First characterization of co-chaperonin protein 10 from hyper-thermophilic Aquifex aeolicus

All known co-chaperonin protein 10 (cpn10) molecules are heptamers of seven identical subunits that are linked together by beta-strand interactions. Here, we report the first characterization of a cpn10 protein from a thermophilic organism: Aquifex aeolicus. Primary-structure alignment of A. aeolicus cpn10 (Aaecpn10) shows high homology with mesophilic cpn10 sequences, except for a unique 25-residue C-terminal extension not found in any other cpn10. Recombinant Aaecpn10 adopts a heptameric structure in solution at pH values above 4 (20 degrees C). Both monomers and heptamers are folded at 20 degrees C, although the thermal stability of the monomers (pH 3; Tm approximately 58 degrees C) is lower than that of the heptamers (pH 7; Tm approximately 115 degrees C). Aaecpn10 functions in a GroEL-dependent in vitro activity assay. Taken together, Aaecpn10 appears similar in secondary, tertiary, and quaternary structure, as well as in many biophysical features, to its mesophilic counterparts despite a functional temperature of 90 degrees C.     

Kinetic folding and assembly mechanisms differ for two homologous heptamers

Here we investigate the time-resolved folding and assembly mechanism of the heptameric co-chaperonin protein 10 (cpn10) in vitro. The structure of cpn10 is conserved throughout nature: seven beta-barrel subunits are non-covalently assembled through beta-strand pairings in an overall doughnut-like shape. Kinetic folding/assembly experiments of chemically denatured cpn10 from Homo sapiens (hmcpn10) and Aquifex aeolicus (Aacpn10) were monitored by far-UV circular dichroism and fluorescence. We find the processes to be complex, involving several kinetic steps, and to differ between the mesophilic and hyper-thermophilic proteins. The hmcpn10 molecules partition into two parallel pathways, one involving polypeptide folding before protein-protein assembly and another in which inter-protein interactions take place prior to folding. In contrast, the Aacpn10 molecules follow a single sequential path that includes initial monomer misfolding, relaxation to productive intermediates and, subsequently, final folding and heptamer assembly. An A. aeolicus variant lacking the unique C-terminal extension of Aacpn10 displays the same kinetic mechanism as Aacpn10, signifying that the tail is not responsible for the rapid misfolding step. This study demonstrates that molecular details can overrule similarity of native-state topology in defining apparent protein-biophysical properties.     

Folding and assembly pathways of co-chaperonin proteins 10: Origin of bacterial thermostability

To compare folding/assembly processes of heptameric co-chaperonin proteins 10 (cpn10) from different species and search for the origin of thermostability in hyper-thermostable Aquifex aeolicus cpn10 (Aacpn10), we have studied two bacterial variants-Aacpn10 and Escherichia coli cpn10 (GroES)-and compared the results to data on Homo sapiens cpn10 (hmcpn10). Equilibrium denaturation of GroES by urea, guanidine hydrochloride (GuHCl) and temperature results in coupled heptamer-to-monomer transitions in all cases. This is similar to the behavior of Aacpn10 but differs from hmcpn10 denaturation in urea. Time-resolved experiments reveal that GroES unfolds before heptamer dissociation, whereas refolding/reassembly begins with folding of individual monomers; these assemble in a slower step. The sequential folding/assembly mechanism for GroES is rather similar to that observed for Aacpn10 but contradicts the parallel paths of hmcpn10. We reveal that Aacpn10's stability profile is shifted upwards, broadened, and also moved horizontally to higher temperatures, as compared to that of GroES.     

Location and flexibility of the unique C-terminal tail of Aquifex aeolicus co-chaperonin protein 10 as derived by cryo-electron microscopy and biophysical techniques

