Structural and functional conservation of Mycobacterium tuberculosis GroEL paralogs suggests that GroEL1 Is a chaperonin

GroEL is a group I chaperonin that facilitates protein folding and prevents protein aggregation in the bacterial cytosol. Mycobacteria are unusual in encoding two or more copies of GroEL in their genome. While GroEL2 is essential for viability and likely functions as the general housekeeping chaperonin, GroEL1 is dispensable, but its structure and function remain unclear.Here, we present the 2.2-Å resolution crystal structure of a 23-kDa fragment of Mycobacterium tuberculosis GroEL1 consisting of an extended apical domain. Our X-ray structure of the GroEL1 apical domain closely resembles those of Escherichia coli GroEL and M. tuberculosis GroEL2, thus highlighting the remarkable structural conservation of bacterial chaperonins. Notably, in our structure, the proposed substrate-binding site of GroEL1 interacts with the N-terminal region of a symmetry-related neighboring GroEL1 molecule. The latter is consistent with the known GroEL apical domain function in substrate binding and is supported by results obtained from using peptide array technology. Taken together, these data show that the apical domains of M. tuberculosis GroEL paralogs are conserved in three-dimensional structure, suggesting that GroEL1, like GroEL2, is a chaperonin.     

The 13 angstroms structure of a chaperonin GroEL-protein substrate complex by cryo-electron microscopy

The 13 angstroms resolution structures of GroEL bound to a single monomer of the protein substrate glutamine synthetase (GS(m)), as well as that of unliganded GroEL have been determined from a heterogeneous image population using cryo-electron microscopy (cryo-EM) coupled with single-particle image classification and reconstruction techniques. We combined structural data from cryo-EM maps and dynamic modeling, taking advantage of the known X-ray crystallographic structure and normal mode flexible fitting (NMFF) analysis, to describe the changes that occur in GroEL structure induced by GS(m) binding. The NMFF analysis reveals that the molecular movements induced by GS(m) binding propagate throughout the GroEL structure. The modeled molecular motions show that some domains undergo en bloc movements, while others show more complex independent internal movements. Interestingly, the substrate-bound apical domains of both the cis (GS(m)-bound ring) and trans (the opposite substrate-free ring) show counterclockwise rotations, in the same direction (though not as dramatic) as those documented for the ATP-GroEL-induced structure changes. The structural changes from the allosteric substrate protein-induced negative cooperativity between the GroEL rings involves upward concerted movements of both cis and trans equatorial domains toward the GS(m)-bound ring, while the inter-ring distances between the heptamer contact residues are maintained. Furthermore, the NMFF analysis identifies the secondary structural elements that are involved in the observed approximately 5 angstroms reduction in the diameter of the cavity opening in the unbound trans ring. Understanding the molecular basis of these substrate protein-induced structural changes across the heptamer rings provides insight into the origins of the allosteric negative cooperative effects that are transmitted over long distances (approximately 140 angstroms).     

A thermophilic mini-chaperonin contains a conserved polypeptide-binding surface: combined crystallographic and NMR studies of the GroEL apical domain with implications for substrate interactions

A homologue of the Escherichia coli GroEL apical domain was obtained from thermophilic eubacterium Thermus thermophilus. The domains share 70 % sequence identity (101 out of 145 residues). The thermal stability of the T. thermophilus apical domain (Tm>100 degrees C as evaluated by circular dichroism) is at least 35 degrees C greater than that of the E. coli apical domain (Tm=65 degrees C). The crystal structure of a selenomethione-substituted apical domain from T. thermophilus was determined to a resolution of 1.78 A using multiwavelength-anomalous-diffraction phasing. The structure is similar to that of the E. coli apical domain (root-mean-square deviation 0.45 A based on main-chain atoms). The thermophilic structure contains seven additional salt bridges of which four contain charge-stabilized hydrogen bonds. Only one of the additional salt bridges would face the "Anfinsen cage" in GroEL. High temperatures were exploited to map sites of interactions between the apical domain and molten globules. NMR footprints of apical domain-protein complexes were obtained at elevated temperature using 15N-1H correlation spectra of 15N-labeled apical domain. Footprints employing two polypeptides unrelated in sequence or structure (an insulin monomer and the SRY high-mobility-group box, each partially unfolded at 50 degrees C) are essentially the same and consistent with the peptide-binding surface previously defined in E. coli GroEL and its apical domain-peptide complexes. An additional part of this surface comprising a short N-terminal alpha-helix is observed. The extended footprint rationalizes mutagenesis studies of intact GroEL in which point mutations affecting substrate binding were found outside the "classical" peptide-binding site. Our results demonstrate structural conservation of the apical domain among GroEL homologues and conservation of an extended non-polar surface recognizing diverse polypeptides.     

