Setting the chaperonin timer: The effects of K + and substrate protein on ATP hydrolysis

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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:GroES 2 "football" particles regardless of the presence or absence of substrate protein (SP). In the absence of SP, these symmetric particles revert to asymmetric GroEL:GroES 1 "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.G roEL plays a central role in the function of the GroEL/ GroES nanomachine. It binds unfolded proteins, transiently encapsulates them under the GroES "lid," and then releases them to the external medium, this cycle of events being driven by the hydrolysis of ATP (1, 2). GroEL, however, consists of two heptameric rings, and some controversy has arisen as to whether each ring operates alternately via an asymmetric cycle or simultaneously via a symmetric cycle. In the former case, GroEL engages one GroES at a time and the predominant species is the asymmetric GroEL:GroES 1 "bullet" complex whereas, in the latter, symmetric GroEL:GroES 2 "football" particles predominate in the symmetric cycle (figure 9 in ref. 4) (3, 4). Nevertheless, the asymmetric cycle, championed by leading authorities (5, 6), has become the widely accepted model of chaperonin function, promulgated in recent textbooks (7, 8) despite much evidence (9-21) that assigns a role to the symmetric football particles. However, the involvement of these symmetric particles in chaperonin function has been questioned on the basis of three items of chaperonin dogma: (i) that the formation of symmetric GroEL:GroES 2 particles and polypeptide binding are mutually exclusive (22, 23); (ii) the belief that, because of negative cooperativity, "when one GroEL ring binds ATP, the other ring cannot also do so" (5); and (iii) that the chaperonin ATPase cycle turns over at the same rate in the presence of substrate protein as it does in its absence. Here...

Section: Methodsmentioning

confidence: 99%

Symmetric GroEL:GroES ₂ complexes are the protein-folding functional form of the chaperonin nanomachine

Yang¹,

Ye²,

Lorimer

2013

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“…The mechanism by which this occurs is not known. It has been shown, however, that protein substrates enhance the steady-state ATPase activity of GroEL by increasing the rate of ADP release (10,11). Hence, it is likely that tethering shifts the equilibrium after hydrolysis in favor of the T state, which has lower affinity for nucleotides, thereby increasing the rate of ADP release and stimulating ATPase activity.…”

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confidence: 99%

Putting handcuffs on the chaperonin GroEL

Horovitz

2013

Oligomeric, ring-shaped nano-machines that are fueled by ATP are ubiquitous in all three kingdoms of life and are involved in a wide range of processes that include, for example, protein folding, protein degradation, DNA and RNA remodeling, and protein insertion into membranes (for review, see ref. 1). These assemblies are true machines because they carry out work by undergoing movements that are coordinated in time and space and are driven by energy consumption (i.e., ATP binding and hydrolysis). One fundamental question regarding such machines relates to the coupling between the order of their ATPpromoted conformational changes ( Fig. 1) and their biological function. These conformational changes can take place in a concerted fashion according to the MonodWyman-Changeux (MWC) model (2), as proposed and recently demonstrated in the case of the chaperonin GroEL (3, 4). Alternatively, the changes can occur in a domino-like sequential fashion according to the Koshland-Némethy-Filmer model (5) or in a probabilistic manner, as suggested in the case of the proteolytic machine ClpP (6). A second fundamental question regarding such machines concerns the mechanisms by which ATP consumption is converted into work. Both questions are elegantly addressed, in the case of the chaperonin GroEL, in the report by Corsepius and Lorimer (7) published in PNAS.GroEL consists of two back-to-back stacked heptameric rings, with a cavity at each end in which protein folding can take place under confining conditions. GroEL assists protein folding by cycling between protein substrate acceptor and release states upon ATP binding and hydrolysis. ATP binding to GroEL occurs with positive cooperativity within rings and negative cooperativity between rings, both with respect to ATP. A nested allosteric model that accounts for these observations was put forward (3) in which each ring of GroEL switches in a concerted MWC fashion between a T state, with low affinity for ATP and high affinity for protein substrates, and an R state, with high affinity for ATP and low affinity for protein substrates. In the presence of ATP, the GroEL double-ring can, therefore, exist in three states: TT, TR, and RR. Because of the negative interring cooperativity, the TR→RR transition is less favored than the TT→TR transition, thus ensuring that the two rings can operate out of phase with each other.Complexes of protein substrates with GroEL are heterogeneous because bound nonfolded substrates can exist in multiple conformations that interact with different configurations of GroEL subunits (8). It has, therefore, been difficult to quantitate the effects of substrate binding on the allosteric properties of GroEL. Corsepius and Lorimer (7) have neatly circumvented this heterogeneity problem by labeling GroEL with tetramethylrhodamine (TMR) at position 242 in the apical domain near the protein substrate-binding site. This position was chosen so that TMR molecules attached to adjacent subunits can form noncovalently "stacked" dimers in the T state but not in the R stat...

“…The bound SP serves as a noncovalent tether between two adjacent subunits. This stabilizes the T state, which has weak affinity for nucleotide, accelerating the rate of product release, and increasing the rate of ATP consumption (10,11). Likewise, the apical domains of the EL242C-TMR are tethered by the noncovalent stacking of the dye molecules.…”

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confidence: 99%

“…In the absence of SP, the release of ADP is the rate-limiting step in the catalytic cycle (10). The addition of SP increases the rate of ATP consumption by GroEL by accelerating ADP/ATP exchange.…”

mentioning

confidence: 99%

“…As originally formulated (25,26), the NCM contained a simplifying assumption that ATP bound exclusively to the R state. Subsequent work, however, showed that the T state could also bind and hydrolyze ATP (10,26). An expanded version of the NCM, without the exclusive binding assumption was developed by Gresham (27) and was used to quantitatively analyze the turnover rate of GroEL (Eq.…”

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confidence: 99%

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Measuring how much work the chaperone GroEL can do

Corsepius

Lorimer

2013

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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 crosslinks 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.allostery | substrate protein binding problem