The GroE chaperonins assist substrate protein (SP) folding by cycling through several conformational states. With each cycle the SP is, in turn, captured, unfolded, briefly encapsulated (t_1/2 ∼1 s), and released by the chaperonin complex. The protein-folding functional form is the US-football–shaped GroEL:GroES₂ complex. We report structures of two such “football” complexes to ∼3.7-Å resolution; one is empty whereas the other contains encapsulated SP in both chambers. Although encapsulated SP is not visible on the electron density map, using calibrated FRET and order-of-addition experiments we show that owing to SP-catalyzed ADP/ATP exchange both chambers of the football complex encapsulate SP efficiently only if the binding of SP precedes that of ATP. The two rings of GroEL thus behave as a parallel processing machine, rather than functioning alternately. Compared with the bullet-shaped GroEL:GroES₁ complex, the GroEL:GroES₂ football complex differs conformationally at the GroEL–GroES interface and also at the interface between the two GroEL rings. We propose that the electrostatic interactions between the ε-NH³⁺ of K105 of helix D in one ring with the negatively charged carboxyl oxygen of A109 at the carboxyl end of helix D of the other ring provide the structural basis for negative inter-ring cooperativity.

The chaperonin proteins GroEL and GroES are cellular nanomachines driven by the hydrolysis of ATP that facilitate the folding of structurally diverse substrate proteins. In response to ligand binding, the subunits of a ring cycle in a concerted manner through a series of allosteric states (T, R, and R″), enabling work to be performed on the substrate protein. Removing two salt bridges that ordinarily break during the allosteric transitions of the WT permitted the structure of GroEL-ADP in the R state to be solved to 2.7 Å resolution. Whereas the equatorial domain displays almost perfect sevenfold symmetry, the apical domains, to which substrate proteins bind, and to a lesser extent, the intermediate domains display a remarkable asymmetry. Freed of intersubunit contacts, the apical domain of each subunit adopts a different conformation, suggesting a flexibility that permits interaction with diverse substrate proteins. This result contrasts with a previous cryo-EM study of a related allosteric ATP-bound state at lower resolution. After artificially imposing sevenfold symmetry it was concluded that a GroEL ring in the R-ATP state existed in six homogeneous but slightly different states. By imposing sevenfold symmetry on each of the subunits of the crystal structure of GroEL-ADP, we showed that the synthetic rings of (X-ray) GroEL-ADP and (cryo-EM) GroEL-ATP are structurally closely related. A deterministic model, the click stop mechanism, that implied temporal transitions between these states was proposed. Here, however, these conformational states are shown to exist as a structurally heterogeneous ensemble within a single ring.

Noncovalently “stacked” tetramethylrhodamine (TMR) dimers have been used to both report and perturb the allosteric equilibrium in GroEL. A GroEL mutant (K242C) has been labeled with TMR, close to the peptide-binding site in the apical domain, such that TMR molecules on adjacent subunits are able to form dimers in the T allosteric state. Addition of ATP induces the transition to the R state and the separation of the peptide-binding sites, with concomitant unstacking of the TMR dimers. A statistical analysis of the spectra allowed us to compute the number and orientation of TMR dimers per ring as a function of the average number of TMR molecules per ring. The TMR dimers thus serve as quantitative reporter of the allosteric state of the system. The TMR dimers also serve as a surrogate for substrate protein, substituting in a more homogeneous, quantifiable manner for the heterogeneous intersubunit, intraring, noncovalent cross-links provided by the substrate protein. The characteristic stimulation of the ATPase activity by substrate protein is also mimicked by the TMR dimers. Using an expanded version of the nested cooperativity model, we determine values for the free energy of the TT to TR and TR to RR allosteric equilibria to be 27 ± 11 and 46 ± 2 kJ/mol, respectively. The free energy of unstacking of the TMR dimers was estimated at 2.6 ± 1.0 kJ/mol dimer. These results demonstrate that GroEL can perform work during the T to R transition, supporting the iterative annealing model of chaperonin function.

