Multiple chaperonins in bacteria – why so many?

A significant proportion of bacteria express two or more chaperonin genes. Chaperonins are a group of molecular chaperones, defined by sequence similarity, required for the folding of some cellular proteins. Chaperonin monomers have a mass of c . 60 kDa, and are typically found as large protein complexes containing 14 subunits arranged in two rings. The mechanism of action of the Escherichia coli GroEL protein has been studied in great detail. It acts by binding to unfolded proteins and enabling them to fold in a protected environment where they do not interact with any other proteins. GroEL can assist the folding of many proteins of different sizes, sequences, and structures, and homologues from many different bacteria can functionally replace GroEL in E. coli . What then are the functions of multiple chaperonins? Do they provide a mechanism for cells to increase their general chaperoning ability, or have they become specialized to take on specific novel cellular roles? Here I will review the genetic, biochemical, and phylogenetic evidence that has a bearing on this question, and show that there is good evidence for at least some specificity of function in multiple chaperonin genes.     

Regulation of translation of the head protein of T4 bacteriophage by specific binding of EF-Tu to a leader sequence

Recent evidence indicates that translation elongation factor Tu (EF-Tu) has a role in the cell in addition to its well established role in translation. The translation factor binds to a specific region called the Gol region close to the N terminus of the T4 bacteriophage major head protein as the head protein emerges from the ribosome. This binding was discovered because EF-Tu bound to Gol peptide is the specific substrate of the Lit protease that cleaves the EF-Tu between amino acid residues Gly59 and lle60, blocking phage development. These experiments raised the question of why the Gol region of the incipient head protein binds to EF-Tu, as binding to incipient proteins is not expected from the canonical role of EF-Tu. Here, we use gol-lacZ translational fusions to show that cleavage of EF-Tu in the complex with Gol peptide can block translation of a lacZ reporter gene fused translationally downstream of the Gol peptide that activated the cleavage. We propose a model to explain how binding of EF-Tu to the emerging Gol peptide could cause translation to pause temporarily and allow time for the leader polypeptide to bind to the GroEL chaperonin before translation continues, allowing cotranslation of the head protein with its insertion into the GroEL chaperonin chamber, and preventing premature synthesis and precipitation of the head protein. Cleavage of EF-Tu in the complex would block translation of the head protein and therefore development of the infecting phage. Experiments are presented that confirm two predictions of this model. Considering the evolutionary conservation of the components of this system, this novel regulatory mechanism could be used in other situations, both in bacteria and eukaryotes, where proteins are cotranslated with their insertion into cellular structures.     

Directed evolution of substrate-optimized GroEL/S chaperonins

GroEL/S chaperonin ring complexes fold many unrelated proteins. To understand the basis and extent of the chaperonin substrate spectrum, we used rounds of selection and DNA shuffling to obtain GroEL/S variants that dramatically enhanced folding of a single substrate-green fluorescent protein (GFP). Changes in the substrate-optimized chaperonins increase the polarity of the folding cavity and alter the ATPase cycle. These findings reveal a surprising plasticity of GroEL/S, which can be exploited to aid folding of recombinant proteins. Our studies also reveal a conflict between specialization and generalization of chaperonins as increased GFP folding comes at the expense of the ability of GroEL/S to fold its natural substrates. This conflict and the nature of the ring structure may help explain the evolution of cellular chaperone systems.     

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.     

Active Cage Mechanism of Chaperonin-Assisted Protein Folding Demonstrated at Single-Molecule Level

The cylindrical chaperonin GroEL and its lid-shaped cofactor GroES of Escherichia coli have an essential role in assisting protein folding by transiently encapsulating non-native substrate in an ATP-regulated mechanism. It remains controversial whether the chaperonin system functions solely as an infinite dilution chamber, preventing off-pathway aggregation, or actively enhances folding kinetics by modulating the folding energy landscape. Here we developed single-molecule approaches to distinguish between passive and active chaperonin mechanisms. Using low protein concentrations (100 pM) to exclude aggregation, we measured the spontaneous and GroEL/ES-assisted folding of double-mutant maltose binding protein (DM-MBP) by single-pair fluorescence resonance energy transfer and fluorescence correlation spectroscopy. We find that GroEL/ES accelerates folding of DM-MBP up to 8-fold over the spontaneous folding rate. Accelerated folding is achieved by encapsulation of folding intermediate in the GroEL/ES cage, independent of repetitive cycles of protein binding and release from GroEL. Moreover, photoinduced electron transfer experiments provided direct physical evidence that the confining environment of the chaperonin restricts polypeptide chain dynamics. This effect is mediated by the net-negatively charged wall of the GroEL/ES cavity, as shown using the GroEL mutant EL(KKK2) in which the net-negative charge is removed. EL(KKK2)/ES functions as a passive cage in which folding occurs at the slow spontaneous rate. Taken together our findings suggest that protein encapsulation can accelerate folding by entropically destabilizing folding intermediates, in strong support of an active chaperonin mechanism in the folding of some proteins. Accelerated folding is biologically significant as it adjusts folding rates relative to the speed of protein synthesis.     