Co-chaperonin protein 10 (cpn10, GroES in Escherichia coli) is a ring-shaped heptameric protein that facilitates substrate folding when in complex with cpn60 (GroEL in E. coli). The cpn10 from the hyperthermophilic, ancient bacterium Aquifex aeolicus (Aacpn10) has a 25-residue C-terminal extension in each monomer not found in any other cpn10 protein. Earlier in vitro work has shown that this tail is not needed for heptamer assembly or protein function. Without the tail, however, the heptamers (Aacpn10del-25) readily aggregate into fibrillar stacked rings. To explain this phenomenon, we performed binding experiments with a peptide construct of the tail to establish its specificity for Aacpn10del-25 and used cryo-electron microscopy to determine the three-dimensional (3D) structure of the GroEL-Aacpn10-ADP complex at an 8-Å resolution. We found that the GroEL-Aacpn10 structure is similar to the GroEL-GroES structure at this resolution, suggesting that Aacpn10 has molecular interactions with cpn60 similar to other cpn10s. The cryo-electron microscopy density map does not directly reveal the density of the Aacpn10 25-residue tail. However, the 3D statistical variance coefficient map computed from multiple 3D reconstructions with randomly selected particle images suggests that the tail is located at the Aacpn10 monomer-monomer interface and extends toward the cis-ring apical domain of GroEL. The tail at this location does not block the formation of a functional co-chaperonin/chaperonin complex but limits self-aggregation into linear fibrils at high temperatures. In addition, the 3D variance coefficient map identifies several regions inside the GroEL-Aacpn10 complex that have flexible conformations. This observation is in full agreement with the structural properties of an effective chaperonin.     

Monomer topology defines folding speed of heptamer

Small monomeric proteins often fold in apparent two-state processes with folding speeds dictated by their native-state topology. Here we test, for the first time, the influence of monomer topology on the folding speed of an oligomeric protein: the heptameric cochaperonin protein 10 (cpn10), which in the native state has seven beta-barrel subunits noncovalently assembled through beta-strand pairing. Cpn10 is a particularly useful model because equilibrium-unfolding experiments have revealed that the denatured state in urea is that of a nonnative heptamer. Surprisingly, refolding of the nonnative cpn10 heptamer is a simple two-state kinetic process with a folding-rate constant in water (2.1 sec(-1); pH 7.0, 20 degrees C) that is in excellent agreement with the prediction based on the native-state topology of the cpn10 monomer. Thus, the monomers appear to fold as independent units, with a speed that correlates with topology, although the C and N termini are trapped in beta-strand pairing with neighboring subunits. In contrast, refolding of unfolded cpn10 monomers is dominated by a slow association step.     

Reversible denaturation of oligomeric human chaperonin 10: denatured state depends on chemical denaturant

Chaperonins cpn60/cpn10 (GroEL/GroES in Escherichia coli) assist folding of nonnative polypeptides. Folding of the chaperonins themselves is distinct in that it entails assembly of a sevenfold symmetrical structure. We have characterized denaturation and renaturation of the recombinant human chaperonin 10 (cpn10), which forms a heptamer. Denaturation induced by chemical denaturants urea and guanidine hydrochloride (GuHCl) as well as by heat was monitored by tyrosine fluorescence, far-ultraviolet circular dichroism, and cross-linking; all denaturation reactions were reversible. GuHCl-induced denaturation was found to be cpn10 concentration dependent, in accord with a native heptamer to denatured monomer transition. In contrast, urea-induced denaturation was not cpn10 concentration dependent, suggesting that under these conditions cpn10 heptamers denature without dissociation. There were no indications of equilibrium intermediates, such as folded monomers, in either denaturant. The different cpn10 denatured states observed in high [GuHCl] and high [urea] were supported by cross-linking experiments. Thermal denaturation revealed that monomer and heptamer reactions display the same enthalpy change (per monomer), whereas the entropy-increase is significantly larger for the heptamer. A thermodynamic cycle for oligomeric cpn10, combining chemical denaturation with the dissociation constant in absence of denaturant, shows that dissociated monomers are only marginally stable (3 kJ/mol). The thermodynamics for co-chaperonin stability appears conserved; therefore, instability of the monomer could be necessary to specify the native heptameric structure.     

