Repetitive Protein Unfolding by the trans Ring of the GroEL-GroES Chaperonin Complex Stimulates Folding

A key constraint on the growth of most organisms is the slow and inefficient folding of many essential proteins. To deal with this problem, several diverse families of protein folding machines, known collectively as molecular chaperones, developed early in evolutionary history. The functional role and operational steps of these remarkably complex nanomachines remain subjects of active debate. Here we present evidence that, for the GroEL-GroES chaperonin system, the non-native substrate protein enters the folding cycle on the trans ring of the double-ring GroEL-ATP-GroES complex rather than the ADP-bound complex. The properties of this ATP complex are designed to ensure that non-native substrate protein binds first, followed by ATP and finally GroES. This binding order ensures efficient occupancy of the open GroEL ring and allows for disruption of misfolded structures through two phases of multiaxis unfolding. In this model, repeated cycles of partial unfolding, followed by confinement within the GroEL-GroES chamber, provide the most effective overall mechanism for facilitating the folding of the most stringently dependent GroEL substrate proteins.     

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.     

Stimulating the substrate folding activity of a single ring GroEL variant by modulating the cochaperonin GroES

In mediating protein folding, chaperonin GroEL and cochaperonin GroES form an enclosed chamber for substrate proteins in an ATP-dependent manner. The essential role of the double ring assembly of GroEL is demonstrated by the functional deficiency of the single ring GroEL(SR). The GroEL(SR)-GroES is highly stable with minimal ATPase activity. To restore the ATP cycle and the turnover of the folding chamber, we sought to weaken the GroEL(SR)-GroES interaction systematically by concatenating seven copies of groES to generate groES(7). GroES Ile-25, Val-26, and Leu-27, residues on the GroEL-GroES interface, were substituted with Asp on different groES modules of groES(7). GroES(7) variants activate ATP activity of GroEL(SR), but only some restore the substrate folding function of GroEL(SR), indicating a direct role of GroES in facilitating substrate folding through its dynamics with GroEL. Active GroEL(SR)-GroES(7) systems may resemble mammalian mitochondrial chaperonin systems.     

Transient conformational remodeling of folding proteins by GroES—individually and in concert with GroEL

The commonly accepted dogma of the bacterial GroE chaperonin system entails protein folding mediated by cycles of several ATP-dependent sequential steps where GroEL interacts with the folding client protein. In contrast, we herein report GroES-mediated dynamic remodeling (expansion and compression) of two different protein substrates during folding: the endogenous substrate MreB and carbonic anhydrase (HCAII), a well-characterized protein folding model. GroES was also found to influence GroEL binding induced unfolding and compression of the client protein underlining the synergistic activity of both chaperonins, even in the absence of ATP. This previously unidentified activity by GroES should have important implications for understanding the chaperonin mechanism and cellular stress response. Our findings necessitate a revision of the GroEL/ES mechanism.     

Expansion and compression of a protein folding intermediate by GroEL

The GroEL-GroES chaperonin system is required for the assisted folding of many essential proteins. The precise nature of this assistance remains unclear, however. Here we show that denatured RuBisCO from Rhodospirillum rubrum populates a stable, nonaggregating, and kinetically trapped monomeric state at low temperature. Productive folding of this nonnative intermediate is fully dependent on GroEL, GroES, and ATP. Reactivation of the trapped RuBisCO monomer proceeds through a series of GroEL-induced structural rearrangements, as judged by resonance energy transfer measurements between the amino- and carboxy-terminal domains of RuBisCO. A general mechanism used by GroEL to push large, recalcitrant proteins like RuBisCO toward their native states thus appears to involve two steps: partial unfolding or rearrangement of a nonnative protein upon capture by a GroEL ring, followed by spatial constriction within the GroEL-GroES cavity that favors or enforces compact, folding-competent intermediate states.     

Asp-52 in Combination with Asp-398 Plays a Critical Role in ATP Hydrolysis of Chaperonin GroEL

The Escherichia coli chaperonin GroEL is a double-ring chaperone that assists protein folding with the aid of GroES and ATP. Asp-398 in GroEL is known as one of the critical residues on ATP hydrolysis because GroEL(D398A) mutant is deficient in ATP hydrolysis (<2% of the wild type) but not in ATP binding. In the archaeal Group II chaperonin, another aspartate residue, Asp-52 in the corresponding E. coli GroEL, in addition to Asp-398 is also important for ATP hydrolysis. We investigated the role of Asp-52 in GroEL and found that ATPase activity of GroEL(D52A) and GroEL(D52A/D398A) mutants was ∼20% and <0.01% of wild-type GroEL, respectively, indicating that Asp-52 in E. coli GroEL is also involved in the ATP hydrolysis. GroEL(D52A/D398A) formed a symmetric football-shaped GroEL-GroES complex in the presence of ATP, again confirming the importance of the symmetric complex during the GroEL ATPase cycle. Notably, the symmetric complex of GroEL(D52A/D398A) was extremely stable, with a half-time of ∼150 h (∼6 days), providing a good model to characterize the football-shaped complex.     

