Salt bridges at the inter-ring interface regulate the thermostat of GroEL

The chaperonin GroEL consists of a double-ring structure made of identical subunits and displays unusual allosteric properties caused by the interaction between its constituent subunits. Cooperative binding of ATP to a protein ring allows binding of GroES to that ring, and at the same time negative inter-ring cooperativity discharges the ligands from the opposite ring, thus driving the protein-folding cycle. Biochemical and electron microscopy analysis of wild type GroEL, a single-ring mutant (SR1), and two mutants with one inter-ring salt bridge of the chaperonin disrupted (E461K and E434K) indicate that these ion pairs form part of the interactions that allow the inter-ring allosteric signal to be transmitted. The wild type-like activities of the ion pair mutants at 25 degrees C are in contrast with their lack of inter-ring communication and folding activity at physiological temperatures. These salt bridges stabilize the inter-ring interface and maintain the inter-ring spacing so that functional communication between protein heptamers takes place. The characterization of GroEL hybrids containing different amounts of wild type and mutant subunits also indicates that as the number of inter-ring salt bridges increases the functional properties of the hybrids recover. Taken together, these results strongly suggest that inter-ring salt bridges form a stabilizing ring-shaped, ionic zipper that ensures inter-ring communication at the contact sites and therefore a functional protein-folding cycle. Furthermore, they regulate the chaperonin thermostat, allowing GroEL to distinguish physiological (37 degrees C) from stress temperatures (42 degrees C).     

GroEL stability and function. Contribution of the ionic interactions at the inter-ring contact sites

The chaperonin GroEL consists of a double ring structure made of identical subunits that display different modes of allosteric communication. The protein folding cycle requires the simultaneous positive intra-ring and negative inter-ring cooperativities of ATP binding. This ensures GroES binding to one ring and release of the ligands from the opposite one. To better characterize inter-ring allosterism, the thermal stability as well as the temperature dependence of the functional and conformational properties of wild type GroEL, a single ring mutant (SR1) and two single point mutants suppressing one interring salt bridge (E434K and E461K) were studied. The results indicate that ionic interactions at the two interring contact sites are essential to maintain the negative cooperativity for protein substrate binding and to set the protein thermostat at 39 degrees C. These electrostatic interactions contribute distinctly to the stability of the inter-ring interface and the overall protein stability, e.g. the E434K thermal inactivation curve is shifted to lower temperatures, and its unfolding temperature and activation energy are also lowered. An analysis of the ionic interactions at the inter-ring contact sites reveals that at the so called "left site" a network of electrostatic interactions involving three charged residues might be established, in contrast to what is found at the "right site" where only two oppositely charged residues interact. Our data suggest that electrostatic interactions stabilize protein-protein interfaces depending on both the number of ionic interactions and the number of residues engaged in each of these interactions. In the case of GroEL, this combination sets the thermostat of the protein so that the chaperonin distinguishes physiological from stress temperatures.     

On the role of symmetrical and asymmetrical chaperonin complexes in assisted protein folding.

The cylindrical chaperonin GroEL of E. coli and its ring-shaped cofactor GroES cooperate in mediating the ATP-dependent folding of a wide range of polypeptides in vivo and in vitro. By binding to the ends of the GroEL cylinder, GroES displaces GroEL-bound polypeptide into an enclosed folding cage, thereby preventing protein aggregation during folding. The dynamic interaction of GroEL and GroES is regulated by the GroEL ATPase and involves the formation of asymmetrical GroEL:GroES1 and symmetrical GroEL: GroES2 complexes. The proposed role of the symmetrical complex as a catalytic intermediate of the chaperonin mechanism has been controversial. It has also been suggested that the formation of GroEL:GroES2 complexes allows the folding of two polypeptide molecules per GroEL reaction cycle, one in each ring of GroEL. By making use of a procedure to stabilize chaperonin complexes by rapid crosslinking for subsequent analysis by native PAGE, we have quantified the occurrence of GroEL:GroES1 and GroEL:GroES2 complexes in active refolding reactions under a variety of conditions using mitochondrial malate dehydrogenase (mMDH) as a substrate. Our results show that the symmetrical complexes are neither required for chaperonin function nor does their presence significantly increase the rate of mMDH refolding. In contrast, chaperonin-assisted folding is strictly dependent on the formation of asymmetrical GroEL:GroES1 complexes. These findings support the view that GroEL:GroES2 complexes have no essential role in the chaperonin mechanism.     

ATP-bound states of GroEL captured by cryo-electron microscopy.