Chaperonin cofactors, Cpn10 and Cpn20, of green algae and plants function as hetero-oligomeric ring complexes

The chloroplast chaperonin system of plants and green algae is a curiosity as both the chaperonin cage and its lid are encoded by multiple genes, in contrast to the single genes encoding the two components of the bacterial and mitochondrial systems. In the green alga Chlamydomonas reinhardtii (Cr), three genes encode chaperonin cofactors, with cpn10 encoding a single ～10-kDa domain and cpn20 and cpn23 encoding tandem cpn10 domains. Here, we characterized the functional interaction of these proteins with the Escherichia coli chaperonin, GroEL, which normally cooperates with GroES, a heptamer of ～10-kDa subunits. The C. reinhardtii cofactor proteins alone were all unable to assist GroEL-mediated refolding of bacterial ribulose-bisphosphate carboxylase/oxygenase but gained this ability when CrCpn20 and/or CrCpn23 was combined with CrCpn10. Native mass spectrometry indicated the formation of hetero-oligomeric species, consisting of seven ～10-kDa domains. The cofactor "heptamers" interacted with GroEL and encapsulated substrate protein in a nucleotide-dependent manner. Different hetero-oligomer arrangements, generated by constructing cofactor concatamers, indicated a preferential heptamer configuration for the functional CrCpn10-CrCpn23 complex. Formation of heptamer Cpn10/Cpn20 hetero-oligomers was also observed with the Arabidopsis thaliana (At) cofactors, which functioned with the chloroplast chaperonin, AtCpn60α(7)β(7). It appears that hetero-oligomer formation occurs more generally for chloroplast chaperonin cofactors, perhaps adapting the chaperonin system for the folding of specific client proteins.     

ATP induces large quaternary rearrangements in a cage-like chaperonin structure

BACKGROUND: The chaperonins, a family of molecular chaperones, are large oligomeric proteins that bind nonnative intermediates of protein folding. They couple the release and correct folding of their ligands to the binding and hydrolysis of ATP. Chaperonin 60 (cpn60) is a decatetramer (14-mer) of 60 kD subunits. Folding of some ligands also requires the cooperation of cpn10, a heptamer of 10 kD subunits. RESULTS: We have determined the three-dimensional arrangements of subunits in Rhodobacter sphaeroides cpn60 in the nucleotide-free and ATP-bound forms. Negative stain electron microscopy and tilt reconstruction show the cylindrical structure of the decatetramer comprising two rings of seven subunits. The decatetramer consists of two cages joined base-to-base without a continuous central channel. These cages appear to contain bound polypeptide with an asymmetric distribution between the two rings. The two major domains of each subunit are connected on the exterior of the cylinder by a narrower bridge of density that could be a hinge region. Binding of ATP to cpn60 causes a major rearrangement of the protein density, which is reversed upon the hydrolysis of the ATP. Cpn10 binds to only one end of the cpn60 structure and is visible as an additional layer of density forming a cap on one end of the cpn60 cylinder. CONCLUSIONS: The observed rearrangement is consistent with an inward 5-10 degrees rotation of subunits, pivoting about the subunit contacts between the two heptamers, and thus bringing cpn60 domains towards the position occupied by the bound polypeptide. This change could explain the stimulation of ATPase activity by ligands, and the effects of ATP on lowering the affinity of cpn60 for ligands and on triggering the release of folding polypeptides.     

Structural stability of oligomeric chaperonin 10: the role of two beta-strands at the N and C termini in structural stabilization

Chaperonin 10 (cpn10) is a well-conserved subgroup of the molecular chaperone family. GroES, the cpn10 from Escherichia coli, is composed of seven 10kDa subunits, which form a dome-like oligomeric ring structure. From our previous studies, it was found that GroES unfolded completely through a three-state unfolding mechanism involving a partly folded monomer and that this reaction was reversible. In order to study whether these unfolding-refolding characteristics were conserved in other cpn10 proteins, we have examined the structural stabilities of cpn10s from rat mitochondria (RatES) and from hyperthermophilic eubacteria Thermotoga maritima (TmaES), and compared the values to those of GroES. From size-exclusion chromatography experiments in the presence of various concentrations of Gdn-HCl at 25 degrees C, both cpn10s showed unfolding-refolding characteristics similar to those of GroES, i.e. two-stage unfolding reactions that include formation of a partially folded monomer. Although the partially folded monomer of TmaES was considerably more stable compared to GroES and RatES, it was found that the overall stabilities of all three cpn10s were achieved significantly by inter-subunit interactions. We studied this contribution of inter-subunit interactions to overall stability in the GroES heptamer by introducing a mutation that perturbed subunit association, specifically the interaction between the two anti-parallel beta-strands at the N and C termini of this protein. From analyses of the mutants' stabilities, it was revealed that the anti-parallel beta-strands at the subunit interface are crucial for subunit association and stabilization of the heptameric GroES protein.     

