Equilibrium and kinetics of the allosteric transition of GroEL studied by solution X-ray scattering and fluorescence spectroscopy

We have studied the ATP-induced allosteric structural transition of GroEL using small angle X-ray scattering and fluorescence spectroscopy in combination with a stopped-flow technique. With X-ray scattering one can clearly distinguish the three allosteric states of GroEL, and the kinetics of the transition of GroEL induced by 85 microM ATP have been observed directly by stopped-flow X-ray scattering for the first time. The rate constant has been found to be 3-5s(-1) at 5 degrees C, indicating that this process corresponds to the second phase of the ATP-induced kinetics of tryptophan-inserted GroEL measured by stopped-flow fluorescence. Based on the ATP concentration dependence of the fluorescence kinetics, we conclude that the first phase represents bimolecular non-cooperative binding of ATP to GroEL with a bimolecular rate constant of 5.8 x 10(5)M(-1)s(-1) at 25 degrees C. Considering the electrostatic repulsion between negatively charged GroEL (-18 of the net charge per monomer at pH 7.5) and ATP, the rate constant is consistent with a diffusion-controlled bimolecular process. The ATP-induced fluorescence kinetics (the first and second phases) at various ATP concentrations (< 400 microM) occur before ATP hydrolysis by GroEL takes place and are well explained by a kinetic allosteric model, which is a combination of the conventional transition state theory and the Monod-Wyman-Changeux model, and we have successfully evaluated the equilibrium and kinetic parameters of the allosteric transition, including the binding constant of ATP in the transition state of GroEL.     

The allosteric transition of GroEL induced by metal fluoride-ADP complexes

To understand the mechanism of a functionally important ATP-induced allosteric transition of GroEL, we have studied the effect of a series of metal fluoride-ADP complexes and vanadate-ADP on GroEL by kinetic fluorescence measurement of pyrene-labeled GroEL and by small-angle X-ray scattering measurement of wild-type GroEL. The metal fluorides and vanadate, complexed with ADP, are known to mimic the gamma-phosphate group of ATP, but they differ in geometry and size; it is expected that these compounds will be useful for investigating the strikingly high specificity of GroEL for ATP that enables the induction of the allosteric transition. The kinetic fluorescence measurement revealed that aluminium, beryllium, and gallium ions, when complexed with the fluoride ion and ADP, induced a biphasic fluorescence change of pyrenyl GroEL, while scandium and vanadate ions did not induce any kinetically observed change in fluorescence. The burst phase and the first phase of the fluorescence kinetics were reversible, while the second phase and subsequent changes were irreversible. The dependence of the burst-phase and the first-phase fluorescence changes on the ADP concentration indicated that the burst phase represents non-cooperative nucleotide binding to GroEL, and that the first phase represents the allosteric transition of GroEL. Both the amplitude and the rate constant of the first phase of the fluorescence kinetics were well understood in terms of a kinetic allosteric model, which is a combination of transition state theory and the Monod-Wyman-Changeux allosteric model. From the kinetic allosteric model analysis, the relative free energy of the transition state in the metal fluoride-ADP-induced allosteric transition of GroEL was found to be larger than the corresponding free energy of the ATP-induced allosteric transition by more than 5.5kcal/mol. However, the X-ray scattering measurements indicated that the allosteric state induced by these metal fluoride-ADP complexes is structurally equivalent to the allosteric state induced by ATP. These results suggested that both the size and coordination geometry of gamma-phosphate (and its analogs) are related to the allosteric transition of GroEL. It was therefore concluded that the tetrahedral geometry of gamma-phosphate (or its analogs) and the inter-atomic distance ( approximately 1.6A) between phosphorus (vanadium, or metal atom) and oxygen (or fluorine) are both important for inducing the allosteric transition of GroEL, leading to the high selectivity of GroEL for ATP about ligand adenine nucleotides, which function as the preferred allosteric ligand.     

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.     