Using calibrated FRET, we show that the simultaneous occupancy of both rings of GroEL by ATP and GroES occurs, leading to the rapid formation of symmetric GroEL:GroES2 “football” particles regardless of the presence or absence of substrate protein (SP). In the absence of SP, these symmetric particles revert to asymmetric GroEL:GroES1 “bullet” particles. The breakage of GroES symmetry requires the stochastic hydrolysis of ATP and the breakage of nucleotide symmetry. These asymmetric particles are both persistent and dynamic; they turnover via the asymmetric cycle. When challenged with SP, however, they revert to symmetric particles within a second. In the presence of SP, the symmetric particles are also persistent and dynamic. They turn over via the symmetric cycle. Under these conditions, the stochastic hydrolysis of ATP and the breakage of nucleotide symmetry also occur within the ensemble of particles. However, on account of SP-catalyzed ADP/ATP exchange, GroES symmetry is rapidly restored. The residence time of both GroES and SP on functional GroEL is reduced to ∼1 s, enabling many more iterations than was previously believed possible, consistent with the iterative annealing mechanism. This result is inconsistent with currently accepted models. Using a foldable SP, we show that as the SP folds to the native state and the population of unfolded SP declines, the population of symmetric particles reverts to asymmetric particles in parallel, a result that is consistent with the former being the folding functional form.

The complex kinetics of Pi and ADP release by the chaperonin GroEL/GroES is influenced by the presence of unfolded substrate protein (SP). Without SP, the kinetics of Pi release are described by four phases: a “lag,” a “burst” of ATP hydrolysis by the nascent cis ring, a “delay” caused by ADP release from the nascent trans ring, and steady-state ATP hydrolysis. The release of Pi precedes the release of ADP. The rate-determining step of the asymmetric cycle is the release of ADP from the trans ring of the GroEL-GroES1 “bullet” complex that is, consequently, the predominant species. In the asymmetric cycle, the two rings of GroEL function alternately, 180° out of phase. In the presence of SP, a change in the kinetic mechanism occurs. With SP present, the kinetics of ADP release are also described by four phases: a lag, a “surge” of ADP release attributable to SP-induced ADP/ATP exchange, and a “pause” during which symmetrical “football” particles are formed, followed by steady-state ATP hydrolysis. SP catalyzes ADP/ATP exchange on the trans ring. Now ADP release precedes the release of Pi, and the rate-determining step of the symmetric cycle becomes the hydrolysis of ATP by the symmetric GroEL-GroES2 football complex that is, consequently, the predominant species. A FRET-based analysis confirms that asymmetric GroEL-GroES1 bullets predominate in the absence of SP, whereas symmetric GroEL-GroES2 footballs predominate in the presence of SP. This evidence suggests that symmetrical football particles are the folding functional form of the chaperonin machine in vivo.

In a study of the timing mechanism of the chaperonin nanomachine we show that the hemicycle time (HCT) is determined by the mean residence time (MRT) of GroES on the cis ring of GroEL. In turn, this is governed by allosteric interactions within the trans ring of GroEL. Ligands that enhance the R (relaxed) state (residual ADP, the product of the previous hemicycle, and K+) extend the MRT and the HCT, whereas ligands that enhance the T (taut) state (unfolded substrate protein, SP) decrease the MRT and the HCT. In the absence of SP, the chaperonin machine idles in the resting state, but in the presence of SP it operates close to the speed limit, set by the rate of ATP hydrolysis by the cis ring. Thus, the conformational states of the trans ring largely control the speed of the complete chaperonin cycle.

