Review: allostery in chaperonins

Chaperonins mediate protein folding in an ATP-dependent manner. ATP binding and hydrolysis by chaperonins are subject to both homotropic and heterotropic allosteric regulation. In the case of GroEL and CCT, homotropic regulation by ATP is manifested in nested cooperativity, which involves positive intra-ring cooperativity and negative inter-ring cooperativity in ATP binding. Both types of cooperativity are modulated by various heterotropic allosteric effectors, which include nonfolded proteins, ADP, Mg2+, monovalent ions such as K+, and cochaperonins in the case of type I chaperonins such as GroEL. Here, the allosteric properties of chaperonins are reviewed and new results of ours are presented with regard to allosteric effects of ADP. The role of allostery in the reaction cycle and folding function of chaperonins is discussed.     

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.     

Nested cooperativity and salt dependence of the ATPase activity of the archaeal chaperonin Mm-cpn

The properties of the ATPase activity of the type II chaperonin from Methanococcus maripaludis (Mm-cpn) were examined. Mm-cpn can hydrolyze not only ATP, but also CTP, UTP, and GTP, albeit with different effectiveness. The ATPase activity is dependent on magnesium and potassium ions, and is effectively inhibited by sodium ions. Maximal rates of ATP hydrolysis are achieved at 600 mM potassium. Initial rates of ATP hydrolysis by Mm-cpn were determined at various ATP concentrations, revealing for the first time the presence of both positive intra-ring and negative inter-ring cooperativity in the archaeal chaperonin.     

In vivo and in vitro function of GroEL mutants with impaired allosteric properties

Escherichia coli cells that produce only plasmid-encoded wild-type or mutant GroEL were generated by bacteriophage P1 transduction. Effects of mutations that affect the allosteric properties of GroEL were characterized in vivo. Cells containing only GroEL(R197A), which has reduced intra-ring positive cooperativity and inter-ring negative cooperativity in ATP binding, grow poorly upon a temperature shift from 25 to 42 degrees C. This strain supports the growth of phages T4 and T5 but not phage lambda and produces light at 28 degrees C when transformed with a second plasmid containing the lux operon. In contrast, cells containing only GroEL(R13G, A126V) which lacks negative cooperativity between rings but has intact intra-ring positive cooperativity grow normally and support phage growth but do not produce light at 28 degrees C. In vitro refolding of luciferase in the presence of this mutant is found to be less efficient compared with wild-type GroEL or other mutants tested. Our results show that allostery in GroEL is important in vivo in a manner that depends on the physiological conditions and is protein substrate specific.     

Ionic interactions at both inter-ring contact sites of GroEL are involved in transmission of the allosteric signal: a time-resolved infrared difference study

The biological activity of the double-ring chaperonin GroEL is regulated by complex allosteric interactions, which include positive intra-ring and negative inter-ring cooperativity. To further characterize inter-ring communication, the nucleotide-induced absorbance changes in the vibrational spectrum of the chaperonin GroEL, of two single-point mutants suppressing one inter-ring ionic contact (E461K and E434K) and of a single-ring version of this protein, were investigated by time-resolved infrared difference spectroscopy. Interaction of the nucleotide with the proteins was triggered by its photochemical release from a biologically inactive caged precursor [P3-1-(2-nitro) phenylethyl nucleotide]. The results indicate that (1) ATP binding to the protein induces a conformational change that affects concomitantly both intra-ring and inter-ring communication, and (2) the experimental absorbance changes are sensitive to the double-ring structure of the protein. The characterization of the single-point, inter-ring mutants demonstrates that ionic interactions at both contact sites are involved in the transmission of the allosteric signal. However, both mutations have different effects on the inter-ring interface. While that of E461K still retains ionic contacts sensitive to ATP binding, E434K shows spectroscopic features similar to those of the single-ring version of the protein, therefore suggesting that electrostatic interactions at these contact sites contribute differently to the stability of the inter-ring interface.     