Mycobacterium tuberculosis chaperonin 10 heptamers self-associate through their biologically active loops

The crystal structure of Mycobacterium tuberculosis chaperonin 10 (cpn10(Mt)) has been determined to a resolution of 2.8 A. Two dome-shaped cpn10(Mt) heptamers complex through loops at their bases to form a tetradecamer with 72 symmetry and a spherical cage-like structure. The hollow interior enclosed by the tetradecamer is lined with hydrophilic residues and has dimensions of 30 A perpendicular to and 60 A along the sevenfold axis. Tetradecameric cpn10(Mt) has also been observed in solution by dynamic light scattering. Through its base loop sequence cpn10(Mt) is known to be the agent in the bacterium responsible for bone resorption and for the contribution towards its strong T-cell immunogenicity. Superimposition of the cpn10(Mt) sequences 26 to 32 and 66 to 72 and E. coli GroES 25 to 31 associated with bone resorption activity shows them to have similar conformations and structural features, suggesting that there may be a common receptor for the bone resorption sequences. The base loops of cpn10s in general also attach to the corresponding chaperonin 60 (cpn60) to enclose unfolded protein and to facilitate its correct folding in vivo. Electron density corresponding to a partially disordered protein subunit appears encapsulated within the interior dome cavity of each heptamer. This suggests that the binding of substrates to cpn10 is possible in the absence of cpn60.     

Interface mutation in heptameric co-chaperonin protein 10 destabilizes subunits but not interfaces

We here report on a human mitochondrial co-chaperonin protein 10 (cpn10) variant in which the conserved interface residue leucine-96 is replaced with glycine (Leu96Gly cpn10). According to analytical ultracentrifugation, the mutation does not perturb the ability to assemble into a heptamer and electron microscopy reveals that Leu96Gly cpn10 is ring-shaped like wild-type cpn10. Despite elimination of a hydrophobic residue, the subunit-subunit affinity is essentially identical in Leu96Gly cpn10 and in wild-type cpn10. This is explained by a compensating rearrangement in Leu96Gly cpn10, evident from cross-linking and gel-filtration experiments. As a direct result of lower monomer stability, Leu96Gly cpn10 is dramatically less stable towards chemical and thermal perturbations as compared to wild-type cpn10. We conclude that leucine-96 is an interface residue preserved to guarantee stable cpn10 monomers. Our study demonstrates that the cpn10 interfaces can adapt to structural alterations without loss of either subunit-subunit affinity or heptamer specificity.     

On the oligomeric state of chloroplast chaperonin 10 and chaperonin 20

Type I chaperonins are fundamental protein folding machineries that function in eubacteria, mitochondria and chloroplasts. Eubacteria and mitochondria contain chaperonin systems comprised of homo-oligomeric chaperonin 60 tetradecamers and co-chaperonin 10 heptamers. In contrast, the chloroplast chaperonins are heterooligomeric tetradecamers that are composed of two subunit types, alpha and beta. Additionally, chloroplasts contain two structurally distinct co-chaperonins. One, ch-cpn10, is probably similar to the mitochondrial and bacterial co-chaperonins, and is composed of 10 kDa subunits. The other, termed ch-cpn20 is composed of two cpn10-like domains that are held together by a short linker. While the oligomeric structure of ch-cpn10 remains to be elucidated, it was previously suggested that ch-cpn20 forms tetramers in solution, and that this is the functional oligomer. In the present study, we investigated the properties of purified ch-cpn10 and ch-cpn20. Using bifunctional cross-linking reagents, gel filtration chromatography and analytical ultracentrifugation, we show that ch-cpn10 is a heptamer in solution. In contrast, ch-cpn20 forms multiple oligomers that are in dynamic equilibrium with each other and cover a broad spectrum of molecular weights in a concentration-dependent manner. However, upon association with GroEL, only one type of co-chaperonin-GroEL complex is formed.     