Domain motions in GroEL upon binding of an oligopeptide

GroEL assists protein folding by preventing the interaction of partially folded molecules with other non-native proteins. It binds them, sequesters them, and then releases them so that they can fold in an ATP-driven cycle. Previous studies have also shown that protein substrates, GroES, and oligopeptides bind to partially overlapped sites on the apical domain surfaces of GroEL. In this study, we have determined the crystal structure at 3.0A resolution of a symmetric (GroEL-peptide)(14) complex. The binding of each of these small 12 amino acid residue peptides to GroEL involves interactions between three adjacent apical domains of GroEL. Each peptide interacts primarily with a single GroEL subunit. Residues R231 and R268 from adjacent subunits isolate each substrate-binding pocket, and prevent bound substrates from sliding into adjacent binding pockets. As a consequence of peptide binding, domains rotate and inter-domain interactions are greatly enhanced. The direction of rotation of the apical domain of each GroEL subunit is opposite to that of its intermediate domain. Viewed from outside, the apical domains rotate clockwise within one GroEL ring, while the ATP-induced apical domain rotation is counter-clockwise.     

Probing the functional mechanism of Escherichia coli GroEL using circular permutation

Background

The Escherichia coli chaperonin GroEL subunit consists of three domains linked via two hinge regions, and each domain is responsible for a specific role in the functional mechanism. Here, we have used circular permutation to study the structural and functional characteristics of the GroEL subunit.

Methodology/Principal Findings

Three soluble, partially active mutants with polypeptide ends relocated into various positions of the apical domain of GroEL were isolated and studied. The basic functional hallmarks of GroEL (ATPase and chaperoning activities) were retained in all three mutants. Certain functional characteristics, such as basal ATPase activity and ATPase inhibition by the cochaperonin GroES, differed in the mutants while at the same time, the ability to facilitate the refolding of rhodanese was roughly equal. Stopped-flow fluorescence experiments using a fluorescent variant of the circularly permuted GroEL CP376 revealed that a specific kinetic transition that reflects movements of the apical domain was missing in this mutant. This mutant also displayed several characteristics that suggested that the apical domains were behaving in an uncoordinated fashion.

Conclusions/Significance

The loss of apical domain coordination and a concomitant decrease in functional ability highlights the importance of certain conformational signals that are relayed through domain interlinks in GroEL. We propose that circular permutation is a very versatile tool to probe chaperonin structure and function.     

The crystal structure of a GroEL/peptide complex: plasticity as a basis for substrate diversity

The chaperonin GroEL is a double toriodal assembly that with its cochaperonin GroES facilitates protein folding with an ATP-dependent mechanism. Nonnative conformations of diverse protein substrates bind to the apical domains surrounding the opening of the double toroid's central cavity. Using phage display, we have selected peptides with high affinity for the isolated apical domain. We have determined the crystal structures of the complexes formed by the most strongly bound peptide with the isolated apical domain, and with GroEL. The peptide interacts with the groove between paired alpha helices in a manner similar to that of the GroES mobile loop. Our structural analysis, combined with other results, suggests that various modes of molecular plasticity are responsible for tight promiscuous binding of nonnative substrates and their release into the shielded cis assembly.     

Toward a detailed description of the pathways of allosteric communication in the GroEL chaperonin through atomistic simulation