Transient kinetic analysis of ATP-induced allosteric transitions in the eukaryotic chaperonin containing TCP-1

The chaperonin CCT (chaperonin containing t-complex polypeptide 1 (TCP-1)) from bovine testis was mixed rapidly with different concentrations of ATP and the time-resolved change in fluorescence emission, upon excitation at 280 nm, was followed. Two kinetic phases were observed and assigned by (i) analyzing the dependence of the corresponding observed rate constants on ATP concentration; and (ii) by carrying out mixing experiments also with ADP, ATPgammaS and ATP without K(+). The values of the observed rate constants corresponding to both phases are found to be dependent on ATP concentration. The observed rate constant corresponding to the fast phase displays a bi-sigmoidal dependence on ATP concentration with Hill coefficients that are similar to those determined in steady-state ATPase experiments. This phase most likely reflects ATP binding-induced conformational changes. The rate constant of the conformational change in the presence of excess ATP is about 17s(-1) (at 25 degrees C) and is tenfold slower than the corresponding rate constant of GroEL. The observed rate constant corresponding to the second slower phase displays a hyperbolic dependence on ATP concentration. This phase is not observed in mixing experiments of CCT with ADP, ATPgammaS or ATP without K(+) and it, therefore, reflects a conformational change associated with ATP hydrolysis. Taken together, our results indicate that the kinetic mechanism of the allosteric transitions of CCT differs considerably from that of GroEL.     

Effect of inosine 5' -(beta, gamma-imido) triphosphate and other nucleotides on beef heart mitochondrial ATPase.

The effects of various substrates and alternative substrates on the hydrolytic activity of beef heart mitochondrial ATPase was examined. It was found that ATP or ADP, ITP hydrolysis showed positive cooperativity. IDP inhibited ITP hydrolysis and caused positive cooperativity. When ITP was present during an ATP hydrolysis assay, the rate of ATP hydrolysis was stimulated. IDP had no effect on ATP hydrolysis rates. A nonhydrolyzable ITP analog, inosine 5'-(beta, gamma-imido)triphosphate (IMP-P(NH)P), was synthesized and purified. It was found to be a potent competitive inhibitor of ITP and GTP hydrolytic activity. However, this beta-gamma-imido-bridged ITP analog was found to change the ITP and GTP hydrolysis kinetics from linear to positively cooperative. This compound inhibited ATP hydrolysis at substrate concentrations of 100 muM and lower, and stimulated ATP hydrolysis at substrate concentrations between 100 muM and 2 mM. IMP-P(NH)P had no effect on ATP hydrolysis when the substrate concentration was above 2 mM. In the presence of the activating anion, bicarbonate, IMP-P(NH)P inhibited ATP hydrolysis competitively, and induced positive cooperativity. IMP-P(NH)P had no effect on the ATP equilibrium Pi exchange, the ITP equilibrium Pi exchange, or ATP synthesis catalyzed by beef heart submitochondrial particles.     

Sequential ATP-induced allosteric transitions of the cytoplasmic chaperonin containing TCP-1 revealed by EM analysis

The eukaryotic cytoplasmic chaperonin containing TCP-1 (CCT) is a hetero-oligomeric complex that assists the folding of actins, tubulins and other proteins in an ATP-dependent manner. To understand the allosteric transitions that occur during the functional cycle of CCT, we imaged the chaperonin complex in the presence of different ATP concentrations. Labeling by monoclonal antibodies that bind specifically to the CCTalpha and CCTdelta subunits enabled alignment of all the CCT subunits of a given type in different particles. The analysis shows that the apo state of CCT has considerable apparent conformational heterogeneity that decreases with increasing ATP concentration. In contrast with the concerted allosteric switch of GroEL, ATP-induced conformational changes in CCT are found to spread around the ring in a sequential fashion that may facilitate domain-by-domain substrate folding. The approach described here can be used to unravel the allosteric mechanisms of other ring-shaped molecular machines.     

Spin-labelled phosphofructokinase. A simple and direct approach to the study of allosteric equilibria under near-physiological conditions.

Rabbit muscle phosphofructokinase, spin-labelled at its most reactive thiol group, has an electron spin resonance spectrum which is very sensitive to the binding of substrates and allosteric effectors. The spectral changes have been interpreted in terms of a concerted allosteric transition between two conformational states with non-exclusive binding of effectors. On this basis MgATP, fructose 6-phosphate plus ATP, and NH+4ions behave as potent positive effectors, inorganic phosphate, sulphate, AMP, fructose 6-phosphate and fructose 1,6-bisphosphate are less potent activators, and free ATP and H+ions are negative effectors, in agreement with the kinetic behaviour, but citrate behaves anomalously. In addition, the allosteric equilibrium can be displaced towards the inhibited state by selectively modifying two further thiol groups. Strong positive cooperativity occurs under suitable conditions with ATP, metal-ATP and fructose 6-phosphate. Biphasic changes of conformation, attributed to binding at the catalytic and inhibitory sites, have been observed in titrations with ATP. The differentiation of the two ATP binding sites arises in the presence of fructose 6-phosphate because of a distinct concerted effect on conformation between the two substrates at the active site. A similar effect occurs between ATP and citrate. Other heterotropic effects are more consistent with simple models; phosphates favour the binding, and reduce the cooperativity, of fructose 6-phosphate and metal-ATP, whereas excess ATP and H+ ions antagonise the binding and increase the cooperativity of fructose 6-phosphate. The observations are related to existing kinetic and binding studies where possible. Anomalous features of the behaviour suggest that the model should be regarded only as a first approximation.     