Kinetic analysis of ATP-dependent inter-ring communication in GroEL

Different concentrations of ATP were mixed rapidly with single-ring or double-ring forms of GroEL containing the Phe44-->Trp mutation and the time-resolved changes in fluorescence emission, upon excitation at 295 nm, were followed. Two kinetic phases that were previously found for double-ring GroEL are also observed in the case of the single-ring version: (i) a fast phase with a relatively large amplitude that is associated with the T-->R allosteric transition; (ii) and a slow phase with a smaller amplitude that is associated with ATP hydrolysis. In the case of weak intra-ring positive cooperativity, the rate constant corresponding to the T-->R allosteric switch of single-ring GroEL displays a bi-sigmoidal dependence on ATP concentration that may reflect parallel pathways of the allosteric transition. The slow phase is absent when double-ring or single-ring forms of GroEL are mixed with ADP or ATP without K(+), and it has a rate constant that is independent of ATP concentration. A third fast phase that is still unassigned is observed for both single-ring and double-ring GroEL when a large amount of data is collected. Finally, a fourth phase is observed in the case of double-ring GroEL that is found to be absent in the case of single-ring GroEL. This phase is here assigned to inter-ring communication by (i) determining its dependence on ATP concentration and (ii) based on its absence from single-ring GroEL and the Arg13-->Gly, Ala126-->Val GroEL mutant, which is defective in inter-ring negative cooperativity. The value of the rate constant corresponding to this phase is found to increase with increasing intra-ring positive cooperativity, with respect to ATP. This is the first report of the rate of ATP-mediated inter-ring communication in GroEL, in the presence of ATP alone, which is crucial for the cycling of this molecular machine between different functional states.     

On the relationship between the Hill coefficients for steady-state and transient kinetic data: A criterion for concerted transitions in allosteric proteins

A frequently used measure for the extent of cooperativity in ligand binding by allosteric proteins is the Hill coefficient. Hill coefficients can be measured for steady-state kinetic data and also for transient kinetic data. Here, the relationship between the two types of Hill coefficients is analysed. It is shown that a value of 1 for the ratio of the two Hill coefficients is a test for a concerted ligand-induced transition between two conformations of the protein, in accordance with the Monod-Wyman-Changeux model. A value of 1 for this ratio has recently been observed for a series of chaperonin GroEL mutants suggesting that ATP-induced allosteric transitions in this protein are concerted.     

Analysis of the allosteric properties of rabbit muscle phosphofructokinase by means of affinity labeling with a reactive ATP analog.

A reactive ATP analog, N6-(6-bromoacetamidohexyl)-AMP-PCP, reacted specifically with the ATP inhibitory site of rabbit skeletal muscle phosphofructokinase without affecting the active site. Modification resulted in the incorporation of 1.01 mol of the reagent per mol of enzyme subunit. The modified enzyme was insensitive to allosteric inhibition by ATP and to activation by AMP at pH 7.2, where the native enzyme exhibits allosteric kinetic behavior. These observations demonstrate that we had succeeded in obtaining PFK fixed in the T state. Using the kinetic parameters of this modified enzyme, the kinetic properties of native enzyme can be quantitatively accounted for by the allosteric model of Monod-Wyman-Changeux. Further, the reagent was shown to have reacted with a specific cysteine residue near or at the ATP inhibitory site, and the sequence around the cysteine was determined as Cys-Lys-Asp-Phe-Arg.     

Kinetic model for the coupling between allosteric transitions in GroEL and substrate protein folding and aggregation

The bacterial chaperonin GroEL and the co-chaperonin GroES assist in the folding of a number of structurally unrelated substrate proteins (SPs). In the absence of chaperonins, SP folds by the kinetic partitioning mechanism (KPM), according to which a fraction of unfolded molecules reaches the native state directly, while the remaining fraction gets trapped in a potentially aggregation-prone misfolded state. During the catalytic reaction cycle, GroEL undergoes a series of allosteric transitions (T<-->R-->R"-->T) triggered by SP capture, ATP binding and hydrolysis, and GroES binding. We developed a general kinetic model that takes into account the coupling between the rates of the allosteric transitions and the folding and aggregation of the SP. Our model, in which the GroEL allosteric rates and SP-dependent folding and aggregation rates are independently varied without prior assumption, quantitatively fits the GroEL concentration-dependent data on the yield of native ribulose bisphosphate carboxylase/oxygenase (Rubisco) as a function of time. The extracted kinetic parameters for the GroEL reaction cycle are consistent with the available values from independent experiments. In addition, we also obtained physically reasonable parameters for the kinetic steps in the reaction cycle that are difficult to measure. If experimental values for GroEL allosteric rates are used, the time-dependent changes in native-state yield at eight GroEL concentrations can be quantitatively fit using only three SP-dependent parameters. The model predicts that the differences in the efficiencies (as measured by yields of the native state) of GroEL, single-ring mutant (SR1), and variants of SR1, in the rescue of mitochondrial malate dehydrogenase, citrate synthase, and Rubisco, are related to the large variations in the allosteric transition rates. We also show that GroEL/S mutants that efficiently fold one SP at the expense of all others are due to a decrease in the rate of a key step in the reaction cycle, which implies that wild-type GroEL has evolved as a compromise between generality and specificity. We predict that, under maximum loading conditions and saturating ATP concentration, the efficiency of GroEL (using parameters for Rubisco) depends predominantly on the rate of R-->R" transition, while the equilibrium constant of the T<-->R has a small effect only. Both under sub- and superstoichiometric GroEL concentrations, enhanced efficiency is achieved by rapid turnover of the reaction cycle, which is in accord with the predictions of the iterative annealing mechanism. The effects are most dramatic at substoichiometric conditions (most relevant for in vivo situations) when SP aggregation can outcompete capture of SP by chaperonins.     