GroEL, along with its coprotein GroES, is essential for ensuring the correct folding of unfolded or newly synthesized proteins in bacteria. GroEL is a complex, allosteric molecule, composed of two heptameric rings stacked back to back, that undergoes large structural changes during its reaction cycle. These structural changes are driven by the cooperative binding and subsequent hydrolysis of ATP, by GroEL. Despite numerous previous studies, the precise mechanisms of allosteric communication and the associated structural changes remain elusive. In this paper, we describe a series of all-atom, unbiased, molecular dynamics simulations over relatively long (50-100 ns) time scales of a single, isolated GroEL subunit and also a heptameric GroEL ring, in the presence and absence of ATP. Combined with results from a distance restraint-biased simulation of the single ring, the atomistic details of the earliest stages of ATP-driven structural changes within this complex molecule are illuminated. Our results are in broad agreement with previous modeling studies of isolated subunits and with a coarse-grained, forcing simulation of the single ring. These are the first reported all-atom simulations of the GroEL single-ring complex and provide a unique insight into the role of charged residues K80, K277, R284, R285, and E388 at the subunit interface in transmission of the allosteric signal. These simulations also demonstrate the feasibility of performing all-atom simulations of very large systems on sufficiently long time scales on typical high performance computing facilities to show the origins of the earliest events in biologically relevant processes.     

Crystal structure of the native chaperonin complex from Thermus thermophilus revealed unexpected asymmetry at the cis-cavity

The chaperonins GroEL and GroES are essential mediators of protein folding. GroEL binds nonnative protein, ATP, and GroES, generating a ternary complex in which protein folding occurs within the cavity capped by GroES (cis-cavity). We determined the crystal structure of the native GroEL-GroES-ADP homolog from Thermus thermophilus, with substrate proteins in the cis-cavity, at 2.8 A resolution. Twenty-four in vivo substrate proteins within the cis-cavity were identified from the crystals. The structure around the cis-cavity, which encapsulates substrate proteins, shows significant differences from that observed for the substrate-free Escherichia coli GroEL-GroES complex. The apical domain around the cis-cavity of the Thermus GroEL-GroES complex exhibits a large deviation from the 7-fold symmetry. As a result, the GroEL-GroES interface differs considerably from the previously reported E. coli GroEL-GroES complex, including a previously unknown contact between GroEL and GroES.     

Crystal structure of the temperature-sensitive and allosteric-defective chaperonin GroELE461K

The chaperonin GroEL adopts a double-ring structure with various modes of allosteric communication. The simultaneous positive intra-ring and negative inter-ring co-operativities alternate the functionality of the folding cavities in both protein rings. Negative inter-ring co-operativity is maintained through different inter-ring interactions, including a salt bridge involving Glu 461. Replacement of this residue by Lys modifies the temperature sensitivity of the substrate-folding activity of this protein, most likely as a result of the loss of inter-ring co-operativity. The crystal structure of the mutant chaperonin GroELE461K has been determined at 3.3A and compared with other structures: the wild-type GroEL, an allosteric defective GroEL double mutant and the GroEL-GroES-(ADP)7 complex. The inter-ring region of the mutant exhibits the following characteristics: (i) no salt-bridge stabilizes the inter-ring interface; (ii) the mutated residue plays a central role in defining the relative ring rotation (of about 22 degrees) around the 7-fold axis; (iii) an increase in the inter-ring distance and solvent accessibility of the inter-ring interface; and (iv) a 2-fold reduction in the stabilization energy of the inter-ring interface, due to the modification of inter-ring interactions. These characteristics explain how the thermal sensitivity of the protein's fundamental properties permits GroEL to distinguish physiological (37 degrees C) from stress (42 degrees C) temperatures.     