Influence of phosphate on the allosteric behavior of yeast phosphofructokinase.

Initial rate data obtained with purified yeast phosphofructokinase (PFK) show an ATP dependent kinetic cooperativity with respect to fructose-6-phosphate. In the presence of 25 mM phosphate, the cooperativity index (Hill number) is related to the half saturation concentration of fructose-6-phosphate as predicted by the concerted allosteric model in the case of a “K-system”. In the absence of phosphate, however, the kinetic behavior of yeast PFK is more complex and the cooperativity index is invariant with respect to the half saturation concentration of fructose-6-phosphate which is increased by ATP. In both cases, 5′AMP behaves as a strong activator of the enzyme. These kinetic data suggest that the two distinct functions of ATP as phosphate donnor and as allosteric inhibitor, respectively, are supported by different binding sites. These regulatory properties of yeast PFK are discussed in relation to glycolytic oscillations.     

Inhibition of E. coli CTP synthase by the "positive" allosteric effector GTP

Cytidine 5'-triphosphate (CTP) synthase catalyzes the ATP-dependent formation of CTP from UTP using either ammonia or l-glutamine as the source of nitrogen. When glutamine is the substrate, GTP is required as a positive allosteric effector to promote catalysis of glutamine hydrolysis. We show that at concentrations exceeding approximately 0.15 mM, GTP actually behaves as a negative allosteric effector of E. coli CTP synthase, inhibiting glutamine-dependent CTP formation. In addition, GTP inhibits NH(3)-dependent CTP formation in a concentration-dependent manner. However, GTP does not inhibit the enzyme's intrinsic glutaminase activity. Although the activation of CTP synthase by GTP does not display cooperative behavior, inhibition of both CTP synthase-catalyzed ammonia- and glutamine-dependent CTP synthesis by GTP do exhibit positive cooperativity. These results suggest that GTP binding affects CTP synthase catalysis in two ways: it activates enzyme-catalyzed glutamine hydrolysis and it inhibits the utilization of NH(3) as a substrate by the synthase domain.     

Is there a toxicological advantage for non-hyperbolic kinetics in cytochrome P450 catalysis? Functional allostery from "distributive catalysis"

The cytochrome P450s (CYPs) are the major enzymatic detoxification and drug metabolism system. Recently, it has become clear that several CYP isoforms exhibit positive and negative homotropic cooperativity. However, the toxicological implications of allosteric kinetics have not been considered, nor understood. The allosteric kinetics are particularly enigmatic in several respects. In many cases, CYPs bioactivate substrates to more toxic products, thus making it difficult to rationalize a functional advantage for positive cooperativity. Also, CYPs exhibit cooperativity with many structurally diverse ligands, in marked contrast to the specificity observed with other allosteric systems. Here, kinetic simulations are used to compare the probabilistic time- and concentration-dependent integrated toxicity function during conversion of substrate to product for CYP models exhibiting Michaelis-Menten (non-cooperative) kinetics, positive cooperativity, or negative cooperativity. The results demonstrate that, at low substrate concentrations, the slower substrate turnover afforded by cooperative CYPs compared with Michaelis-Menten enzymes can be a significant toxicological advantage, when toxic thresholds exist. When present, the advantage results from enhanced "distribution" of toxin in two pools, substrate and product, for an extended period, thus minimizing the chance that either exceeds its toxic threshold. At intermediate concentrations, the allosteric kinetics can be a modest advantage or modest disadvantage, depending on the kinetic parameters. However, at high substrate concentrations associated with a high probability of toxicity, fast turnover is desirable, and this advantage is provided also by the cooperative enzymes. For the positive homotropic cooperativity, the allosteric kinetics minimize the probability of toxicity over the widest range of system parameters. Furthermore, this apparent functional cooperativity is achieved without specific molecular recognition that is the hallmark of "traditional" allostery.     