The C-terminal Tails of the Bacterial Chaperonin GroEL Stimulate Protein Folding by Directly Altering the Conformation of a Substrate Protein

Many essential cellular proteins fold only with the assistance of chaperonin machines like the GroEL-GroES system of Escherichia coli. However, the mechanistic details of assisted protein folding by GroEL-GroES remain the subject of ongoing debate. We previously demonstrated that GroEL-GroES enhances the productive folding of a kinetically trapped substrate protein through unfolding, where both binding energy and the energy of ATP hydrolysis are used to disrupt the inhibitory misfolded states. Here, we show that the intrinsically disordered yet highly conserved C-terminal sequence of the GroEL subunits directly contributes to substrate protein unfolding. Interactions between the C terminus and the non-native substrate protein alter the binding position of the substrate protein on the GroEL apical surface. The C-terminal tails also impact the conformational state of the substrate protein during capture and encapsulation on the GroEL ring. Importantly, removal of the C termini results in slower overall folding, reducing the fraction of the substrate protein that commits quickly to a productive folding pathway and slowing several kinetically distinct folding transitions that occur inside the GroEL-GroES cavity. The conserved C-terminal tails of GroEL are thus important for protein folding from the beginning to the end of the chaperonin reaction cycle.     

Closing the folding chamber of the eukaryotic chaperonin requires the transition state of ATP hydrolysis

Chaperonins use ATPase cycling to promote conformational changes leading to protein folding. The prokaryotic chaperonin GroEL requires a cofactor, GroES, which serves as a "lid" enclosing substrates in the central cavity and confers an asymmetry on GroEL required for cooperative transitions driving the reaction. The eukaryotic chaperonin TRiC/CCT does not have such a cofactor but appears to have a "built-in" lid. Whether this seemingly symmetric chaperonin also operates through an asymmetric cycle is unclear. We show that unlike GroEL, TRiC does not close its lid upon nucleotide binding, but instead responds to the trigonal-bipyramidal transition state of ATP hydrolysis. Further, nucleotide analogs inducing this transition state confer an asymmetric conformation on TRiC. Similar to GroEL, lid closure in TRiC confines the substrates in the cavity and is essential for folding. Understanding the distinct mechanisms governing eukaryotic and bacterial chaperonin function may reveal how TRiC has evolved to fold specific eukaryotic proteins.     

An expanded protein folding cage in the GroEL-gp31 complex

Bacteriophage T4 produces a GroES analogue, gp31, which cooperates with the Escherichia coli GroEL to fold its major coat protein gp23. We have used cryo-electron microscopy and image processing to obtain three-dimensional structures of the E.coli chaperonin GroEL complexed with gp31, in the presence of both ATP and ADP. The GroEL-gp31-ADP map has a resolution of 8.2 A, which allows accurate fitting of the GroEL and gp31 crystal structures. Comparison of this fitted structure with that of the GroEL-GroES-ADP structure previously determined by cryo-electron microscopy shows that the folding cage is expanded. The enlarged volume for folding is consistent with the size of the bacteriophage coat protein gp23, which is the major substrate of GroEL-gp31 chaperonin complex. At 56 kDa, gp23 is close to the maximum size limit of a polypeptide that is thought to fit inside the GroEL-GroES folding cage.     

Designing a High Throughput Refolding Array Using a Combination of the GroEL Chaperonin and Osmolytes

