Both the Escherichia coli chaperone systems, GroEL/GroES and DnaK/DnaJ/GrpE, can reactivate heat-treated RNA polymerase. Different mechanisms for the same activity

In this work we show that the GroEL (Hsp60 equivalent) chaperone protein can protected purified Escherichia coli RNA polymerase (RNAP) holoenzyme from heat inactivation better than the DnaK (Hsp70 equivalent) chaperone can. In this protection reaction, the GroES protein is not essential, but its presence reduces the amount of GroEL required. GroEL and GroES can also reactivate heat-inactivated RNAP in the presence of ATP. The mutant GroEL673 protein, with or without GroES, is incapable of reactivating heat-inactivated RNAP. GroEL673 can only protect RNAP, and this protecting ability is not stimulated by GroES. The mechanism by which the DnaJ and GrpE heat shock proteins contribute to DnaK's ability to reactivate heat-inactivated RNAP GroEL673 has also been investigated. We found that the DnaJ protein substantially reduces the levels of DnaK protein needed in this reactivation assay. However, the observed lag in reactivation is diminished only in the additional presence of the GrpE protein. Hence, DnaJ and GrpE are involved in both steps of this reactivation reaction (recognition of substrate and release of chaperone from the substrate-chaperone complex) while, in the case of the GroEL-dependent reaction, GroES is involved only during the release of chaperone from the substrate-chaperone complex.     

Identification of amino acid residues at nucleotide-binding sites of chaperonin GroEL/GroES and cpn10 by photoaffinity labeling with 2-azido-adenosine 5'-triphosphate

Although the chaperonin GroEL/GroES complex binds and hydrolyzes ATP, its structure is unlike other known ATPases. In order to better characterize its nucleotide binding sites, we have photolabeled the complex with the affinity analog 2-azido-ATP. Three residues of GroEL, Pro137, Cys138 and Thr468, are labeled by the probe. The location of these residues in the GroEL crystal structure [Braig, K., Otwinowski, Z., Hedge, R., Boisvert, D., Joachimiak, A., Horwich, A. & Sigler, P. (1994) Nature 371, 578-586: Boisvert, D. C., Wang, J., Otwinowski, Z., Horwich, A. L. & Sigler, P. B. (1996) Nat. Struct. Biol. 3, 170-177] suggests that 2-azido-ATP binds to an alternative conformer of GroEL in the presence of GroES. The labeled site appears to be located at the GroEL/GroEL subunit interface since modification of Pro137 and Cys138 is most readily explained by attack of a probe molecule bound to the adjacent GroEL subunit. Labeling of the co-chaperonin, GroES, is clearly demonstrated on gels and the covalent tethering of nucleotide allows detection of a GroES dimer in the presence of SDS. However, no stable peptide derivative of GroES could be purified for sequencing. In contrast, the GroES homolog, yeast cpn10, does give a stable derivative. The modified amino acid is identified as the conserved Pro13, which corresponds to Pro5 in Escherichia coli GroES.     

Spinach chloroplast cpn21 co-chaperonin possesses two functional domains fused together in a toroidal structure and exhibits nucleotide-dependent binding to plastid chaperonin 60

Chloroplasts contain a 21-kDa co-chaperonin polypeptide (cpn21) formed by two GroES-like domains fused together in tandem. Expression of a double-domain spinach cpn21 in Escherichia coli groES mutant strains supports growth of bacteriophages lambda and T5, and will also suppress a temperature-sensitive growth phenotype of a groES619 strain. Each domain of cpn21 expressed separately can function independently to support bacteriophage lambda growth, and the N-terminal domain will additionally suppress the temperature-sensitive growth phenotype. These results indicate that chloroplast cpn21 has two functional domains, either of which can interact with GroEL in vivo to facilitate bacteriophage morphogenesis. Purified spinach cpn21 has a ring-like toroidal structure and forms a stable complex with E. coli GroEL in the presence of ADP and is functionally interchangeable with bacterial GroES in the chaperonin-facilitated refolding of denatured ribulose-1,5-bisphosphate carboxylase. Cpn21 also inhibits the ATPase activity of GroEL. Cpn21 binds with similar efficiency to both the alpha and beta subunits of spinach cpn60 in the presence of adenine nucleotides, with ATP being more effective than ADP. The tandemly fused domains of cpn21 evolved early and are present in a wide range of photosynthetic eukaryotes examined, indicating a high degree of conservation of this structure in chloroplasts.     