Oxygen binding and its allosteric control in hemoglobin of the primitive branchiopod crustacean Triops cancriformis

Branchiopod crustaceans are endowed with extracellular, high-molecular-mass hemoglobins (Hbs), the functional and allosteric properties of which have largely remained obscure. The Hb of the phylogenetically ancient Triops cancriformis (Notostraca) revealed moderate oxygen affinity, cooperativity and pH dependence (Bohr effect) coefficients: P(50) = 13.3 mmHg, n(50) = 2.3, and Phi = -0.18, at 20 degrees C and pH 7.44 in Tris buffer. The in vivo hemolymph pH was 7.52. Bivalent cations increased oxygen affinity, Mg(2+) exerting a greater effect than Ca(2+). Analysis of cooperative oxygen binding in terms of the nested Monod-Wyman-Changeux (MWC) model revealed an allosteric unit of four oxygen-binding sites and functional coupling of two to three allosteric units. The predicted 2 x 4 and 3 x 4 nested structures are in accord with stoichiometric models of the quarternary structure. The allosteric control mechanism of protons comprises a left shift of the upper asymptote of extended Hill plots which is ascribable to the displacement of the equilibrium between (at least) two high-affinity (relaxed) states, similar to that found in extracellular annelid and pulmonate molluscan Hbs. Remarkably, Mg(2+) ions increased oxygen affinity solely by displacing the equilibrium between the tense and relaxed conformations towards the relaxed states, which accords with the original MWC concept, but appears to be unique among Hbs. This effect is distinctly different from those of ionic effectors (bivalent cations, protons and organic phosphates) on annelid, pulmonate and vertebrate Hbs, which involve changes in the oxygen affinity of the tense and/or relaxed conformations.     

Kinetic and thermodynamic parameters for oxygen binding to the allosteric states of Panulirus interruptus hemocyanin

The temperature dependence of the oxygen binding equilibria and kinetics of Panulirus interruptus hemocyanin has been analyzed within the context of the two-state allosteric model. Oxygenation of the T-state is characterized by a more negative value of DeltaH than that of the R-state; therefore, cooperative effects in oxygen binding to P. interruptus hemocyanin are thermodynamically governed by favorable entropy changes. The allosteric transition in the unliganded derivative shows an enthalpy-entropy compensation effect. The activation enthalpies for oxygenation and deoxygenation of the T-state are larger than those for the R-state, while the activation entropies are favorable for the T-state and unfavorable for the R-state. Thus, the activation free energies for oxygen binding to the T- and R-states are similar, while for the deoxygenation reaction DeltaG++ is smaller for the T-state. The analysis reported confirms the applicability of the Monod-Wyman-Changeux two-state allosteric model to P. interruptus hemocyanin and yields a complete thermodynamic characterization of oxygen binding under both equilibrium and dynamic regimes.     

The regulatory design of an allosteric feedback loop: the effect of saturation by pathway substrate

Aspects of metabolic regulation can be fruitfully studied with a combination of generic modelling, control analysis and graphical analysis using rate characteristics. This paper analyses a prototypical supply-demand system consisting of a biosynthetic subsystem subject to allosteric inhibition by its product and a demand process that consumes this product. The effect of changes in affinity of the committing supply enzyme for the pathway substrate on the regulatory properties of the supply subsystem is compared for the Monod-Wyman-Changeux and the reversible Hill allosteric enzyme models. We found that the Hill model has a distinct advantage in that the steady-state concentration at which it maintains the product is set by the half-saturating product concentration and is independent of changes in the degree of saturation for substrate. In contrast, with the Monod-Wyman-Changeux model this set point varies with affinity for substrate. Explicitly incorporating reversibility in all rate equations made it possible to distinguish between kinetic and thermodynamic aspects of regulation. Combining the supply and demand rate characteristics allows us to explore both the control distribution at steady state and the regulatory performance of the system over a wide range of demand activities.     

