Mechanism of the eukaryotic chaperonin: protein folding in the chamber of secrets

Chaperonins are key components of the cellular chaperone machinery. These large, cylindrical complexes contain a central cavity that binds to unfolded polypeptides and sequesters them from the cellular environment. Substrate folding then occurs in this central cavity in an ATP-dependent manner. The eukaryotic chaperonin TCP-1 ring complex (TRiC, also called CCT) is indispensable for cell survival because the folding of an essential subset of cytosolic proteins requires TRiC, and this function cannot be substituted by other chaperones. This specificity indicates that TRiC has evolved structural and mechanistic features that distinguish it from other chaperones. Although knowledge of this unique complex is in its infancy, we review recent advances that open the way to understanding the secrets of its folding chamber.     

Dual action of ATP hydrolysis couples lid closure to substrate release into the group II chaperonin chamber

Group II chaperonins are ATP-dependent ring-shaped complexes that bind nonnative polypeptides and facilitate protein folding in archaea and eukaryotes. A built-in lid encapsulates substrate proteins within the central chaperonin chamber. Here, we describe the fate of the substrate during the nucleotide cycle of group II chaperonins. The chaperonin substrate-binding sites are exposed, and the lid is open in both the ATP-free and ATP-bound prehydrolysis states. ATP hydrolysis has a dual function in the folding cycle, triggering both lid closure and substrate release into the central chamber. Notably, substrate release can occur in the absence of a lid, and lid closure can occur without substrate release. However, productive folding requires both events, so that the polypeptide is released into the confined space of the closed chamber where it folds. Our results show that ATP hydrolysis coordinates the structural and functional determinants that trigger productive folding.     

Molecular determinants of the ATP hydrolysis asymmetry of the CCT chaperonin complex

The eukaryotic cytosolic chaperonin CCT is a molecular machine involved in assisting the folding of proteins involved in important cellular processes. Like other chaperonins, CCT is formed by a double‐ring structure but, unlike all of them, each ring is composed of eight different, albeit homologous subunits. This complexity has probably to do with the specificity in substrate interaction and with the mechanism of protein folding that takes place during the chaperonin functional cycle, but its detailed molecular basis remains unknown. We have analyzed the known proteomes in search of residues that are differentially conserved in the eight subunits, as predictors of functional specificity (specificity‐determining positions; SDPs). We have found that most of these SDPs are located near the ATP binding site, and that they define four CCT clusters, corresponding to subunits CCT3, CCT6, CCT8 and CCT1/2/4/5/7. Our results point to a spatial organisation of the CCT subunits in two opposite areas of the ring and provide a molecular explanation for the previously described asymmetry in the hydrolysis of ATP. Proteins 2014; 82:703–707. © 2014 Wiley Periodicals, Inc.     

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.     

Structure and function of a protein folding machine: the eukaryotic cytosolic chaperonin CCT

Coupled translocation of tRNA and mRNA in the ribosome during protein synthesis is one of the most challenging and intriguing problems in the field of translation. We highlight several key questions regarding the mechanism of translocation, and discuss possible mechanistic models in light of the recent crystal structures of the ribosome and its subunits.     

Yeast cell-free nuclear protein import requires ATP hydrolysis

Saccharomyces cerevisiae nuclear proteins are shown to localize specifically to isolated yeast nuclei under conditions selective for nuclear proteins. Nuclear association is time- and temperature-dependent, requires ATP hydrolysis, and is abolished by protease pretreatment of nuclei. The nucleus-localized protein is translocated across the nuclear envelope as determined by inaccessibility to externally added immobilized protease. This cell-free system, consisting of components from an organism amenable to genetic analysis, will facilitate the study of the poorly understood mechanism of nuclear protein localization. The finding that ATP hydrolysis is required for nuclear import is the most direct evidence that nuclear localization is energy-dependent.     

Maltose-binding protein is open in the catalytic transition state for ATP hydrolysis during maltose transport

The maltose transport complex of Escherichia coli, a member of the ATP-binding cassette superfamily, mediates the high affinity uptake of maltose at the expense of ATP. The membrane-associated transporter consists of two transmembrane subunits, MalF and MalG, and two copies of the cytoplasmic ATP-binding cassette subunit, MalK. Maltose-binding protein (MBP), a soluble periplasmic protein, delivers maltose to the MalFGK(2) transporter and stimulates hydrolysis by the transporter. Site-directed spin labeling electron paramagnetic resonance spectroscopy is used to monitor binding of MBP to MalFGK(2) and conformational changes in MBP as it interacts with MalFGK(2). Cysteine residues and spin labels have been introduced into the two lobes of MBP so that spin-spin interaction will report on ligand-induced closure of the protein (Hall, J. A., Thorgeirsson, T. E., Liu, J., Shin, Y. K., and Nikaido, H. (1997) J. Biol. Chem. 272, 17610-17614). At least two different modes of interaction between MBP and MalFGK(2) were detected. Binding of MBP to MalFGK(2) in the absence of ATP resulted in a decrease in motion of spin label at position 41 in the C-terminal domain of MBP. In a vanadate-trapped transition state intermediate, all free MBP became tightly bound to MalFGK(2), spin label in both lobes became completely immobilized, and spin-spin interactions were lost, suggesting that MBP was in an open conformation. Binding of non-hydrolyzable MgATP analogs or ATP in the absence of Mg is sufficient to stabilize a complex of open MBP and MalFGK(2). Taken together, these data suggest that closure of the MalK dimer interface coincides with opening of MBP and maltose release to the transporter.     