The chaperonin GroEL drives its protein-folding cycle by cooperatively binding ATP to one of its two rings, priming that ring to become folding-active upon GroES binding, while simultaneously discharging the previous folding chamber from the opposite ring. The GroEL-ATP structure, determined by cryo-EM and atomic structure fitting, shows that the intermediate domains rotate downward, switching their intersubunit salt bridge contacts from substrate binding to ATP binding domains. These observations, together with the effects of ATP binding to a GroEL-GroES-ADP complex, suggest structural models for the ATP-induced reduction in affinity for polypeptide and for cooperativity. The model for cooperativity, based on switching of intersubunit salt bridge interactions around the GroEL ring, may provide general insight into cooperativity in other ring complexes and molecular machines.     

GroEL-GroES cycling: ATP and nonnative polypeptide direct alternation of folding-active rings.

The double-ring chaperonin GroEL mediates protein folding in the central cavity of a ring bound by ATP and GroES, but it is unclear how GroEL cycles from one folding-active complex to the next. We observe that hydrolysis of ATP within the cis ring must occur before either nonnative polypeptide or GroES can bind to the trans ring, and this is associated with reorientation of the trans ring apical domains. Subsequently, formation of a new cis-ternary complex proceeds on the open trans ring with polypeptide binding first, which stimulates the ATP-dependent dissociation of the cis complex (by 20- to 50-fold), followed by GroES binding. These results indicate that, in the presence of nonnative protein, GroEL alternates its rings as folding-active cis complexes, expending only one round of seven ATPs per folding cycle.     

BeF(x) stops the chaperonin cycle of GroEL-GroES and generates a complex with double folding chambers

Coupling with ATP hydrolysis and cooperating with GroES, the double ring chaperonin GroEL assists the folding of other proteins. Here we report novel GroEL-GroES complexes formed in fluoroberyllate (BeF(x)) that can mimic the phosphate part of the enzyme-bound nucleotides. In ATP, BeF(x) stops the functional turnover of GroEL by preventing GroES release and produces a symmetric 1:2 GroEL-GroES complex in which both GroEL rings contain ADP.BeF(x) and an encapsulated substrate protein. In ADP, the substrate protein-loaded GroEL cannot bind GroES. In ADP plus BeF(x), however, it can bind GroES to form a stable 1:1 GroEL-GroES complex in which one of GroEL rings contains ADP.BeF(x) and an encapsulated substrate protein. This 1:1 GroEL-GroES complex is converted into the symmetric 1:2 GroEL-GroES complex when GroES is supplied in ATP plus BeF(x). Thus, BeF(x) stabilizes two GroEL-GroES complexes; one with a single folding chamber and the other with double folding chambers. These results shed light on the intermediate ADP.P(i) nucleotide states in the functional cycle of GroEL.     

Mapping pathways of allosteric communication in GroEL by analysis of correlated mutations

An interesting example of an allosteric protein is the chaperonin GroEL. It undergoes adenosine 5'-triphosphate-induced conformational changes that are reflected in binding of adenosine 5'-triphosphate with positive cooperativity within rings and negative cooperativity between rings. Herein, correlated mutations in chaperonins are analyzed to unravel routes of allosteric communication in GroEL and in its complex with its co-chaperonin GroES. It is shown that analysis of correlated mutations in the chaperonin family can provide information about pathways of allosteric communication within GroEL and between GroEL and GroES. The results are discussed in the context of available structural, genetic, and biochemical data concerning short- and long-range interactions in the GroE system.     

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.     

Synchronized domain-opening motion of GroEL is essential for communication between the two rings

Escherichia coli chaperonin GroEL consists of two stacked rings of seven identical subunits each. Accompanying binding of ATP and GroES to one ring of GroEL, that ring undergoes a large en bloc domain movement, in which the apical domain twists upward and outward. A mutant GroEL(AEX) (C138S,C458S,C519S,D83C,K327C) in the oxidized form is locked in a closed conformation by an interdomain disulfide cross-link and cannot hydrolyze ATP (Murai, N., Makino, Y., and Yoshida, M. (1996) J. Biol. Chem. 271, 28229-28234). By reconstitution of GroEL complex from subunits of both wild-type GroEL and oxidized GroEL(AEX), hybrid GroEL complexes containing various numbers of oxidized GroEL(AEX) subunits were prepared. ATPase activity of the hybrid GroEL containing one or two oxidized GroEL(AEX) subunits per ring was about 70% higher than that of wild-type GroEL. Based on the detailed analysis of the ATPase activity, we concluded that inter-ring negative cooperativity was lost in the hybrid GroEL, indicating that synchronized opening of the subunits in one ring is necessary for the negative cooperativity. Indeed, hybrid GroEL complex reconstituted from subunits of wild-type and GroEL mutant (D398A), which is ATPase-deficient but can undergo domain opening motion, retained the negative cooperativity of ATPase. In contrast, the ability of GroEL to assist protein folding was impaired by the presence of a single oxidized GroEL(AEX) subunit in a ring. Taken together, cooperative conformational transitions in GroEL rings ensure the functional communication between the two rings of GroEL.     