Allostery Wiring Diagrams in the Transitions that Drive the GroEL Reaction Cycle

Determining the network of residues that transmit allosteric signals is crucial to understanding the function of biological nanomachines. During the course of a reaction cycle, biological machines in general, and Escherichia coli chaperonin GroEL in particular, undergo large-scale conformational changes in response to ligand binding. Normal mode analyses, based on structure-based coarse-grained models where each residue is represented by an α carbon atom, have been widely used to describe the motions encoded in the structures of proteins. Here, we propose a new C^α-side chain elastic network model of proteins that includes information about the physical identity of each residue and accurately accounts for the side-chain topology and packing within the structure. Using the C^α-side chain elastic network model and the structural perturbation method, which probes the response of a local perturbation at a given site at all other sites in the structure, we determine the network of key residues (allostery wiring diagram) responsible for the T → R and R″ → T transitions in GroEL. A number of residues, both within a subunit and at the interface of two adjacent subunits, are found to be at the origin of the positive cooperativity in the ATP-driven T → R transition. Of particular note are residues G244, R58, D83, E209, and K327. Of these, R38, D83, and K327 are highly conserved. G244 is located in the apical domain at the interface between two subunits; E209 and K327 are located in the apical domain, toward the center of a subunit; R58 and D83 are equatorial domain residues. The allostery wiring diagram shows that the network of residues are interspersed throughout the structure. Residues D83, V174, E191, and D359 play a critical role in the R″ → T transition, which implies that mutations of these residues would compromise the ATPase activity. D83 and E191 are also highly conserved; D359 is moderately conserved. The negative cooperativity between the rings in the R″ → T transition is orchestrated through several interface residues within a single ring, including N10, E434, D435, and E451. Signal from the trans ring that is transmitted across the interface between the equatorial domains is responsible for the R″ → T transition. The cochaperonin GroES plays a passive role in the R″ → T transition. Remarkably, the binding affinity of GroES for GroEL is allosterically linked to GroEL residues 350-365 that span helices K and L. The movements of helices K and L alter the polarity of the cavity throughout the GroEL functional cycle and undergo large-scale motions that are anticorrelated with the other apical domain residues. The allostery wiring diagrams for the T → R and R″ → T transitions of GroEL provide a microscopic foundation for the cooperativity (anticooperativity) within (between) the ring (rings). Using statistical coupling analysis, we extract evolutionarily linked clusters of residues in GroEL and GroES. We find that several substrate protein binding residues as well as sites related to ATPase activity belong to a single functional network in GroEL. For GroES, the mobile loop residues and GroES/GroES interface residues are linked.     

Structural basis for GroEL-assisted protein folding from the crystal structure of (GroEL-KMgATP)14 at 2.0A resolution

Nucleotide regulates the affinity of the bacterial chaperonin GroEL for protein substrates. GroEL binds protein substrates with high affinity in the absence of ATP and with low affinity in its presence. We report the crystal structure of (GroEL-KMgATP)(14) refined to 2.0 A resolution in which the ATP triphosphate moiety is directly coordinated by both K(+) and Mg(2+). Upon the binding of KMgATP, we observe previously unnoticed domain rotations and a 102 degrees rotation of the apical domain surface helix I. Two major consequences are a large lateral displacement of, and a dramatic reduction of hydrophobicity in, the apical domain surface. These results provide a basis for the nucleotide-dependent regulation of protein substrate binding and suggest a mechanism for GroEL-assisted protein folding by forced unfolding.     

Phi value analysis of an allosteric transition of GroEL based on a single-pathway model

There are currently two contradictory models for the kinetics of the ATP-induced GroEL allosteric transition occurring around 20 microM ATP. One model, proposed by Horovitz et al. demonstrates the existence of two parallel pathways for the allosteric transition and an abrupt ATP-dependent switch from one pathway to the other. The other model, which was proposed by the present authors, shows no need to assume the parallel pathways, and a combination of the transition-state theory and the Monod-Wyman-Changeux model of allostery can explain the kinetics as well as the equilibrium of the transition. The discrepancy appears to be due to whether we regard the transition as reversible or irreversible. Thus, here we have investigated the reversibility of the allosteric transition between 0 microM and 70 microM ATP by the use of a stopped-flow double-jump technique, which has allowed us to monitor the kinetics of the reverse reaction from the relaxed state at a high ATP concentration to the tense state at a low ATP concentration. The tryptophan fluorescence of a tryptophan-inserted variant of GroEL was used to follow the kinetics. As a result, the allosteric transition was shown to be a reversible process, supporting the validity of our model. We also show that the structural environment around the ATP-binding site of GroEL in the transition state is very similar to that in the relaxed state (Phi=0.9) by using a Phi value analysis in the kinetic Monod-Wyman-Changeux model, which is analogous to the mutational Phi value analysis in protein folding.     

Structural plasticity and noncovalent substrate binding in the GroEL apical domain. A study using electrospay ionization mass spectrometry and fluorescence binding studies

Advances in understanding how GroEL binds to non-native proteins are reported. Conformational flexibility in the GroEL apical domain, which could account for the variety of substrates that GroEL binds, is illustrated by comparison of several independent crystallographic structures of apical domain constructs that show conformational plasticity in helices H and I. Additionally, ESI-MS indicates that apical domain constructs have co-populated conformations at neutral pH. To assess the ability of different apical domain conformers to bind co-chaperone and substrate, model peptides corresponding to the mobile loop of GroES and to helix D from rhodanese were studied. Analysis of apical domain-peptide complexes by ESI-MS indicates that only the folded or partially folded apical domain conformations form complexes that survive gas phase conditions. Fluorescence binding studies show that the apical domain can fully bind both peptides independently. No competition for binding was observed, suggesting the peptides have distinct apical domain-binding sites. Blocking the GroES-apical domain-binding site in GroEL rendered the chaperonin inactive in binding GroES and in assisting the folding of denatured rhodanese, but still capable of binding non-native proteins, supporting the conclusion that GroES and substrate proteins have, at least partially, distinct binding sites even in the intact GroEL tetradecamer.     