Residue lysine-34 in GroES modulates allosteric transitions in GroEL

The conserved residue Lys-34 in GroES was replaced by alanine and glutamic acid using site-directed mutagenesis. This residue is near the carboxy terminus of the mobile loop in GroES (residues 17-32) which becomes immobilized upon formation of the GroEL/GroES complex [Landry et al. (1993) Nature 364, 255-258]. Both charge neutralization (Lys-34-->Ala) and charge reversal (Lys-34-->Glu) at this position have little effect on the binding constant of GroES to GroEL, but they increase the enhancement by GroES of cooperativity in ATP hydrolysis by GroEL. This is reflected by a change in the Hill coefficient (at 10 mM K+) from 4.10 (+/- 0.22) in the presence of wild-type GroES to 5.17 (+/- 0.24) and 4.46 (+/- 0.14) in the presence of the GroES mutants Lys-34-->Ala and Lys-34-->Glu, respectively. The results are interpreted using the Monod-Wyman-Changeux (MWC) model for cooperativity [Monod et al. (1965) J. Mol. Biol. 12, 88-118]. They suggest that Lys-34 in GroES modulates the allosteric transition in GroEL by stabilizing a relaxed (R)-like state.     

Mechanism of chaperonin action: GroES binding and release can drive GroEL-mediated protein folding in the absence of ATP hydrolysis

As a basic principle, assisted protein folding by GroEL has been proposed to involve the disruption of misfolded protein structures through ATP hydrolysis and interaction with the cofactor GroES. Here, we describe chaperonin subreactions that prompt a re-examination of this view. We find that GroEL-bound substrate polypeptide can induce GroES cycling on and off GroEL in the presence of ADP. This mechanism promotes efficient folding of the model protein rhodanese, although at a slower rate than in the presence of ATP. Folding occurs when GroES displaces the bound protein into the sequestered volume of the GroEL cavity. Resulting native protein leaves GroEL upon GroES release. A single-ring variant of GroEL is also fully functional in supporting this reaction cycle. We conclude that neither the energy of ATP hydrolysis nor the allosteric coupling of the two GroEL rings is directly required for GroEL/GroES-mediated protein folding. The minimal mechanism of the reaction is the binding and release of GroES to a polypeptide-containing ring of GroEL, thereby closing and opening the GroEL folding cage. The role of ATP hydrolysis is mainly to induce conformational changes in GroEL that result in GroES cycling at a physiologically relevant rate.     

Asymmetrical interaction of GroEL and GroES in the ATPase cycle of assisted protein folding

The chaperonins GroEL and GroES of Escherichia coli facilitate protein folding in an adenosine triphosphate (ATP)-dependent reaction cycle. The kinetic parameters for the formation and dissociation of GroEL-GroES complexes were analyzed by surface plasmon resonance. Association of GroES and subsequent ATP hydrolysis in the interacting GroEL toroid resulted in the formation of a stable GroEL:ADP:GroES complex. The complex dissociated as a result of ATP hydrolysis in the opposite GroEL toroid, without formation of a symmetrical GroEL:(GroES)2 intermediate. Dissociation was accelerated by the addition of unfolded polypeptide. Thus, the functional chaperonin unit is an asymmetrical GroEL:GroES complex, and substrate protein plays an active role in modulating the chaperonin reaction cycle.     

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In the presence of MgATP or MgADP the E. coli chaperonin proteins, GroEL and GroES, form a stable asymmetric complex with a stoichiometry of two GroEL7:one GroES7: seven MgADP. The distribution of the ligands between the two heptameric GroEL rings is crucial to our understanding of the mechanism of chaperonin-assisted folding, being either cis (i.e. [GroEL7.MgADP7.GroES7]-[GroEL7]) or trans (i.e. [GroEL7.MgADP7]-[GroEL7.GroES7]. On the basis of cross-linking experiments with 8-azido-ATP and the heterobifunctional reagent, N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP), it was suggested that GroES and MgADP are bound to the same GroEL ring which resists proteinase K digestion [Nature 366 (1993) 228-233]. However, we find that the SPDP-promoted cross linking of GroES and GroEL occurs in the absence of Mg2+, ADP or ATP, which are required for the formation of the asymmetric complex. Cross-linking is shown to occur only when the SPDP-modified GroES is co-precipitated with GroEL by trichloracetic acid. Furthermore, there are structural grounds for questioning whether SPDP can crosslink, in a physiologically relevant manner, an amino group of GroES with any of the cysteinyl groups of GroEL.     