Single-molecule Observation of Protein Folding in Symmetric GroEL-(GroES)2 Complexes

The chaperonin, GroEL, is an essential molecular chaperone that mediates protein folding together with its cofactor, GroES, in Escherichia coli. It is widely believed that the two rings of GroEL alternate between the folding active state coupled to GroES binding during the reaction cycle. In other words, an asymmetric GroEL-GroES complex (the bullet-shaped complex) is formed throughout the cycle, whereas a symmetric GroEL-(GroES)₂ complex (the football-shaped complex) is not formed. We have recently shown that the football-shaped complex coexists with the bullet-shaped complex during the reaction cycle. However, how protein folding proceeds in the football-shaped complex remains poorly understood. Here, we used GFP as a substrate to visualize protein folding in the football-shaped complex by single-molecule fluorescence techniques. We directly showed that GFP folding occurs in both rings of the football-shaped complex. Remarkably, the folding was a sequential two-step reaction, and the kinetics were in excellent agreement with those in the bullet-shaped complex. These results demonstrate that the same reactions take place independently in both rings of the football-shaped complex to facilitate protein folding.     

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.     

Crystal Structure of a Symmetric Football-Shaped GroEL:GroES2-ATP14 Complex Determined at 3.8 Å Reveals Rearrangement between Two GroEL Rings

The chaperonin GroEL is an essential chaperone that assists in protein folding with the aid of GroES and ATP. GroEL forms a double-ring structure, and both rings can bind GroES in the presence of ATP. Recent progress on the GroEL mechanism has revealed the importance of a symmetric 1:2 GroEL:GroES₂ complex (the “football”-shaped complex) as a critical intermediate during the functional GroEL cycle. We determined the crystal structure of the football GroEL:GroES₂-ATP₁₄ complex from Escherichia coli at 3.8 Å, using a GroEL mutant that is extremely defective in ATP hydrolysis. The overall structure of the football complex resembled the GroES-bound GroEL ring of the asymmetric 1:1 GroEL:GroES complex (the “bullet” complex). However, the two GroES-bound GroEL rings form a modified interface by an ~ 7° rotation about the 7-fold axis. As a result, the inter-ring contacts between the two GroEL rings in the football complex differed from those in the bullet complex. The differences provide a structural basis for the apparently impaired inter-ring negative cooperativity observed in several biochemical analyses.     

Ionic interactions at both inter-ring contact sites of GroEL are involved in transmission of the allosteric signal: a time-resolved infrared difference study

The biological activity of the double-ring chaperonin GroEL is regulated by complex allosteric interactions, which include positive intra-ring and negative inter-ring cooperativity. To further characterize inter-ring communication, the nucleotide-induced absorbance changes in the vibrational spectrum of the chaperonin GroEL, of two single-point mutants suppressing one inter-ring ionic contact (E461K and E434K) and of a single-ring version of this protein, were investigated by time-resolved infrared difference spectroscopy. Interaction of the nucleotide with the proteins was triggered by its photochemical release from a biologically inactive caged precursor [P3-1-(2-nitro) phenylethyl nucleotide]. The results indicate that (1) ATP binding to the protein induces a conformational change that affects concomitantly both intra-ring and inter-ring communication, and (2) the experimental absorbance changes are sensitive to the double-ring structure of the protein. The characterization of the single-point, inter-ring mutants demonstrates that ionic interactions at both contact sites are involved in the transmission of the allosteric signal. However, both mutations have different effects on the inter-ring interface. While that of E461K still retains ionic contacts sensitive to ATP binding, E434K shows spectroscopic features similar to those of the single-ring version of the protein, therefore suggesting that electrostatic interactions at these contact sites contribute differently to the stability of the inter-ring interface.     

Football- and bullet-shaped GroEL-GroES complexes coexist during the reaction cycle

GroEL is an Escherichia coli chaperonin that is composed of two heptameric rings stacked back-to-back. GroEL assists protein folding with its cochaperonin GroES in an ATP-dependent manner in vitro and in vivo. However, it is still unclear whether GroES binds to both rings of GroEL simultaneously under physiological conditions. In this study, we monitored the GroEL-GroES interaction in the reaction cycle using fluorescence resonance energy transfer. We found that nearly equivalent amounts of symmetric GroEL-(GroES)(2) (football-shaped) complex and asymmetric GroEL-GroES (bullet-shaped) complex coexist during the functional reaction cycle. We also found that D398A, an ATP hydrolysis defective mutant of GroEL, forms a football-shaped complex with ATP bound to the two rings. Furthermore, we showed that ADP prevents the association of ATP to the trans-ring of GroEL, and as a consequence, the second GroES cannot bind to GroEL. Considering the concentrations of ADP and ATP in E. coli, ADP is expected to have a small effect on the inhibition of GroES binding to the trans-ring of GroEL in vivo. These results suggest that we should reconsider the chaperonin-mediated protein-folding mechanism that involves the football-shaped complex.     