Stopped-flow fluorescence analysis of the conformational changes in the GroEL apical domain: relationships between movements in the apical domain and the quaternary structure of GroEL

GroEL undergoes numerous conformational alterations in the course of facilitating the folding of various proteins, and the specific movements of the GroEL apical domain are of particular importance in the molecular mechanism. In order to monitor in detail the numerous movements of the GroEL apical domain, we have constructed a mutant chaperonin (GroEL R231W) with wild type-like function and a fluorescent probe introduced into the apical domain. By monitoring the tryptophan fluorescence changes of GroEL R231W upon ATP addition in the presence and absence of the co-chaperonin GroES, we detected a total of four distinct kinetic phases that corresponded to conformational changes of the apical domain and GroES binding. By introducing this mutation into a single ring variant of GroEL (GroEL SR-1), we determined the extent of inter-ring cooperation that was involved in apical domain movements. Surprisingly, we found that the apical domain movements of GroEL were affected only slightly by the change in quaternary structure. Our experiments provide a number of novel insights regarding the dynamic movements of this protein.     

Crystal Structure of the Extracellular Domain of a Bacterial Ligand-Gated Ion Channel

The crystal structure of the extracellular domain (ECD) of the pentameric ligand-gated ion-channel from Gloeobacter violaceus (GLIC) was solved at neutral pH at 2.3 Å resolution in two crystal forms, showing a surprising hexameric quaternary structure with a 6-fold axis replacing the expected 5-fold axis. While each subunit retains the usual β-sandwich immunoglobulin-like fold, small deviations from the whole GLIC structure indicate zones of differential flexibility. The changes in interface between two adjacent subunits in the pentamer and the hexamer can be described in a downward translation by one inter-strand distance and a global rotation of the second subunit, using the first one for superposition. While global characteristics of the interface, such as the buried accessible surface area, do not change very much, most of the atom-atom interactions are rearranged. It thus appears that the transmembrane domain is necessary for the proper oligomeric assembly of GLIC and that there is an intrinsic plasticity or polymorphism in possible subunit-subunit interfaces at the ECD level, the latter behaving as a monomer in solution. Possible functional implications of these novel structural data are discussed in the context of the allosteric transition of this family of proteins. In addition, we propose a novel way to quantify elastic energy stored in the interface between subunits, which indicates a tenser interface for the open form than for the closed form (rest state). The hexameric or pentameric forms of the ECD have a similar negative curvature in their subunit-subunit interface, while acetylcholine binding proteins have a smaller and positive curvature that increases from the apo to the holo form.     

GroEL recognises sequential and non-sequential linear structural motifs compatible with extended beta-strands and alpha-helices.

The chaperonin GroEL binds a variety of polypeptides that share no obvious sequence similarity. The precise structural, chemical and dynamic features that are recognised remain largely unknown. Structural models of the complex between GroEL and its co-chaperonin GroES, and of the isolated apical domain of GroEL (minichaperone; residues 191-376) with a 17 residue N-terminal tag show that a linear sequential sequence (extended beta-strand) can be bound. We have analysed characteristics of the motifs that bind to GroEL by using affinity panning of immobilised GroEL minichaperones for a library of bacteriophages that display the fungal cellulose-binding domain of the enzyme cellobiohydrolase I. This protein has seven non-sequential residues in its binding site that form a linear binding motif with similar dimensions and characteristics to the peptide tag that was bound to the minichaperone GroEL(191-376). The seven residues thus form a constrained scaffold. We find that GroEL does bind suitable mutants of these seven residues. The side-chains recognised do not have to be totally hydrophobic, but polar and positively charged chains can be accommodated. Further, the spatial distribution of the side-chains is also compatible with those in an alpha-helix. This implies that GroEL can bind a wide range of structures, from extended beta-strands and alpha-helices to folded states, with exposed side-chains. The binding site can accommodate substrates of approximately 18 residues when in a helical or seven when in an extended conformation. The data support two activities of GroEL: the ability to act as a temporary parking spot for sticky intermediates by binding many motifs; and an unfolding activity of GroEL by binding an extended sequential conformation of the substrate.     