Although GroE chaperonins and osmolytes had been used separately as protein folding aids, combining these two methods provides a considerable advantage for folding proteins that cannot fold with either osmolytes or chaperonins alone. This technique rapidly identifies superior folding solution conditions for a broad array of proteins that are difficult or impossible to fold by other methods. While testing the broad applicability of this technique, we have discovered that osmolytes greatly simplify the chaperonin reaction by eliminating the requirement for the co-chaperonin GroES which is normally involved in encapsulating folding proteins within the GroEL–GroES cavity. Therefore, combinations of soluble or immobilized GroEL, osmolytes and ATP or even ADP are sufficient to refold the test proteins. The first step in the chaperonin/osmolyte process is to form a stable long-lived chaperonin–substrate protein complex in the absence of nucleotide. In the second step, different osmolyte solutions are added along with nucleotides, thus forming a ‘folding array’ to identify superior folding conditions. The stable chaperonin–substrate protein complex can be concentrated or immobilized prior to osmolyte addition. This procedure prevents-off pathway aggregation during folding/refolding reactions and more importantly allows one to refold proteins at concentrations (~mg/ml) that are substantially higher than the critical aggregation concentration for given protein. This technique can be used for successful refolding of proteins from purified inclusion bodies. Recently, other investigators have used our chaperonin/osmolyte method to demonstrate that a mutant protein that misfolds in human disease can be rescued by GroEL/osmolyte system. Soluble or immobilized GroEL can be easily removed from the released folded protein using simple separation techniques. The method allows for isolation of folded monomeric or oligomeric proteins in quantities sufficient for X-ray crystallography or NMR structural determinations.     

Do Chaperonins Boost Protein Yields by Accelerating Folding or Preventing Aggregation?

The GroEL chaperonin has the ability to behave as an unfoldase, repeatedly denaturing proteins upon binding, which in turn can free them from kinetic traps and increase their folding rates. The complex formed by GroEL+GroES+ATP can also act as an infinite dilution cage, enclosing proteins within a protective container where they can fold without danger of aggregation. Controversy remains over which of these two properties is more critical to the GroEL/ES chaperonin's function. We probe the importance of the unfoldase nature of GroEL under conditions where aggregation is the predominant protein degradation pathway. We consider the effect of a hypothetical mutation to GroEL which increases the cycle frequency of GroEL/ES by increasing the rate of hydrolysis of GroEL-bound ATP. Using a simple kinetic model, we show that this modified chaperonin would be self-defeating: any potential reduction in folding time would be negated by an increase in time spent in the bulk, causing an increase in aggregation and a net decrease in protein folding yields.     

On the evolutionary origin of the chaperonins

An analysis of the apical domain of the Group-I and Group-II chaperonins shows that they have structural similarities to two different protein folds: a "swivel-domain" phosphotransferase and a thioredoxin-like peroxiredoxin. There is no significant sequence similarity that supports either similarity and the degree of similarity based on structure is comparable but weak for both relationships. Based on possible evolutionary transitions, we deduced that a phosphotransferase origin would require both a large insertion and deletion of structure whereas a peroxiredoxin origin requires only a peripheral rearrangement, similar to an internal domain-swap. We postulate that this change could have been triggered by the insertion of a peroxiredoxin into the ATPase domain that led to the modern chaperonin domain arrangement. The peroxidoxin fold is the most highly embellished member of the thioredoxin super-family and the insertion event may have "overloaded" the core, leading to a rearrangement. A peroxiredoxin origin for the domain also provides a functional explanation, as the peroxiredoxins can act as chaperones when they adopt a multimeric ring complex, similar to the chaperonin subunit configuration. In addition, several of the GroEL apical domain hydrophobic residues which interact with the unfolded protein are located in a position that corresponds to the protein substrate binding region of the peroxiredoxin fold. We suggest that the origin of the ur-chaperonin from a thioredoxin/peroxiredoxin fold might also account for the number of thioredoxin-fold containing proteins that interact with chaperonins, such as tubulin and phosducin-like proteins.     

Physicochemical Determinants of Chaperone Requirements

We describe a series of stringent relationships between abundance, solubility and chaperone usage of proteins. Based on these relationships, we show that the need of Escherichia coli proteins for the chaperonin GroEL can be predicted with 86% accuracy. Furthermore, from the observation that the abundance and solubility of proteins depend on the physicochemical properties of their amino acid sequences, we demonstrate that the requirement for GroEL can also be predicted directly from the sequences with 90% accuracy. These results indicate that the physicochemical properties of the amino acid sequences represent an essential component of the cellular quality control system that ensures the maintenance of protein homeostasis in living systems.     

Mechanisms of protein folding

The strong correlation between protein folding rates and the contact order suggests that folding rates are largely determined by the topology of the native structure. However, for a given topology, there may be several possible low free energy paths to the native state and the path that is chosen (the lowest free energy path) may depend on differences in interaction energies and local free energies of ordering in different parts of the structure. For larger proteins whose folding is assisted by chaperones, such as the Escherichia coli chaperonin GroEL, advances have been made in understanding both the aspects of an unfolded protein that GroEL recognizes and the mode of binding to the chaperonin. The possibility that GroEL can remove non-native proteins from kinetic traps by unfolding them either during polypeptide binding to the chaperonin or during the subsequent ATP-dependent formation of folding-active complexes with the co-chaperonin GroES has also been explored.     