Co-expression of chaperonin GroEL/GroES enhances in vivo folding of yeast mitochondrial aconitase and alters the growth characteristics of Escherichia coli

Over last two decades many researchers have demonstrated the mechanisms of how the Escherichia coli chaperonin GroEL and GroES work in the binding and folding of different aggregation prone substrate proteins both in vivo and in vitro. However, preliminary aspects, such as influence of co-expressing GroEL and GroES on the over expression of other recombinant proteins in E. coli cells and subsequent growth aspects, as well as the conditions for optimum production of recombinant proteins in presence of recombinant chaperones have not been properly investigated. In the present study we have demonstrated the temperature dependent growth characteristics of E. coli cells, which are over expressing recombinant aconitase and how the co-expression of E. coli chaperonin GroEL and GroES influence the growth rate of the cells and in vivo folding of recombinant aconitase. Presence of co-expressed GroEL reduces the aconitase over-expression drastically; however, exogenous GroEL & GroES together compensate this reduction. For the aconitase over-expressing cells the growth rate decreases by 30% at 25 degrees C when compared with the M15 E. coli cells, however, there is an increase of 20% at 37 degrees C indicating the participation of endogenous chaperonin in the folding of a fraction of over expressed aconitase. However, in presence of co-expressed GroEL and GroES the growth rate of aconitase producing cells was enhanced by 30% at 37 degrees C confirming the assistance of exogenous chaperone system for the folding of recombinant aconitase. Optimum in vivo folding of aconitase requires co-production of complete E. coli chaperonin machinery GroEL and GroES together.     

Identification of Elements That Dictate the Specificity of Mitochondrial Hsp60 for Its Co-Chaperonin

Type I chaperonins (cpn60/Hsp60) are essential proteins that mediate the folding of proteins in bacteria, chloroplast and mitochondria. Despite the high sequence homology among chaperonins, the mitochondrial chaperonin system has developed unique properties that distinguish it from the widely-studied bacterial system (GroEL and GroES). The most relevant difference to this study is that mitochondrial chaperonins are able to refold denatured proteins only with the assistance of the mitochondrial co-chaperonin. This is in contrast to the bacterial chaperonin, which is able to function with the help of co-chaperonin from any source. The goal of our work was to determine structural elements that govern the specificity between chaperonin and co-chaperonin pairs using mitochondrial Hsp60 as model system. We used a mutagenesis approach to obtain human mitochondrial Hsp60 mutants that are able to function with the bacterial co-chaperonin, GroES. We isolated two mutants, a single mutant (E321K) and a double mutant (R264K/E358K) that, together with GroES, were able to rescue an E. coli strain, in which the endogenous chaperonin system was silenced. Although the mutations are located in the apical domain of the chaperonin, where the interaction with co-chaperonin takes place, none of the residues are located in positions that are directly responsible for co-chaperonin binding. Moreover, while both mutants were able to function with GroES, they showed distinct functional and structural properties. Our results indicate that the phenotype of the E321K mutant is caused mainly by a profound increase in the binding affinity to all co-chaperonins, while the phenotype of R264K/E358K is caused by a slight increase in affinity toward co-chaperonins that is accompanied by an alteration in the allosteric signal transmitted upon nucleotide binding. The latter changes lead to a great increase in affinity for GroES, with only a minor increase in affinity toward the mammalian mitochondrial co-chaperonin.     

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.     

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.     

Dual function of protein confinement in chaperonin-assisted protein folding.

The GroEL/GroES chaperonin system mediates the folding of a range of newly synthesized polypeptides in the bacterial cytosol. Using a rapid biotin-streptavidin-based inhibition of chaperonin function, we show that the cage formed by GroEL and its cofactor GroES can have a dual role in promoting folding. First, enclosure of nonnative protein in the GroEL:GroES complex is essential for folding to proceed unimpaired by aggregation. Second, folding inside the cage can be significantly faster than folding in free solution, independently of ATP-driven cycles of GroES binding and release. This suggests that confinement of unfolded protein in the narrow hydrophilic space of the chaperonin cage smoothes the energy landscape for the folding of some proteins, increasing the flux of folding intermediates toward the native state.