Cooperative kinetics of both Hsp104 ATPase domains and interdomain communication revealed by AAA sensor-1 mutants.

AAA proteins share a conserved active site for ATP hydrolysis and regulate many cellular processes. AAA proteins are oligomeric and often have multiple ATPase domains per monomer, which is suggestive of complex allosteric kinetics of ATP hydrolysis. Here, using wild-type Hsp104 in the hexameric state, we demonstrate that its two AAA modules (NBD1 and NBD2) have very different catalytic activities, but each displays cooperative kinetics of hydrolysis. Using mutations in the AAA sensor-1 motif of NBD1 and NBD2 that reduce the rate of ATP hydrolysis without affecting nucleotide binding, we also examine the consequences of keeping each site in the ATP-bound state. In vitro, reducing k(cat) at NBD2 significantly alters the steady-state kinetic behavior of NBD1. Thus, Hsp104 exhibits allosteric communication between the two sites in addition to homotypic cooperativity at both NBD1 and NBD2. In vivo, each sensor-1 mutation causes a loss-of-function phenotype in two assays of Hsp104 function (thermotolerance and yeast prion propagation), demonstrating the importance of ATP hydrolysis as distinct from ATP binding at each site for Hsp104 function.     

Negative homotropic cooperativity in rat muscle AMP deaminase. A kinetic study on the inhibition of the enzyme by ATP

1. Rat skeletal muscle AMP deaminase (AMP aminohydrolase, EC 3.5.4.6) at optimal KCl concentrations shows a biphasic response to increasing levels of the allosteric inhibitor ATP. 2. Up to 10 micrometer, ATP appears to convert the enzyme to a form exhibiting sigmoidal kinetics while at higher concentrations its inhibitory effect is manifested by an alteration of AMP binding to AMP deaminase indicative of negative homotropic cooperativity at about 50% saturation. 3. AMP deaminase is inactivated by incubation with the periodate oxidation product of ATP. The (oxidized ATP)--AMP deaminase complex stabilized by NaBH4 reduction shows kinetic properties similar to those of the native enzyme in the presence of high ATP concentrations. 4. A plausible explanation of the observed cooperativity is that ATP induces different conformational state of AMP deaminase subunits, causing the substrate to follow a sequential mechanism of binding to enzyme. 5. Binding of the radioactive oxidized ATP shows that 3.2 mol of this reagent bind per mol AMP deaminase.     

GroEL stability and function. Contribution of the ionic interactions at the inter-ring contact sites

The chaperonin GroEL consists of a double ring structure made of identical subunits that display different modes of allosteric communication. The protein folding cycle requires the simultaneous positive intra-ring and negative inter-ring cooperativities of ATP binding. This ensures GroES binding to one ring and release of the ligands from the opposite one. To better characterize inter-ring allosterism, the thermal stability as well as the temperature dependence of the functional and conformational properties of wild type GroEL, a single ring mutant (SR1) and two single point mutants suppressing one interring salt bridge (E434K and E461K) were studied. The results indicate that ionic interactions at the two interring contact sites are essential to maintain the negative cooperativity for protein substrate binding and to set the protein thermostat at 39 degrees C. These electrostatic interactions contribute distinctly to the stability of the inter-ring interface and the overall protein stability, e.g. the E434K thermal inactivation curve is shifted to lower temperatures, and its unfolding temperature and activation energy are also lowered. An analysis of the ionic interactions at the inter-ring contact sites reveals that at the so called "left site" a network of electrostatic interactions involving three charged residues might be established, in contrast to what is found at the "right site" where only two oppositely charged residues interact. Our data suggest that electrostatic interactions stabilize protein-protein interfaces depending on both the number of ionic interactions and the number of residues engaged in each of these interactions. In the case of GroEL, this combination sets the thermostat of the protein so that the chaperonin distinguishes physiological from stress temperatures.     