GroEL mediates protein folding with a two successive timer mechanism

GroEL encapsulates nonnative substrate proteins in a central cavity capped by GroES, providing a safe folding cage. Conventional models assume that a single timer lasting approximately 8 s governs the ATP hydrolysis-driven GroEL chaperonin cycle. We examine single molecule imaging of GFP folding within the cavity, binding release dynamics of GroEL-GroES, ensemble measurements of GroEL/substrate FRET, and the initial kinetics of GroEL ATPase activity. We conclude that the cycle consists of two successive timers of approximately 3 s and approximately 5 s duration. During the first timer, GroEL is bound to ATP, substrate protein, and GroES. When the first timer ends, the substrate protein is released into the central cavity and folding begins. ATP hydrolysis and phosphate release immediately follow this transition. ADP, GroES, and substrate depart GroEL after the second timer is complete. This mechanism explains how GroES binding to a GroEL-substrate complex encapsulates the substrate rather than allowing it to escape into solution.     

Characterisation of a GroEL Single-Ring Mutant that Supports Growth of Escherichia coli and Has GroES-Dependent ATPase Activity

Binding and folding of substrate proteins by the molecular chaperone GroEL alternates between its two seven-membered rings in an ATP-regulated manner. The association of ATP and GroES to a polypeptide-bound ring of GroEL encapsulates the folding proteins in the central cavity of that ring (cis ring) and allows it to fold in a protected environment where the risk of aggregation is reduced. ATP hydrolysis in the cis ring changes the potentials within the system such that ATP binding to the opposite (trans) ring triggers the release of all ligands from the cis ring of GroEL through a complex network of allosteric communication between the rings. Inter-ring allosteric communication thus appears indispensable for the function of GroEL, and an engineered single-ring version (SR1) cannot substitute for GroEL in vivo. We describe here the isolation and characterisation of an active single-ring form of the GroEL protein (SR-A92T), which has an exceptionally low ATPase activity that is strongly stimulated by the addition of GroES. Dissection of the kinetic pathway of the ATP-induced structural changes in this active single ring can be explained by the fact that the mutation effectively blocks progression through the full allosteric pathway of the GroEL reaction cycle, thus trapping an early allosteric intermediate. Addition of GroES is able to overcome this block by binding this intermediate and pulling the allosteric pathway to completion via mass action, explaining how bacterial cells expressing this protein as their only chaperonin are viable.     

Protein substrate binding induces conformational changes in the chaperonin GroEL. A suggested mechanism for unfoldase activity

Chaperonins are molecules that assist proteins during folding and protect them from irreversible aggregation. We studied the chaperonin GroEL and its interaction with the enzyme human carbonic anhydrase II (HCA II), which induces unfolding of the enzyme. We focused on conformational changes that occur in GroEL during formation of the GroEL-HCA II complex. We measured the rate of GroEL cysteine reactivity toward iodo[2-(14)C]acetic acid and found that the cysteines become more accessible during binding of a cysteine free mutant of HCA II. Spin labeling of GroEL with N-(1-oxyl-2,2,5, 5-tetramethyl-3-pyrrolidinyl)iodoacetamide revealed that this additional binding occurred because buried cysteine residues become accessible during HCA II binding. In addition, a GroEL variant labeled with 6-iodoacetamidofluorescein exhibited decreased fluorescence anisotropy upon HCA II binding, which resembles the effect of GroES/ATP binding. Furthermore, by producing cysteine-modified GroEL with the spin label N-(1-oxyl-2,2,5, 5-tetramethyl-3-pyrrolidinyl)iodoacetamide and the fluorescent label 5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid, we detected increases in spin-label mobility and fluorescence intensity in GroEL upon HCA II binding. Together, these results show that conformational changes occur in the chaperonin as a consequence of protein substrate binding. Together with previous results on the unfoldase activity of GroEL, we suggest that the chaperonin opens up as the substrate protein binds. This opening mechanism may induce stretching of the protein, which would account for reported unfoldase activity of GroEL and might explain how GroEL can actively chaperone proteins larger than HCA II.     