Allosteric modulation bypasses the requirement for ATP hydrolysis in regenerating low affinity transition state conformation of human P-glycoprotein

ATP-dependent drug transport by human P-glycoprotein (Pgp, ABCB1) involves a coordinated communication between its drug-binding site (substrate site) and the nucleotide binding/hydrolysis domain (ATP sites). It has been demonstrated that the two ATP sites of Pgp play distinct roles within a single catalytic turnover; whereas ATP binding or/and hydrolysis by one drives substrate translocation and dissociation, the hydrolytic activity of the other resets the transporter for the subsequent cycle (Sauna, Z. E., and Ambudkar, S. V. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 2515-2520; Sauna, Z. E., and Ambudkar, S. V. (2001) J. Biol. Chem. 276, 11653-11661). Trapping of ADP (or 8-azido-ADP) and vanadate (ADP.Vi or 8-azido-ADP.Vi) at the catalytic site, following nucleotide hydrolysis, markedly reduces the affinity of Pgp for its transport substrate [125I]iodoarylazidoprazosin ([125I]IAAP), resulting in dissociation of the latter. Regeneration of the [125I]IAAP site requires an additional round of nucleotide hydrolysis. In this study, we demonstrate that certain thioxanthene-based allosteric modulators, such as cis-(Z)-flupentixol and its closely related analogs, induce regeneration of [125I]IAAP binding to vanadate-trapped (or fluoroaluminate-trapped) Pgp without any further nucleotide hydrolysis. Regeneration was facilitated by dissociation of the trapped nucleotide and vanadate. Once regenerated, the substrate site remains accessible to [125I]IAAP even after removal of the modulator from the medium, suggesting a modulator-induced relaxation of a constrained transition state conformation. Consistent with this, limited trypsin digestion of vanadate-trapped Pgp shows protection by cis-(Z)-flupentixol of two Pgp fragments (approximately 60 kDa) recognizable by a polyclonal antiserum specific for the NH2-terminal half. No regeneration was observed in the Pgp mutant F983A that is impaired in modulation by flupentixols, indicating involvement of the allosteric modulator site in the phenomenon. In summary, the data demonstrate that in the nucleotide-trapped low affinity state of Pgp, the allosteric site remains accessible and responsive to modulation by flupentixol (and its closely related analogs), which can reset the high affinity state for [125I]IAAP binding without any further nucleotide hydrolysis.     

A first-order-like state transition for recombinant protein folding

Normally, proteins will aggregate and precipitate by direct folding processes. In this study, we report that quasi-static processes can restore both the structure and bio-function of two kinds of fish recombinant growth hormones (Plecoglossus altivelis and Epinephelus awoara). The conformational changes and the particle-size-distribution (PSD) of each refolding intermediate can be monitored by circular dichroism spectroscopy (CD) and dynamic light scattering (DLS), respectively. Conformation analysis of the CD spectra of the refolding intermediates indicated that the secondary structures were restored in the initial refolding intermediate. However, the tertiary interactions of the proteins were restored during the last two refolding stages, as elucidated by thermal stability tests. This is consistent with a sequential model. DLS analysis suggested that the average hydrodynamic radii of the refolding intermediates shrank to their native-like sizes after the first refolding stage. This is consistent with a collapse model. After comparison with the data on the direct folding process, it is concluded that the denaturant-containing protein folding reaction is a first-order-like state transition process.     