Chaperones GroEL/GroES Accelerate the Refolding of a Multidomain Protein through Modulating On-pathway Intermediates

Despite a vast amount information on the interplay of GroEL, GroES, and ATP in chaperone-assisted folding, the molecular details on the conformational dynamics of folding polypeptide during its GroEL/GroES-assisted folding cycle is quite limited. Practically no such studies have been reported to date on large proteins, which often have difficulty folding in vitro. The effect of the GroEL/GroES chaperonin system on the folding pathway of an 82-kDa slow folding protein, malate synthase G (MSG), was investigated. GroEL bound to the burst phase intermediate of MSG and accelerated the slowest kinetic phase associated with the formation of native topology in the spontaneous folding pathway. GroEL slowly induced conformational changes on the bound burst phase intermediate, which was then transformed into a more folding-compatible form. Subsequent addition of ATP or GroES/ATP to the GroEL-MSG complex led to the formation of the native state via a compact intermediate with the rate several times faster than that of spontaneous refolding. The presence of GroES doubled the ATP-dependent reactivation rate of bound MSG by preventing multiple cycles of its GroEL binding and release. Because GroES bound to the trans side of GroEL-MSG complex, it may be anticipated that confinement of the substrate underneath the co-chaperone is not required for accelerating the rate in the assisted folding pathway. The potential role of GroEL/GroES in assisted folding is most likely to modulate the conformation of MSG intermediates that can fold faster and thereby eliminate the possibility of partial aggregation caused by the slow folding intermediates during its spontaneous refolding pathway.     

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.     

Triggering protein folding within the GroEL-GroES complex

The folding of many proteins depends on the assistance of chaperonins like GroEL and GroES and involves the enclosure of substrate proteins inside an internal cavity that is formed when GroES binds to GroEL in the presence of ATP. Precisely how assembly of the GroEL-GroES complex leads to substrate protein encapsulation and folding remains poorly understood. Here we use a chemically modified mutant of GroEL (EL43Py) to uncouple substrate protein encapsulation from release and folding. Although EL43Py correctly initiates a substrate protein encapsulation reaction, this mutant stalls in an intermediate allosteric state of the GroEL ring, which is essential for both GroES binding and the forced unfolding of the substrate protein. This intermediate conformation of the GroEL ring possesses simultaneously high affinity for both GroES and non-native substrate protein, thus preventing escape of the substrate protein while GroES binding and substrate protein compaction takes place. Strikingly, assembly of the folding-active GroEL-GroES complex appears to involve a strategic delay in ATP hydrolysis that is coupled to disassembly of the old, ADP-bound GroEL-GroES complex on the opposite ring.     

Chaperonin-mediated protein folding: fate of substrate polypeptide

Chaperonins are megadalton ring assemblies that mediate essential ATP-dependent assistance of protein folding to the native state in a variety of cellular compartments, including the mitochondrial matrix, the eukaryotic cytosol, and the bacterial cytoplasm. Structural studies of the bacterial chaperonin, GroEL, both alone and in complex with its co-chaperonin, GroES, have resolved the states of chaperonin that bind and fold non-native polypeptides. Functional studies have resolved the action of ATP binding and hydrolysis in driving the GroEL-GroES machine through its folding-active and binding-active states, respectively. Yet the exact fate of substrate polypeptide during these steps is only poorly understood. For example, while binding involves multivalent interactions between hydrophobic side-chains facing the central cavity of GroEL and exposed hydrophobic surfaces of the non-native protein, the structure of any polypeptide substrate while bound to GroEL remains unknown. It is also unclear whether binding to an open GroEL ring is accompanied by structural changes in the non-native substrate, in particular whether there is an unfolding action. As a polypeptide-bound ring becomes associated with GroES, do the large rigid-body movements of the GroEL apical domains serve as another source of a potential unfolding action? Regarding the encapsulated folding-active state, how does the central cavity itself influence the folding trajectory of a substrate? Finally, how do GroEL and GroES serve, as recently recognized, to assist the folding of substrates too large to be encapsulated inside the machine? Here, such questions are addressed with the findings available to date, and means of further resolving the states of chaperonin-associated polypeptide are discussed.     