Characterization of a functional GroEL14(GroES7)2 chaperonin hetero-oligomer

Chaperonins GroEL and GroES form two types of hetero-oligomers in vitro that can mediate the folding of proteins. Chemical cross-linking and electron microscopy showed that in the presence of adenosine triphosphate (ATP), two GroES7 rings can successively bind a single GroEL14 core oligomer. The symmetric GroEL14(GroES7)2 chaperonin, whose central cavity appears obstructed by two GroES7 rings, can nonetheless stably bind and assist the ATP-dependent refolding of RuBisCO enzyme. Thus, unfolded proteins first bind and possibly fold on the external envelope of the chaperonin hetero-oligomer.     

Preformed GroES oligomers are not required as functional cochaperonins

We have previously shown that the C-terminal sequence of GroES is required for oligomerization [Seale and Horowitz (1995), J. Biol. Chem. 270, 30268-30270]. In this report, we have generated a C-terminal deletion mutant of GroES with a significantly destabilized oligomer and have investigated its function in the chaperonin-assisted protein folding cycle. Removal of the two C-terminal residues of GroES results in a cochaperonin [GroESD(96-97)] that is monomeric at concentrations where GroES function is assessed. Using equilibrium ultracentrifugation, we measured the dissociation constant for the oligomer-monomer equilibrium to be 7.3 x 10(-34)M6. The GroESD(96-97) is fully active as a cochaperonin. This mutant is able to inhibit the ATPase activity of GroEL to levels comparable to wild-type GroES. It is also able to assist the refolding of urea-denatured rhodanese by GroEL. While GroESD(96-97) can function at levels comparable to wild-type GroES, higher concentrations of mutant are required to produce the same effect. These results support the idea that the performed GroES heptamer is not required for function, but they suggest that the oligomeric cochaperonin is most efficient.     

SCL 90-R interpretation and brain tumour: a correction factor?

Two models are being considered for the mechanism of chaperonin-assisted protein folding in E. coli: (i) GroEL/GroES act primarily by enclosing substrate polypeptide in a folding cage in which aggregation is prevented during folding. (ii) GroEL mediates the repetitive unfolding of misfolded polypeptides, returning them onto a productive folding track. Both models are not mutually exclusive, but studies with the polypeptide-binding domain of GroEL have suggested that unfolding is the primary mechanism, enclosure being unnecessary. Here we investigate the capacity of the isolated apical polypeptide-binding domain to functionally replace the complete GroEL/GroES system. We show that the apical domain binds aggregation-sensitive polypeptides but cannot significantly assist their refolding in vitro and fails to replace the groEL gene or to complement defects of groEL mutants in vivo. A single-ring version of GroEL cannot substitute for GroEL. These results strongly support the view that sequestration of aggregation-prone intermediates in a folding cage is an important element of the chaperonin mechanism.     

One founder/one gene hypothesis in a new expanding population: Saguenay (Quebec, Canada)

Here we report a method of immobilising the chaperonins GroEL and GroES to a glass matrix. The immobilised chaperone system has been used to successfully refold target proteins denatured by guanidine hydrochloride and produce substantially higher levels of active protein than occur on dilution into aqueous solution alone. The chaperone system has been shown to refold proteins from each of the three categories of GroEL substrate. The refolding of the enzyme glycerol dehydrogenase from Bacillus stearothermophilus shows a two-fold increase in activity in the presence of immobilised GroEL compared to that in free solution. The lactate dehydrogenase from B. stearothermophilus also shows a two-fold higher yield of activity in the presence of the immobilised GroEL and ATP. The presence of immobilised GroEL in the absence of ATP arrests the refolding of LDH. The enzyme citrate synthetase from porcine heart demonstrates a three-fold increase in activity when refolded in the presence of immobilised GroEL, ATP and free GroES. Similar results are obtained in the presence of free GroEL, immobilised GroES and ATP. The matrix-bound chaperone can be removed from the refolding mixture by centrifugation, producing a reusable system that can be easily isolated and purified from the refolded substrate.     

Trigger factor associates with GroEL in vivo and promotes its binding to certain polypeptides