Multiple equilibria of the Escherichia coli chaperonin GroES revealed by mass spectrometry

Nanospray time-of-flight mass spectrometry has been used to study the assembly of the heptamer of the Escherichia coli cochaperonin protein GroES, a system previously described as a monomer-heptamer equilibrium. In addition to the monomers and heptamers, we have found measurable amounts of dimers and hexamers, the presence of which suggests the following mechanism for heptamer assembly: 2 Monomers <--> Dimer; 3 Dimers <--> Hexamer; Hexamer + Monomer <--> Heptamer. Equilibrium constants for each of these steps, and an overall constant for the Monomer <--> Heptamer equilibrium, have been estimated from the data. These constants imply a standard free-energy change, DeltaG(0), of about 9 kcal/mol for each contact surface formed between GroES subunits, except for the addition of the last subunit, where DeltaG(0) = 6 kcal/mol. This lower value probably reflects the loss of entropy when the heptamer ring is formed. These experiments illustrate the advantages of electrospray mass spectrometry as a method of measuring all components of a multiple equilibrium system.     

Gas channels for NH(3): proteins from hyperthermophiles complement an Escherichia coli mutant

Ammonium transport (Amt) proteins appear to be bidirectional channels for NH(3). The amt genes of the hyperthermophiles Aquifex aeolicus and Methanococcus jannaschii complement enteric amtB mutants for growth at 25 nM NH(3) at 37 degrees C. To our knowledge, Amt proteins are the first hyperthermophilic membrane transport proteins shown to be active in a mesophilic bacterium. Despite low expression levels, His-tagged Aquifex Amt could be purified by heating and nickel chelate affinity chromatography. It could be studied genetically in Escherichia coli.     

Intermediates in the chaperonin-assisted refolding of rhodanese are trapped at low temperature and show a small stoichiometry.

In vitro refolding of the urea-unfolded, monomeric, mitochondrial enzyme rhodanese (thiosulfate sulfur-transferase; EC 2.8.1.1) is facilitated by the chaperonin proteins cpn60 and cpn10 from Escherichia coli at 37 degrees C, but the refolding is strongly inhibited at 10 degrees C. In contrast, the unassisted refolding of rhodanese is efficient at 10 degrees C, but the refolding efficiency decreases as the temperature is raised. These observations provided two measures of the cpn60-rhodanese complex. Thus, we monitored either 1) the cpn60-dependent inhibition of spontaneous folding at 10 degrees C or 2) the recovery of active rhodanese in the complete chaperonin system at 25 degrees C, after first forming a cpn60-rhodanese complex at 10 degrees C. These procedures minimized the aggregation of interactive folding intermediates that tend to overestimate the apparent number of cpn60 14-mers in determining the stoichiometry of protein-cpn60 14-mer interactions. Both procedures used here gave results that were consistent with there being 1 rhodanese binding site/cpn60 tetradecamer. This stoichiometry is significantly less than might be expected from the fact that cpn60 is composed of 14 identical subunits, and it may indicate that rhodanese interacts with a restricted region that is formed when the cpn60 tetradecamer is assembled. The ability to stabilize chaperonin-protein complexes that can subsequently be reactivated will aid studies of the mode of action of the ubiquitous chaperonin proteins.     

Macromolecular crowding extended to a heptameric system: the Co-chaperonin protein 10

Experiments on monomeric proteins have shown that macromolecular crowding can stabilize toward heat perturbation and also modulate native-state structure. To assess the effects of macromolecular crowding on unfolding of an oligomeric protein, we here tested the effects of the synthetic crowding agent Ficoll 70 on human cpn10 (GroES in E. coli), a heptameric protein consisting of seven identical β-barrel subunits assembling into a ring. Using far-UV circular dichroism (CD), tyrosine fluorescence, nuclear magnetic resonance (NMR), and cross-linking experiments, we investigated thermal and chemical stability, as well as the heptamer-monomer dissociation constant, without and with crowding agent. We find that crowding shifts the heptamer-monomer equilibrium constant in the direction of the heptamer. The cpn10 heptamer is both thermally and thermodynamically stabilized in 300 mg/mL Ficoll 70 as compared to regular buffer conditions. Kinetic unfolding experiments show that the increased stability in crowded conditions, in part, is explained by slower unfolding rates. A thermodynamic cycle reveals that in presence of 300 mg/mL Ficoll the thermodynamic stability of each cpn10 monomer increases by over 30%, whereas the interfaces are stabilized by less than 10%. We also introduce a new approach to analyze the spectroscopic data that makes use of multiple wavelengths: this provides robust error estimates of thermodynamic parameters.     