Vanadate-induced trapping of nucleotides by purified maltose transport complex requires ATP hydrolysis

The maltose transport system in Escherichia coli is a member of the ATP-binding cassette superfamily of transporters that is defined by the presence of two nucleotide-binding domains or subunits and two transmembrane regions. The bacterial import systems are unique in that they require a periplasmic substrate-binding protein to stimulate the ATPase activity of the transport complex and initiate the transport process. Upon stimulation by maltose-binding protein, the intact MalFGK(2) transport complex hydrolyzes ATP with positive cooperativity, suggesting that the two nucleotide-binding MalK subunits interact to couple ATP hydrolysis to transport. The ATPase activity of the intact transport complex is inhibited by vanadate. In this study, we investigated the mechanism of inhibition by vanadate and found that incubation of the transport complex with MgATP and vanadate results in the formation of a stably inhibited species containing tightly bound ADP that persists after free vanadate and nucleotide are removed from the solution. The inhibited species does not form in the absence of MgCl(2) or of maltose-binding protein, and ADP or another nonhydrolyzable analogue does not substitute for ATP. Taken together, these data conclusively show that ATP hydrolysis must precede the formation of the vanadate-inhibited species in this system and implicate a role for a high-energy, ADP-bound intermediate in the transport cycle. Transport complexes containing a mutation in a single MalK subunit are still inhibited by vanadate during steady-state hydrolysis; however, a stably inhibited species does not form. ATP hydrolysis is therefore necessary, but not sufficient, for vanadate-induced nucleotide trapping.     

Appropriate initiation of the strand exchange reaction promoted by RecA protein requires ATP hydrolysis

The DNA-dependent ATPase activity of the Escherichia coli RecA protein has been recognized for more than two decades. Yet, the role of ATP hydrolysis in the RecA-promoted strand exchange reaction remains unclear. Here, we demonstrate that ATP hydrolysis is required as part of a proofreading process during homology recognition. It enables the RecA-ssDNA complex, after determining that the strand-exchanged duplex is mismatched, to dissociate from the synaptic complex, which allows it to re-initiate the search for a "true" homologous region. Furthermore, the results suggest that when non-homologous sequences are present at the proximal end, ATP hydrolysis is required to allow ssDNA-RecA to reinitiate the strand exchange from an internal homologous region.     

RecA-mediated SOS induction requires an extended filament conformation but no ATP hydrolysis

The Escherichia coli SOS response to DNA damage is modulated by the RecA protein, a recombinase that forms an extended filament on single-stranded DNA and hydrolyzes ATP. The RecA K72R ( recA2201 ) mutation eliminates the ATPase activity of RecA protein. The mutation also limits the capacity of RecA to form long filaments in the presence of ATP. Strains with this mutation do not undergo SOS induction in vivo . We have combined the K72R variant of RecA with another mutation, RecA E38K ( recA730 ). In vitro , the double mutant RecA E38K/K72R ( recA730,2201 ) mimics the K72R mutant protein in that it has no ATPase activity. The double mutant protein will form long extended filaments on ssDNA and facilitate LexA cleavage almost as well as wild-type, and do so in the presence of ATP. Unlike recA K72R, the recA E38K/K72R double mutant promotes SOS induction in vivo after UV treatment. Thus, SOS induction does not require ATP hydrolysis by the RecA protein, but does require formation of extended RecA filaments. The RecA E38K/K72R protein represents an improved reagent for studies of the function of ATP hydrolysis by RecA in vivo and in vitro .     

Essential role of the chaperonin folding compartment in vivo

The GroEL/GroES chaperonin system of Escherichia coli forms a nano-cage allowing single protein molecules to fold in isolation. However, as the chaperonin can also mediate folding independently of substrate encapsulation, it remained unclear whether the folding cage is essential in vivo. To address this question, we replaced wild-type GroEL with mutants of GroEL having either a reduced cage volume or altered charge properties of the cage wall. A stepwise reduction in cage size resulted in a gradual loss of cell viability, although the mutants bound non-native protein efficiently. Strikingly, a mild reduction in cage size increased the yield and the apparent rate of green fluorescent protein folding, consistent with the view that an effect of steric confinement can accelerate folding. As shown in vitro, the observed acceleration of folding was dependent on protein encapsulation by GroES but independent of GroES cycling regulated by the GroEL ATPase. Altering the net-negative charge of the GroEL cage wall also strongly affected chaperonin function. Based on these findings, the GroEL/GroES compartment is essential for protein folding in vivo.     

Complete change of the protein folding transition state upon circular permutation

Reversing the loop lengths of the small protein S6 by circular permutation has a dramatic effect on the transition state structure: it changes from globally diffuse to locally condensed. The phenomenon arises from a biased dispersion of the contact energies. Stability data derived from point mutations throughout the S6 structure show that interactions between residues that are far apart in sequence are stronger than those that are close. This entropy compensation drives all parts of the protein to fold simultaneously and produces the diffuse transition-state structure typical for two-state proteins. In the circular permutant, where strong contacts and short sequence separations are engineered to concur, the transition state becomes atypically condensed and polarized. Taken together with earlier findings that S6 may also fold by a 'collapsed' trajectory with an intermediate, the results suggest that this protein may fold by a multiplicity of mechanisms. The observations indicate that the diffuse transition state of S6 is not required for folding but could be an evolutionary development to optimize cooperativity.     

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.