Trigger factor (TF) is a putative molecular chaperone recently found to function together with GroEL in the degradation of the fusion protein, CRAG. TF overproduction enhanced the ability of GroEL to form complexes with CRAG, as well as fetuin or histone. To define further this effect on GroEL binding, affinity columns containing a variety of denatured proteins were used. When cell extracts were applied onto a fetuin column, both TF and GroEL bound but not GroES. Upon ATP addition, TF and GroEL were eluted together and remained tightly associated (even in presence of GroES) in complexes containing one TF per GroEL 14-mer. Overproduction of TF enhanced the capacity of GroEL to bind to many denatured proteins. Moreover, GroEL-TF complexes isolated from such cells showed much greater binding capacity than GroEL from TF-deficient cells. Furthermore, the addition of pure TF to pure GroEL also enhanced markedly its binding capacity. The affinity of GroEL for CRAG also rises during heat shock due to GroEL phosphorylation. TF expression, however, did not promote GroEL phosphorylation. Moreover, heat shock and TF overproduction affected GroEL binding to other denatured polypeptides in distinct ways; only TF promoted binding to certain polypeptides, whereas only phosphorylation increased binding to others. Thus, association with TF and phosphorylation are independent regulators of GroEL function. This enhanced affinity of TF-GroEL complexes for unfolded proteins may also be important in protein folding, because TF has prolyl isomerase activity and associates with nascent polypeptides.     

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When chaperonins GroEL and GroES are incubated under functional conditions in the presence of ATP (5 mM) and K+ (150 mM), GroEL-GroES complexes appear in the incubation mixture, that are either asymmetric (1:1 GroEL:GroES oligomer ratio) or symmetric (1:2 GroEL:GroES oligomer ratio). The percentage of symmetric complexes present is directly related to the [ATP]/[ADP] ratio and to the K+ concentration. Kinetic analysis shows that there is a cycle of formation and disappearance of symmetric complexes. A correlation between the presence of symmetric complexes in the incubation mixture and its rhodanese folding activity suggests some active role of these complexes in the protein folding process. Accordingly, under functional conditions, symmetric complexes are found to contain denatured rhodanese. These data suggest that binding of substrate inside the GroEL cavity takes place before the symmetric complex is formed.     

Minimal and optimal mechanisms for GroE-mediated protein folding

We have analyzed the effects of different components of the GroE chaperonin system on protein folding by using a nonpermissive substrate (i.e., one that has very low spontaneous refolding yield) for which rate data can be acquired. In the absence of GroES and nucleotides, the rate of GroEL-mediated refolding of heat- and DTT-denatured mitochondrial malate dehydrogenase was extremely low, but some three times higher than the spontaneous rate. This GroEL-mediated rate was increased 17-fold by saturating concentrations of ATP, 11-fold by ADP and GroES, and 465-fold by ATP and GroES. Optimal refolding activity was observed when the dissociation of GroES from the chaperonin complex was dramatically reduced. Although GroEL minichaperones were able to bind denatured mitochondrial malate dehydrogenase, they were ineffective in enhancing the refolding rate. The spectrum of mechanisms for GroE-mediated protein folding depends on the nature of the substrate. The minimal mechanism for permissive substrates (i.e., having significant yields of spontaneous refolding), requires only binding to the apical domain of GroEL. Slow folding rates of nonpermissive substrates are limited by the transitions between high- and low-affinity states of GroEL alone. The optimal mechanism, which requires holoGroEL, physiological amounts of GroES, and ATP hydrolysis, is necessary for the chaperonin-mediated folding of nonpermissive substrates at physiologically relevant rates under conditions in which retention of bound GroES prevents the premature release of aggregation-prone folding intermediates from the chaperonin complex. The different mechanisms are described in terms of the structural features of mini- and holo-chaperones.     

In vivo activities of GroEL minichaperones

Fragments encompassing the apical domain of GroEL, called minichaperones, facilitate the refolding of several proteins in vitro without requiring GroES, ATP, or the cage-like structure of multimeric GroEL. We have identified the smallest minichaperone that is active in vitro in chaperoning the refolding of rhodanese and cyclophilin A: GroEL(193-335). This finding raises the question of whether the minichaperones are active under more stringent conditions in vivo. The smallest minichaperones complement two temperature-sensitive Escherichia coli groEL alleles, EL44 and EL673, at 43 degreesC. Although they cannot replace GroEL in cells in which the chromosomal groEL gene has been deleted by P1 transduction, GroEL(193-335) enhances the colony-forming ability of such cells when limiting amounts of GroEL are expressed from a tightly regulated plasmid. Surprisingly, we found that overexpression of GroEL prevents plaque formation by bacteriophage lambda and inhibits replication of the lambda origin-dependent plasmid, Lorist6. The minichaperones also inhibit Lorist6 replication, but less markedly. The complex quaternary structure of GroEL, its central cavity, and the structural allosteric changes that take place on the binding of nucleotides and GroES are not essential for all of its functions in vivo.     