Structure of GroEL in Complex with an Early Folding Intermediate of Alanine Glyoxylate Aminotransferase

Primary hyperoxaluria type 1 is a rare autosomal recessive disease caused by mutations in the alanine glyoxylate aminotransferase gene (AGXT). We have previously shown that P11L and I340M polymorphisms together with I244T mutation (AGXT-LTM) represent a conformational disease that could be amenable to pharmacological intervention. Thus, the study of the folding mechanism of AGXT is crucial to understand the molecular basis of the disease. Here, we provide biochemical and structural data showing that AGXT-LTM is able to form non-native folding intermediates. The three-dimensional structure of a complex between the bacterial chaperonin GroEL and a folding intermediate of AGXT-LTM mutant has been solved by cryoelectron microscopy. The electron density map shows the protein substrate in a non-native extended conformation that crosses the GroEL central cavity. Addition of ATP to the complex induces conformational changes on the chaperonin and the internalization of the protein substrate into the folding cavity. The structure provides a three-dimensional picture of an in vivo early ATP-dependent step of the folding reaction cycle of the chaperonin and supports a GroEL functional model in which the chaperonin promotes folding of the AGXT-LTM mutant protein through forced unfolding mechanism.     

GroEL-mediated protein folding.

I. Architecture of GroEL and GroES and the reaction pathway A. Architecture of the chaperonins B. Reaction pathway of GroEL-GroES-mediated folding II. Polypeptide binding A. A parallel network of chaperones binding polypeptides in vivo B. Polypeptide binding in vitro 1. Role of hydrophobicity in recognition 2. Homologous proteins with differing recognition-differences in primary structure versus effects on folding pathway 3. Conformations recognized by GroEL a. Refolding studies b. Binding of metastable intermediates c. Conformations while stably bound at GroEL 4. Binding constants and rates of association 5. Conformational changes in the substrate protein associated with binding by GroEL a. Observations b. Kinetic versus thermodynamic action of GroEL in mediating unfolding c. Crossing the energy landscape in the presence of GroEL III. ATP binding and hydrolysis-driving the reaction cycle IV. GroEL-GroES-polypeptide ternary complexes-the folding-active cis complex A. Cis and trans ternary complexes B. Symmetric complexes C. The folding-active intermediate of a chaperonin reaction-cis ternary complex D. The role of the cis space in the folding reaction E. Folding governed by a "timer" mechanism F. Release of nonnative polypeptides during the GroEL-GroES reaction G. Release of both native and nonnative forms under physiologic conditions H. A role for ATP binding, as well as hydrolysis, in the folding cycle V. Concluding remarks.     

A thermodynamic coupling mechanism can explain the GroEL-mediated acceleration of the folding of barstar

Despite extensive structural and kinetic studies, the mechanism by which the Escherichia coli chaperonin GroEL assists protein folding has remained somewhat elusive. It appears that GroEL might play an active role in facilitating folding, in addition to its role in restricting protein aggregation by secluding folding intermediates. We have investigated the kinetic mechanism of GroEL-mediated refolding of the small protein barstar. GroEL accelerates the observed fast (millisecond) refolding rate, but it does not affect the slow refolding kinetics. A thermodynamic coupling mechanism, in which the concentration of exchange-competent states is increased by the law of mass action, can explain the enhancement of the fast refolding rates. It is not necessary to invoke a catalytic role for GroEL, whereby either the intrinsic refolding rate of a productive folding transition or the unfolding rate of a kinetically trapped off-pathway intermediate is increased by the chaperonin.     

Single-molecule Study on the Decay Process of the Football-shaped GroEL-GroES Complex Using Zero-mode Waveguides

It has been widely believed that an asymmetric GroEL-GroES complex (termed the bullet-shaped complex) is formed solely throughout the chaperonin reaction cycle, whereas we have recently revealed that a symmetric GroEL-(GroES)₂ complex (the football-shaped complex) can form in the presence of denatured proteins. However, the dynamics of the GroEL-GroES interaction, including the football-shaped complex, is unclear. We investigated the decay process of the football-shaped complex at a single-molecule level. Because submicromolar concentrations of fluorescent GroES are required in solution to form saturated amounts of the football-shaped complex, single-molecule fluorescence imaging was carried out using zero-mode waveguides. The single-molecule study revealed two insights into the GroEL-GroES reaction. First, the first GroES to interact with GroEL does not always dissociate from the football-shaped complex prior to the dissociation of a second GroES. Second, there are two cycles, the “football cycle ” and the “bullet cycle,” in the chaperonin reaction, and the lifetimes of the football-shaped and the bullet-shaped complexes were determined to be 3–5 s and about 6 s, respectively. These findings shed new light on the molecular mechanism of protein folding mediated by the GroEL-GroES chaperonin system.     