The effects of potassium ion on the nested allostery of GroEL are due to increases in the affinity for nucleotide. Both positive allosteric transitions, TT-TR and TR-RR, occur at lower [ATP] as [K+] is increased. Negative cooperativity in the double-ringed system is also due to an increase in the affinity of the trans ring for the product ADP as [K+] is increased. Consequently, (i) rates of ATP hydrolysis are inversely proportional to [K+] and (ii) the residence time of GroES bound to the cis ring is prolonged and the hemicycle time extended. Substrate protein suppresses negative cooperativity by decreasing the affinity of the trans ring for ADP, reducing the hemicycle time to a constant minimum. The trans ring thus serves as a variable timer. ATP added to the asymmetric GroEL-GroES resting-state complex lacking trans ring ADP is hydrolyzed in the newly formed cis ring with a presteady-state burst of ≈6 mol of Pi per mole of 14-mer. No burst is observed when the trans ring contains ADP. The amplitude and kinetics of ATP hydrolysis in the cis ring are independent of the presence or absence of encapsulated substrate protein and independent of K+ at concentrations where there are profound effects on the linear steady-state rate. The hydrolysis of ATP by the cis ring constitutes a second, nonvariable timer of the chaperonin cycle.

Escherichia coli chaperonin, GroEL, helps proteins fold under nonpermissive conditions. During the reaction cycle, GroEL undergoes allosteric transitions in response to binding of a substrate protein (SP), ATP, and the cochaperonin GroES. Using coarse-grained representations of the GroEL and GroES structures, we explore the link between allosteric transitions and the folding of a model SP, a de novo-designed four-helix bundle protein, with low spontaneous yield. The ensemble of GroEL-bound SP is less structured than the bulk misfolded structures. Upon binding, which kinetically occurs in two stages, the SP loses not only native tertiary contacts but also experiences a decrease in helical content. During multivalent binding and the subsequent ATP-driven transition of GroEL the SP undergoes force-induced stretching. Upon encapsulation, which occurs upon GroES binding, the SP finds itself in a “hydrophilic” cavity in which it can reach the folded conformation. Surprisingly, we find that the yield of the native state in the expanded GroEL cavity is relatively small even after it remains in it for twice the spontaneous folding time. Thus, in accord with the iterative annealing mechanism, multiple rounds of binding, partial unfolding, and release of the SP are required to enhance the yield of the folded SP.

The chaperonin GroEL-GroES, a machine that helps proteins to fold, cycles through a number of allosteric states, the T state, with high affinity for substrate proteins, the ATP-bound R state, and the R″ (GroEL–ADP–GroES) complex. Here, we use a self-organized polymer model for the GroEL allosteric states and a general structure-based technique to simulate the dynamics of allosteric transitions in two subunits of GroEL and the heptamer. The T → R transition, in which the apical domains undergo counterclockwise motion, is mediated by a multiple salt-bridge switch mechanism, in which a series of salt-bridges break and form. The initial event in the R → R″ transition, during which GroEL rotates clockwise, involves a spectacular outside-in movement of helices K and L that results in K80-D359 salt-bridge formation. In both the transitions there is considerable heterogeneity in the transition pathways. The transition state ensembles (TSEs) connecting the T, R, and R″ states are broad with the TSE for the T → R transition being more plastic than the R → R″ TSE.

We have used a bioinformatic approach to predict the natural substrate proteins for the Escherichia coli chaperonin GroEL based on two simple criteria. Natural substrate proteins should contain binding motifs similar in sequence to the mobile loop peptide of GroES that displaces the binding motif during the chaperonin cycle. Secondly, each substrate protein should contain multiple copies of the binding motif so that the chaperonin can perform “work” on the substrate protein. To validate these criteria, we have used a database of 252 proteins that have been experimentally shown to interact with the chaperonin machinery in vivo. More than 80% are identified by these criteria. The binding motifs of all 79 proteins in the database with a known three-dimensional structure are buried (<50% solvent-accessible surface area) in the native state. Our results show that the binding motifs are inaccessible in the native state but become solvent-exposed in unfolded state, thus enabling GroEL to distinguish between unfolded and native states. The structures of the binding motif in the native states of the substrate proteins include α-helices, β-strands, and random coils. The diversity of secondary structures implies that there are large and varied conformational transitions in the recognition motifs after their displacement by the mobile loops of GroES.