Unfolding and disassembly of the chaperonin GroEL occurs via a tetradecameric intermediate with a folded equatorial domain

The chaperonin GroEL is a homotetradecamer in which the subunits (M(r) 57 000) are joined through noncovalent forces. This study reports on the unfolding and disassembly of GroEL in guanidine hydrochloride and urea. Kinetic and equilibrium measurements were made using amide hydrogen exchange/mass spectrometry, light scattering, and size-exclusion chromatography. Hydrogen exchange in GroEL destabilized in 1.8 M GdHCl (the unfolding midpoint is 1.2 M GdHCl) shows that the apical and intermediate domains unfold 3.1 times faster than the equatorial domain. Light scattering measurements made under the same conditions show that disassembly of the native GroEL tetradecamer occurs at the same rate as unfolding of the equatorial domain. This study of the kinetics of GroEL unfolding and disassembly demonstrates the existence of an intermediate that was identified as a tetradecamer with the apical and intermediate domains unfolded. Although this intermediate was easily detected in dynamic unfolding measurements, its population in equilibrium measurements at the midpoint for GroEL unfolding was too small to be detected. This study of GroEL unfolding and disassembly points to features that may be important in the folding and assembly of the GroEL macroassembly.     

Functional communications between the apical and equatorial domains of GroEL through the intermediate domain

The Escherichia coli GroEL subunit consists of three domains with distinct functional roles. To understand the role of each of the three domains, the effects of mutating a single residue in each domain (Y203C at the apical, T89W at the equatorial, and C138W at the intermediate domain) were studied in detail, using three different enzymes (enolase, lactate dehydrogenase, and rhodanese) as refolding substrates. By analyzing the effects of each mutation, a transfer of signals was detected between the apical domain and the equatorial domain. A signal initiated by the equatorial domain triggers the release of polypeptide from the apical domain. This trigger was independent of nucleotide hydrolysis, as demonstrated using an ATPase-deficient mutant, and, also, the conditions for successful release of polypeptide could be modified by a mutation in the apical domain, suggesting that the polypeptide release mechanism of GroEL is governed by chaperonin-target affinities. Interestingly, a reciprocal signal from the apical domain was suggested to occur, which triggered nucleotide hydrolysis in the equatorial domain. This signal was disrupted by a mutation in the intermediate domain to create a novel ternary complex in which GroES and refolding protein are simultaneously bound in a stable ternary complex devoid of ATPase activity. These results point to a multitude of signals which govern the overall chaperonin mechanism.     

Nucleotide-induced transition of GroEL from the high-affinity to the low-affinity state for a target protein: effects of ATP and ADP on the GroEL-affected refolding of alpha-lactalbumin

We studied the refolding kinetics of alpha-lactalbumin in the presence of wild-type GroEL and its ATPase-deficient mutant D398A at various concentrations of nucleotides (ATP and ADP). We evaluated the apparent binding constant between GroEL and the alpha-lactalbumin refolding intermediate quantitatively by numerical simulation analysis of the alpha-lactalbumin refolding curves in the presence and absence of GroEL. The binding constant showed a co-operative decrease with an increase in ATP concentration, whereas the binding constant decreased in a non-co-operative manner with respect to ADP concentration. For the D398A mutant, the ATP-induced decrease in affinity occurred much faster than the steady-state ATP hydrolysis by this mutant, suggesting that ATP binding to GroEL rather than ATP hydrolysis, was responsible for the co-operative decrease in the affinity for the target protein. We thus analyzed the nucleotide-concentration dependence of affinity of GroEL for the target protein using an allosteric Monod-Wyman-Changeux model in which GroEL underwent an ATP-induced co-operative conformational transition between the high-affinity and low-affinity states of the target protein. The transition midpoint of the ATP-induced transition of GroEL has been found to be around 30 microM, in good agreement with the midpoint evaluated in other structural studies of GroEL. The results show that the observed difference between ATP and ADP-induced transitions of GroEL are brought about by a small difference in an allosteric parameter (the ratio of the nucleotide affinities of GroEL in the high-affinity and the low-affinity states), i.e. 4.1 for ATP and 2.6 for ADP.