000 @default.
• W2782804945 cites W1956301495 @default.
• W2782804945 cites W1970978994 @default.
• W2782804945 cites W1974347829 @default.
• W2782804945 cites W1974725297 @default.
• W2782804945 cites W1978440190 @default.
• W2782804945 cites W1982408965 @default.
• W2782804945 cites W1987770334 @default.
• W2782804945 cites W1989999739 @default.
• W2782804945 cites W1994342014 @default.
• W2782804945 cites W1996645786 @default.
• W2782804945 cites W1997625774 @default.
• W2782804945 cites W2002542088 @default.
• W2782804945 cites W2003824905 @default.
• W2782804945 cites W2004080580 @default.
• W2782804945 cites W2006108571 @default.
• W2782804945 cites W2015951254 @default.
• W2782804945 cites W2016351987 @default.
• W2782804945 cites W2017886114 @default.
• W2782804945 cites W2018884106 @default.
• W2782804945 cites W2024337674 @default.
• W2782804945 cites W2025119181 @default.
• W2782804945 cites W2027454020 @default.
• W2782804945 cites W2028173143 @default.
• W2782804945 cites W2040164455 @default.
• W2782804945 cites W2041370513 @default.
• W2782804945 cites W2046221312 @default.
• W2782804945 cites W2052582745 @default.
• W2782804945 cites W2053779854 @default.
• W2782804945 cites W2057208427 @default.
• W2782804945 cites W2057567541 @default.
• W2782804945 cites W2066777552 @default.
• W2782804945 cites W2068155896 @default.
• W2782804945 cites W2075722785 @default.
• W2782804945 cites W2080906358 @default.
• W2782804945 cites W2085556560 @default.
• W2782804945 cites W2091183731 @default.
• W2782804945 cites W2091191054 @default.
• W2782804945 cites W2093397531 @default.
• W2782804945 cites W2096215703 @default.
• W2782804945 cites W2096858543 @default.
• W2782804945 cites W2098665638 @default.
• W2782804945 cites W2100950477 @default.
• W2782804945 cites W2104438148 @default.
• W2782804945 cites W2107370157 @default.
• W2782804945 cites W2115267975 @default.
• W2782804945 cites W2117318478 @default.
• W2782804945 cites W2130463690 @default.
• W2782804945 cites W2132629607 @default.
• W2782804945 cites W2135046679 @default.
• W2782804945 cites W2138749342 @default.
• W2782804945 cites W2142338805 @default.
• W2782804945 cites W2144081223 @default.
• W2782804945 cites W2147289825 @default.
• W2782804945 cites W2147739915 @default.
• W2782804945 cites W2154714625 @default.
• W2782804945 cites W2156272283 @default.
• W2782804945 cites W2159211495 @default.
• W2782804945 cites W2164897290 @default.
• W2782804945 cites W2165360511 @default.
• W2782804945 cites W2166443214 @default.
• W2782804945 cites W2209257594 @default.
• W2782804945 cites W2214850930 @default.
• W2782804945 cites W2264471836 @default.
• W2782804945 cites W93719713 @default.
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