Chaperone protein GrpE and the GroEL/GroES complex promote the correct folding of tobacco mosaic virus coat protein for ribonucleocapsid assembly in vivo

Several prokaryotic chaperone proteins were shown to promote the correct folding and in vivo assembly of tobacco mosaic virus coat protein (TMV CP) using a chimaeric RNA packaging system in control or chaperone-deficient mutant strains of Escherichia coli. Mutations in groEL or dnaK reduced the amount of both total and soluble TMV CP, and the yield of assembled TMV-like particles, several-fold. Thus both GroEL and DnaK have significant direct or indirect effects on the overall expression, stability, folding and assembly of TMV CP in vivo. In contrast, while cells carrying a mutation in grpE expressed TMV CP to a higher overall level than control E. coli, the amounts of both soluble CP and assembled TMV-like particles were below control levels, suggesting a negative effect of GrpE on overall CP accumulation, but positive role(s) in CP folding and assembly. Curiously, cells with mutations in groES and, to a lesser extent, dnaJ expressed total, soluble and assembled forms of TMV CP significantly above control values, suggesting some form of negative control by these chaperone proteins. To avoid pleiotropic effects or artefacts in chaperone-null mutants, selected chaperone proteins were also over-expressed in control E. coli cells. Overproduction of GroEL or GroES alone had little effect. However, co-overexpression of GroEL and GroES resulted in a two-fold increase in soluble TMV CP and a four-fold rise in assembled TMV-like (pseudovirus) particles in vivo. Moreover, TMV CP was shown to interact directly with GroEL in vivo. Together, these results suggest that GrpE and the GroEL/GroES chaperone complex promote the correct folding and assembly of TMV CP into ribonucleocapsids in vivo.     

Heat-stress response of maize mitochondria

We have identified maize (Zea mays L. inbred B73) mitochondrial homologs of the Escherichia coli molecular chaperones DnaK (HSP70) and GroEL (cpn60) using two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblots. During heat stress (42 degrees C for 4 h), levels of HSP70 and cpn60 proteins did not change significantly. In contrast, levels of two 22-kD proteins increased dramatically (HSP22). Monoclonal antibodies were developed to maize HSP70, cpn60, and HSP22. The monoclonal antibodies were characterized with regard to their cross-reactivity to chloroplastic, cytosolic, and mitochondrial fractions, and to different plant species. Expression of mitochondrial HSP22 was evaluated with regard to induction temperature, time required for induction, and time required for degradation upon relief of stress. Maximal HSP22 expression occurred in etiolated seedling mitochondria after 5 h of a +13 degrees C heat stress. Upon relief of heat stress, the HSP22 proteins disappeared with a half-life of about 4 h and were undetectable after 21 h of recovery. Under continuous heat-stress conditions, the level of HSP22 remained high. A cDNA for maize mitochondrial HSP22 was cloned and extended to full length with sequences from an expressed sequence tag database. Sequence analysis indicated that HSP22 is a member of the plant small heat-shock protein superfamily.     

Role of the GroEL chaperonin intermediate domain in coupling ATP hydrolysis to polypeptide release

Modification of the Escherichia coli chaperonin GroEL with N-ethylmaleimide at residue Cys138 affects the structural and functional integrity of the complex. Nucleotide affinity and ATPase activity of the modified chaperonin are increased, whereas cooperativity of ATP hydrolysis and affinity for GroES are reduced. As a consequence, release and folding of substrate proteins are strongly impaired and uncoupled from ATP hydrolysis in a temperature-dependent manner. Folding of dihydrofolate reductase at 25 degrees C becomes dependent on GroES, whereas folding of typically GroES-dependent proteins is blocked completely. At 37 degrees C, GroES binding is restored to normal levels, and the modified GroEL regains its chaperone activity to some extent. These results assign a central role to the intermediate GroEL domain for transmitting conformational changes between apical and central domains, and for coupling ATP hydrolysis to productive protein release.     

Chaperone activity and structure of monomeric polypeptide binding domains of GroEL

The chaperonin GroEL is a large complex composed of 14 identical 57-kDa subunits that requires ATP and GroES for some of its activities. We find that a monomeric polypeptide corresponding to residues 191 to 345 has the activity of the tetradecamer both in facilitating the refolding of rhodanese and cyclophilin A in the absence of ATP and in catalyzing the unfolding of native barnase. Its crystal structure, solved at 2.5 A resolution, shows a well-ordered domain with the same fold as in intact GroEL. We have thus isolated the active site of the complex allosteric molecular chaperone, which functions as a "minichaperone." This has mechanistic implications: the presence of a central cavity in the GroEL complex is not essential for those representative activities in vitro, and neither are the allosteric properties. The function of the allosteric behavior on the binding of GroES and ATP must be to regulate the affinity of the protein for its various substrates in vivo, where the cavity may also be required for special functions.