Folding of malate dehydrogenase inside the GroEL-GroES cavity

The chaperonin GroEL binds nonnative substrate protein in the hydrophobic central cavity of an open ring. ATP and GroES binding to the same ring converts this cavity into an encapsulated, hydrophilic chamber that mediates productive folding. A 'rack' mechanism of initial protein unfolding proposes that, upon GroES and ATP binding, the polypeptide is stretched between the binding sites on the twisting apical domains of GroEL before complete release into the chamber. Here, the structure of malate dehydrogenase (MDH) subunit during folding is monitored by deuterium exchange, peptic fragment production and mass spectrometry. When bound to GroEL, MDH exhibits a core of partially protected secondary structure that is only modestly deprotected upon ATP and GroES binding. Moreover, deprotection is broadly distributed throughout MDH, suggesting that it results from breaking hydrogen bonds between MDH and the cavity wall or global destabilization, as opposed to forced mechanical unfolding.     

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.     

GroEL Can Unfold Late Intermediates Populated on the Folding Pathways of Monellin

The modulation of the folding mechanism of the small protein single-chain monellin (MNEI) by the Escherichia coli chaperone GroEL has been studied. In the absence of the chaperone, the folding of monellin occurs via three parallel routes. When folding is initiated in the presence of a saturating concentration of GroEL, only 50-60% of monellin molecules fold completely. The remaining 40-50% of the monellin molecules remain bound to the GroEL and are released only upon addition of ATP. It is shown that the basic folding mechanism of monellin is not altered by the presence of GroEL, but that it occurs via only one of the three available routes when folding is initiated in the presence of saturating concentrations of GroEL. Two pathways become nonoperational because GroEL binds very tightly to early intermediates that populate these pathways in a manner that makes the GroEL-bound intermediates incompetent to fold. This accounts for the monellin molecules that remain GroEL-bound at the end of the folding reaction. The third pathway remains operational because the GroEL-bound early intermediate on this pathway is folding-competent, suggesting that this early intermediate binds to GroEL in a manner that is different from that of the binding of the early intermediates on the other two pathways. It appears, therefore, that the same protein can bind GroEL in more than one way. The modulation of the folding energy landscape of monellin by GroEL occurs because GroEL binds folding intermediates on parallel folding pathways, in different ways, and with different affinities. Moreover, when GroEL is added to refolding monellin at different times after commencement of refolding, the unfolding of two late kinetic intermediates on two of the three folding pathways can be observed. It appears that the unfolding of late folding intermediates is enabled by a thermodynamic coupling mechanism, wherein GroEL binds more tightly to an early intermediate than to a late intermediate on a folding pathway, with preferential binding energy being larger than the stability of the late intermediate. Hence, it is shown that GroEL can inadvertently and passively cause, through its ability to bind different folding intermediates differentially, the unfolding of late productive intermediates on folding pathways, and that its unfolding action is not restricted solely to misfolded or kinetically trapped intermediates.     

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.     

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.     

GroEL/GroES-mediated folding of a protein too large to be encapsulated.

The chaperonin GroEL binds nonnative proteins too large to fit inside the productive GroEL-GroES cis cavity, but whether and how it assists their folding has remained unanswered. We have examined yeast mitochondrial aconitase, an 82 kDa monomeric Fe(4)S(4) cluster-containing enzyme, observed to aggregate in chaperonin-deficient mitochondria. We observed that aconitase folding both in vivo and in vitro requires both GroEL and GroES, and proceeds via multiple rounds of binding and release. Unlike the folding of smaller substrates, however, this mechanism does not involve cis encapsulation but, rather, requires GroES binding to the trans ring to release nonnative substrate, which likely folds in solution. Following the phase of ATP/GroES-dependent refolding, GroEL stably bound apoaconitase, releasing active holoenzyme upon Fe(4)S(4) cofactor formation, independent of ATP and GroES.