Allosteric transition and substrate binding are entropy-driven in glucosamine-6-phosphate deaminase from Escherichia coli

Glucosamine-6P-deaminase (EC 3.5.99.6, formerly glucosamine-6-phosphate isomerase, EC 5.3.1.10) from Escherichia coli is an attractive experimental model for the study of allosteric transitions because it is both kinetically and structurally well-known, and follows rapid equilibrium random kinetics, so that the kinetic K(m) values are true thermodynamic equilibrium constants. The enzyme is a typical allosteric K-system activated by N-acetylglucosamine 6-P and displays an allosteric behavior that can be well described by the Monod-Wyman-Changeux model. This thermodynamic study based on the temperature dependence of allosteric parameters derived from this model shows that substrate binding and allosteric transition are both entropy-driven processes in E. coli GlcN6P deaminase. The analysis of this result in the light of the crystallographic structure of the enzyme implicates the active-site lid as the structural motif that could contribute significantly to this entropic component of the allosteric transition because of the remarkable change in its crystallographic B factors.     

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.     

Iterative annealing mechanism explains the functions of the GroEL and RNA chaperones

Molecular chaperones are ATP‐consuming machines, which facilitate the folding of proteins and RNA molecules that are kinetically trapped in misfolded states. Unassisted folding occurs by the kinetic partitioning mechanism according to which folding to the native state, with low probability as well as misfolding to one of the many metastable states, with high probability, occur rapidly. GroEL is an all‐purpose stochastic machine that assists misfolded substrate proteins to fold. The RNA chaperones such as CYT‐19, which are ATP‐consuming enzymes, help the folding of ribozymes that get trapped in metastable states for long times. GroEL does not interact with the folded proteins but CYT‐19 disrupts both the folded and misfolded ribozymes. The structures of GroEL and RNA chaperones are strikingly different. Despite these differences, the iterative annealing mechanism (IAM) quantitatively explains all the available experimental data for assisted folding of proteins and ribozymes. Driven by ATP binding and hydrolysis and GroES binding, GroEL undergoes a catalytic cycle during which it samples three allosteric states, T (apo), R (ATP bound), and R^″ (ADP bound). Analyses of the experimental data show that the efficiency of the GroEL–GroES machinery and mutants is determined by the resetting rate k _{R ″ → T} , which is largest for the wild‐type (WT) GroEL. Generalized IAM accurately predicts the folding kinetics of Tetrahymena ribozyme and its variants. Chaperones maximize the product of the folding rate and the steady‐state native state fold by driving the substrates out of equilibrium. Neither the absolute yield nor the folding rate is optimized.     

Nested allosteric interactions in the cytoplasmic chaperonin containing TCP-1

Initial rates of ATP hydrolysis by the chaperonin containing TCP-1 (CCT) from bovine testis were measured as a function of ATP concentration. Two allosteric transitions are observed: one at relatively low concentrations of ATP (<100 microM) and the second at higher concentrations of ATP. The data suggest that CCT has positive intra-ring cooperativity and negative inter-ring cooperativity in ATP hydrolysis, with respect to ATP, as previously observed in the case of GroEL. It is shown that the relatively weak positive intra-ring cooperativity found in the case of CCT may be due to heterogeneity in its subunit composition. Our results suggest that nested allosteric behavior may be common to chaperone double-ring systems.     

Validity of transfer-function representation of input-output relation in allosteric models

A transfer-function representation of reaction velocity is devised to describe analytically and approximately an input-output response of allosteric enzyme around a steady state. The transfer function is derived on assuming an exponential change in reaction velocity for the indicial response to substrate influx rate. The validity of the representation with variation in the kinetic parameters and flow rates is examined for the response of Koshland-Nemethy-Filmer (KNF) and Monod-Wyman-Changeux (MWC) dimeric models by comparing with the exact response obtained from the computer simulation, that is, by numerical integration of the rate equation. The representation has a wider valid region with a decrease in influx rate than with an increase. For the KNF model the representation is valid for negative cooperativity, but invalid for positive cooperativity. For the MWC model the validity decreases with stronger cooperativity. With the transfer functions valid for the Michaelis-Menten and allosteric reactions, we may derive the transfer-function representation for many metabolic pathways.     