A kinetic analysis of the nucleotide-induced allosteric transitions in a single-ring mutant of GroEL

The function of GroE requires a complex system of allosteric communication driven by protein-nucleotide interactions. These rearrangements couple the binding and hydrolysis of ATP to an overall reaction cycle in which substrate proteins are bound, encapsulated and released. Positive cooperativity with respect to ATP binding occurs within one heptameric ring of GroEL, while negative cooperativity between the two rings generates an inherent asymmetry between the two rings. A previously engineered mutant of GroEL in which the ring-ring contacts are broken gives rise to a single-ring version of the wild-type chaperonin (SR1). We have studied the kinetics of the nucleotide-induced conformational changes in a single-tryptophan variant of SR1 (Y485W-SR1) and compared the resulting data with those we reported previously for the double-ring, single-tryptophan variant of wild-type GroEL (Y485W-GroEL). Remarkably, the parting of the rings does not have a major effect on the conformational changes occurring within the heptameric ring and a kinetic model is presented to describe the sequence of structural rearrangements that occur upon ATP binding to the SR1 molecule. The observation that both the ATP-induced and ADP-induced conformational rearrangements occur more rapidly in the SR1 than they do in wild-type GroEL, indicates that intra-ring conformational changes in the double-ring structure must overcome conformational constraints provided by the presence of the second ring. Lastly, the data presented here imply a role for inter-ring allostery in controlling the dissociation-association behaviour of the GroES co-protein in the overall reaction cycle.     

Energy-dependent changes in conformation and catalytic activity of the chloroplast ATP synthase.

Energy-dependent activation of the chloroplast ATP synthase (CF0CF1) has been elucidated by investigating the conformational changes, the ADP effect, and the catalytic cooperativity of ATP hydrolysis. Conformational change was observed by measuring the reactivity of Lys-109 of the epsilon subunit of chloroplast coupling factor 1 with pyridoxal 5'-phosphate. In the postillumination dark, the Lys-109 reactivity decreased biphasically with half-times of less than 1 and 17 s. NH4Cl accelerated the slow phase decrease. Addition of ADP (0.2 microM) in the postillumination dark inactivated CF0CF1 (0.05 microM) with a half-time of 12 s. At high concentration of CF0CF1 (1.2 microM), inactivation occurred without exogenously added ADP with a half-time of 12 s. Accompanying the inactivation, the positive catalytic cooperativity of ATP hydrolysis decreased. Addition of 10 mM NH4Cl before ADP (0.2 microM) decelerated the ADP-induced inactivation to a half-time of 64 s. Throughout this inactivation, the positive catalytic cooperativity was maintained at a high level. These results suggest three distinct conformations of CF0CF1, EH, EM, and EL, and their ADP binding forms EM-ADP and EL-ADP. EH, EM, and EL have a low affinity for ADP, a high affinity for ADP, and low accessibility to ADP, respectively. EM and EL exhibit highly cooperative ATP hydrolysis. ATP hydrolysis catalyzed by EM-ADP exhibits no cooperativity. EL-ADP is inactive.     

ATPase cycle of an archaeal chaperonin

Recent structural data imply differences in allosteric behavior of the group I chaperonins, typified by GroEL from Escherichia coli, and the group II chaperonins, which comprise archaeal thermosome and eukaryotic TRiC/CCT. Therefore, this study addresses the mechanism of interaction of adenine nucleotides with recombinant alpha-only and native alphabeta-thermosomes from Thermoplasma acidophilum, which also enables us to analyze the role of the heterooligomeric composition of the natural thermosome. Although all subunits of the alpha-only thermosome seem to bind nucleotides tightly and independently, the native chaperonin has two different classes of ATP-binding sites. Furthermore, for the alpha-only thermosome, the steady-state ATPase rate is determined by the cleavage reaction itself, whereas, for the alphabeta-thermosome, the rate-limiting step is associated with a post-hydrolysis isomerisation into a non-covalent ADP*P(i) species prior to the release of the gamma-phosphate group. After half-saturation with ATP, a negative cooperativity in hydrolysis is observed for both thermosomes. The effect of Mg(2+) and K(+) nucleotide cycling is documented. We conclude that archaeal chaperonins have unique allosteric properties and discuss them in the light of the mechanism established for the group I chaperonins.     

ATP-induced allostery in the eukaryotic chaperonin CCT is abolished by the mutation G345D in CCT4 that renders yeast temperature-sensitive for growth

Saccharomyces cerevisiae yeast cells containing the chaperonin CCT (chaperonin-containing t-complex polypeptide 1 (TCP-1)) with the G345D mutation in subunit CCT4 (anc2-1) are temperature-sensitive for growth and display defects in organization of actin structure, budding and cell shape. In this first structure-function analysis of CCT, we show that this mutation abolishes both intra- and inter-ring cooperativity in ATP binding by CCT. The finding that a single mutation in only one subunit in each CCT ring has such drastic effects highlights the importance of allostery for its in vivo function. These results, together with other kinetic data for wild-type CCT reported in this study, provide support for the sequential model for ATP-dependent allosteric transitions in CCT.