Proteome-wide analysis of chaperonin-dependent protein folding in Escherichia coli

The E. coli chaperonin GroEL and its cofactor GroES promote protein folding by sequestering nonnative polypeptides in a cage-like structure. Here we define the contribution of this system to protein folding across the entire E. coli proteome. Approximately 250 different proteins interact with GroEL, but most of these can utilize either GroEL or the upstream chaperones trigger factor (TF) and DnaK for folding. Obligate GroEL-dependence is limited to only approximately 85 substrates, including 13 essential proteins, and occupying more than 75% of GroEL capacity. These proteins appear to populate kinetically trapped intermediates during folding; they are stabilized by TF/DnaK against aggregation but reach native state only upon transfer to GroEL/GroES. Interestingly, substantially enriched among the GroEL substrates are proteins with (betaalpha)8 TIM-barrel domains. We suggest that the chaperonin system may have facilitated the evolution of this fold into a versatile platform for the implementation of numerous enzymatic functions.     

Identifying natural substrates for chaperonins using a sequence-based approach

The Escherichia coli chaperonin machinery, GroEL, assists the folding of a number of proteins. We describe a sequence-based approach to identify the natural substrate proteins (SPs) for GroEL. Our method is based on the hypothesis that natural SPs are those that contain patterns of residues similar to those found in either GroES mobile loop and/or strongly binding peptide in complex with GroEL. The method is validated by comparing the predicted results with experimentally determined natural SPs for GroEL. We have searched for such patterns in five genomes. In the E. coli genome, we identify 1422 (about one-third) sequences that are putative natural SPs. In Saccharomyces cerevisiae, 2885 (32%) of sequences can be natural substrates for Hsp60, which is the analog of GroEL. The precise number of natural SPs is shown to be a function of the number of contacts an SP makes with the apical domain (N(C)) and the number of binding sites (N(B)) in the oligomer with which it interacts. For known SPs for GroEL, we find approximately 4 < N(C) < 5 and 2 相似文献   

Requirement for GroEL/GroES-dependent protein folding under nonpermissive conditions of macromolecular crowding

Macromolecular crowding is a critical parameter affecting the efficiency of cellular protein folding. Here we show that the proteins dihydrofolate reductase, enolase, and green fluorescent protein, which can fold spontaneously in diluted buffer, lose this ability in a crowded environment. Instead, they accumulate as soluble, protease-sensitive non-native species. Their folding becomes dependent on the complete GroEL/GroES chaperonin system and is not affected by trap-GroEL, indicating that folding has to occur in the chaperonin cavity with release of nativelike proteins into the bulk solution. In addition, we demonstrate that efficient folding in the chaperonin cavity requires ATP hydrolysis, as formation of ternary GroEL/GroES complexes with substrate proteins in the presence of ADP results only in very inefficient reactivation. However, protein refolding reactions using ADP-fluoroaluminate complexes, or single-ring GroEL and GroES under conditions where only a single round of ATP hydrolysis occurs, yield large amounts of refolded enzymes. Thus, the mode of initial ternary complex formation appears to be critical for subsequent productive release of substrate into the cavity under certain crowding conditions, and is only efficient when triggered by ATP hydrolysis. Our data indicate that stringent conditions of crowding can impart a stronger dependence of folding proteins on the assistance by chaperonins.     

Chaperonin-assisted folding of glutamine synthetase under nonpermissive conditions: off-pathway aggregation propensity does not determine the co-chaperonin requirement

One of the proposed roles of the GroEL-GroES cavity is to provide an "infinite dilution" folding chamber where protein substrate can fold avoiding deleterious off-pathway aggregation. Support for this hypothesis has been strengthened by a number of studies that demonstrated a mandatory GroES requirement under nonpermissive solution conditions, i.e., the conditions where proteins cannot spontaneously fold. We have found that the refolding of glutamine synthetase (GS) does not follow this pattern. In the presence of natural osmolytes trimethylamine N-oxide (TMAO) or potassium glutamate, refolding GS monomers readily aggregate into very large inactive complexes and fail to reactivate even at low protein concentration. Surprisingly, under these "nonpermissive" folding conditions, GS can reactivate with GroEL and ATP alone and does not require the encapsulation by GroES. In contrast, the chaperonin dependent reactivation of GS under another nonpermissive condition of low Mg2+ (<2 mM MgCl2) shows an absolute requirement of GroES. High-performance liquid chromatography gel filtration analysis and irreversible misfolding kinetics show that a major species of the GS folding intermediates, generated under these "low Mg2+" conditions exist as long-lived metastable monomers that can be reactivated after a significantly delayed addition of the GroEL. Our results indicate that the GroES requirement for refolding of GS is not simply dictated by the aggregation propensity of this protein substrate. Our data also suggest that the GroEL-GroES encapsulated environment is not required under all nonpermissive folding conditions.