Nested MWC model describes hydrolysis of GroEL without assuming negative cooperativity in binding

Folding assistance and ATPase activity of GroEL are based on the existence of different conformations. In order to characterise these conformations, published data on steady state ATPase activity in the absence of GroES were reanalysed simultaneously in terms of the Nested MWC model. This model is a hierarchical extension of the symmetry-model of Monod et al. [J. Mol. Biol. 12 (1965) 88]. An unique set of GroEL specific parameters was obtained. This set was supported by comparison of predictions arising from this set of values with experimental data for hydrolysis of ATP in the presence of ADP and ATPgammaS, binding of ATPgammaS and ADP to GroEL in the absence of ATP, and binding of ATP as monitored by fluorescence labelling. Thus, for the first time, multiple data sets for the interaction of nucleotides with GroEL are described quantitatively by an allosteric model. A noteworthy feature of our model is that no negative cooperativity in ATP binding occurs in accordance to experimental observations. Furthermore, the model also includes the existence of a conformation with very high ATPase activity. Such a conformation might be of importance at a certain stage in the folding cycle.     

Characterisation of a GroEL Single-Ring Mutant that Supports Growth of Escherichia coli and Has GroES-Dependent ATPase Activity

Binding and folding of substrate proteins by the molecular chaperone GroEL alternates between its two seven-membered rings in an ATP-regulated manner. The association of ATP and GroES to a polypeptide-bound ring of GroEL encapsulates the folding proteins in the central cavity of that ring (cis ring) and allows it to fold in a protected environment where the risk of aggregation is reduced. ATP hydrolysis in the cis ring changes the potentials within the system such that ATP binding to the opposite (trans) ring triggers the release of all ligands from the cis ring of GroEL through a complex network of allosteric communication between the rings. Inter-ring allosteric communication thus appears indispensable for the function of GroEL, and an engineered single-ring version (SR1) cannot substitute for GroEL in vivo. We describe here the isolation and characterisation of an active single-ring form of the GroEL protein (SR-A92T), which has an exceptionally low ATPase activity that is strongly stimulated by the addition of GroES. Dissection of the kinetic pathway of the ATP-induced structural changes in this active single ring can be explained by the fact that the mutation effectively blocks progression through the full allosteric pathway of the GroEL reaction cycle, thus trapping an early allosteric intermediate. Addition of GroES is able to overcome this block by binding this intermediate and pulling the allosteric pathway to completion via mass action, explaining how bacterial cells expressing this protein as their only chaperonin are viable.     

Importance of hydrophobic cluster formation through long-range contacts in the folding transition state of two-state proteins

Understanding the folding pathways of proteins is a challenging task. The Phi value approach provides a detailed understanding of transition-state structures of folded proteins. In this work, we have computed the hydrophobicity associated with each residue in the folded state of 16 two-state proteins and compared the Phi values of each mutant residue. We found that most of the residues with high Phi value coincide with local maximum in surrounding hydrophobicity, or have nearby residues that show such maximum in hydrophobicity, indicating the importance of hydrophobic interactions in the transition state. We have tested our approach to different structural classes of proteins, such as alpha-helical, SH3 domains of all-beta proteins, beta-sandwich, and alpha/beta proteins, and we observed a good agreement with experimental results. Further, we have proposed a hydrophobic contact network pattern to relate the Phi values with long-range contacts, which will be helpful to understand the transition-state structures of folded proteins. The present approach could be used to identify potential hydrophobic clusters that may form through long-range contacts during the transition state.     

How ATP inhibits the open K(ATP) channel

ATP-sensitive potassium (K(ATP)) channels are composed of four pore-forming Kir6.2 subunits and four regulatory SUR1 subunits. Binding of ATP to Kir6.2 leads to inhibition of channel activity. Because there are four subunits and thus four ATP-binding sites, four binding events are possible. ATP binds to both the open and closed states of the channel and produces a decrease in the mean open time, a reduction in the mean burst duration, and an increase in the frequency and duration of the interburst closed states. Here, we investigate the mechanism of interaction of ATP with the open state of the channel by analyzing the single-channel kinetics of concatenated Kir6.2 tetramers containing from zero to four mutated Kir6.2 subunits that possess an impaired ATP-binding site. We show that the ATP-dependent decrease in the mean burst duration is well described by a Monod-Wyman-Changeux model in which channel closing is produced by all four subunits acting in a single concerted step. The data are inconsistent with a Hodgkin-Huxley model (four independent steps) or a dimer model (two independent dimers). When the channel is open, ATP binds to a single ATP-binding site with a dissociation constant of 300 microM.