Differential effects of alcohols on conformational switchovers in alpha-helical and beta-sheet protein models

Organic solvents may induce non-native structures of proteins that mimic folding intermediates and/or conformations that occur in proximity to biological membranes. Here we systematically investigate the effects of simple (i.e., MeOH and EtOH) and fluorinated (i.e., trifluoroethanol, TFE) alcohols on the secondary structure and thermodynamic stability of two complementary model proteins using a combination of circular dichroism, fluorescence, and Fourier transform infrared (FTIR) detection methods. The selected proteins are alpha-helical Borrelia burgdorferi VlsE and beta-sheet human mitochondrial co-chaperonin protein 10 (cpn10). We find that switches between VlsE's native and non-native superhelical and beta-sheet structures readily occur (pH 7, 20 degrees C). The pathway depends on the alcohol: addition of MeOH induces a transition to a superhelical structure that is followed by conversion to beta-structure, whereas EtOH only unfolds the protein. TFE unfolds VlsE at low percentages but promotes the formation of a superhelical state upon further additions. For cpn10, both MeOH and TFE additions govern initial unfolding; however, further additions of MeOH result in the formation of a non-native beta-structure, whereas subsequent additions of TFE induce a superhelical structure. EtOH additions promptly unfold and precipitate cpn10. Both VlsE's and cpn10's non-native structures exhibit high stability toward chemical and thermal perturbations. This study demonstrates that in response to different alcohols, polypeptides can readily adopt both alpha- and beta-enriched conformations. The biological significance of these findings is discussed.     

Exceptional stability of a [2Fe-2S] ferredoxin from hyperthermophilic bacterium Aquifex aeolicus

Aquifex aeolicus is the only hyperthermophile that is known to contain a plant- and mammalian-type [2Fe-2S] ferredoxin (Aae Fd1). This unique protein contains two cysteines, in addition to the four that act as ligands of the [2Fe-2S] cluster, which form a disulfide bridge. We have investigated the stability of Aae Fd1 with (wild-type) and without (C87A variant) the disulfide bond, with respect to pH, thermal and chemical perturbation, and compared the results to those for the mesophilic [2Fe-2S] ferredoxin from spinach. Unfolding reactions of all three proteins are irreversible due to cluster decomposition in the unfolded state. Wild-type and C87A Aae Fd1 proteins are extremely stable: unfolding at 20 degrees C requires high concentrations of the chemical denaturant and long incubation times. Moreover, their thermal-unfolding midpoints are 40-50 degrees higher than that for spinach ferredoxin (pH 7). The stability of the Aae Fd1 protein is significantly lower at pH 2.5 than pH 7 and 10, suggesting that ionic interactions play a role in structural integrity. Interestingly, the iron-sulfur cluster in C87A Aae Fd1 rearranges into a transient species with absorption bands at 520 and 610 nm, presumably a linear three-iron cluster, in the high-pH unfolded state.     

Differential regulation by the homologous response regulators NarL and NarP of Escherichia coli K-12 depends on DNA binding site arrangement

The NarL and NarP proteins are homologous response regulators of Escherichia coli that control the expression of several operons in response to nitrate and nitrite. A consensus heptameric NarL DNA-binding sequence has been identified, and previous observations suggest that the NarP protein has a similar sequence specificity. However, some operons are regulated by NarL alone, whereas others are controlled by both NarL and NarP. In this study, DNase I footprinting experiments with the fdnG , nirB and nrfA control regions revealed that NarP only binds to heptamer sequences organized as an inverted repeat with a 2 bp spacing (7–2–7 sites). The NarL protein also binds to these 7–2–7 sites but, unlike NarP, also recognizes heptamers in other arrangements. These results provide an explanation for the regulation of some operons by NarL alone and for the different effects of NarL and NarP at common target operons, such as fdnG and nrfA . To investigate this differential DNA binding further, derivatives of the nrfA control region were constructed in which the spacing of the 7–2–7 heptamers was increased (7– n –7 constructs). Increasing the spacing to four or more basepairs abolished NarP binding and significantly reduced NarL binding. The NarL protein also had a reduced binding affinity for heptamers adjacent to the 7– n –7 heptamer pair, suggesting a decrease in cooperative interactions. In conclusion, we propose that 7–2–7 sites are preferred by both NarL and NarP. NarL can also recognize other binding site arrangements, an ability that appears to be lacking in NarP.